RNA INTERFERENCE-INDUCING NUCLEIC ACID INHIBITING NONCANONICAL TARGETS OF MICRO RNA, AND USE FOR SAME

TECHNICAL FIELD

The present invention relates to an RNA interference-inducing nucleic acid which inhibits gene expression and a use thereof, and particularly, to an interference-inducing nucleic acid having a useful effect exhibited by selectively suppressing a noncanonical target of microRNA and a use thereof.

This application claims priority to and the benefit of Korean Patent Application No. 10-2018-0063054, filed on May 31, 2018, Korean Patent Application No. 10-2018-0063055, filed on May 31, 2018, Korean Patent Application No.10-2019-0064333, filed on May 31, 2019, Korean Patent Application No.10-2019-0064334, filed on May 31, 2019, Korean Patent Application No.10-2019-0064335, filed on May 31, 2019, and Korean Patent Application No. 10-2019-0064386, filed on May 31, 2019, the disclosures of which are incorporated herein by reference in their entirety.

BACKGROUND ART

RNA interference is a phenomenon of inhibiting gene expression at the post-transcription level. The RNA interference phenomenon naturally occurring is caused by microRNA (miRNA). MiRNA consists of 18 to 25 nucleotides, and most miRNAs are small RNA consisting of approximately 21 nucleotides and have base sequences complementary to messenger RNAs (mRNAs) of a target gene by Argonaute protein. Animal miRNAs are associated with the Argonaute protein to have partial base pairing with target mRNAs. In this case, when at least 6 consecutive bases are paired in a seed region defined by nucleotides from positions 1 to 8 based on the 5′ end of miRNA, this sequence is recognized as a target, and most significantly, when at least 6 bases in positions 2 to 7 from the 5′ end consecutively pair with target mRNA, the expression of the mRNA is suppressed by sufficiently degrading the corresponding target mRNA or inhibiting translation (Lewis B P, et. al, 2003, Cell, 115 (7), 787-98). Since the miRNA recognizes mRNA of a target gene through pair base pairing, one miRNA may usually affect the expression of hundreds to thousands of genes.

The suppressive action of miRNA on target gene expression is one of the major mechanisms of gene expression, is involved in differentiation and growth of cells under normal circumstances, and causes cancer, a degenerative disease or diabetes when there is a functional abnormality, and therefore, miRNA attracts attention as a key to life. Accordingly, to artificially induce the gene expression suppressive action of miRNA, an RNA interference material (siRNA or shRNA) having a seed region of miRNA is designed to be transfected into cells, thereby artificially differentiating cells or changing their functions, and in some cases, being used as a therapeutic for diseases. Therefore, complementary base pairing in the miRNA seed region which recognizes target mRNA by Argonaute is important for exhibiting a proper function of an RNA interference material such as miRNA. Particularly, to apply such an RNA interference material, it is required to identify each function of miRNA. Here, the function of miRNA is determined according to which target gene is suppressed, and thus analysis for all target genes (target) of miRNA is required.

At the transcriptome level, research on a miRNA target complex was first conducted through Ago HITS-CLIP (or called CLIP-Seq) by the inventors. Ago HITS CLIP assay is a method of forming a complex by covalent bonds between RNA and Argonaute protein (Ago) in cells through UV irradiation of cells or a tissue sample, isolating the RNA-Ago complex through immunoprecipitation using an Ago-specific recognition antibody, and analyzing the isolated RNA through next-generation sequencing. Accordingly, Ago-bound miRNA, a target mRNA group complementarily pairing therewith and its position can be exactly analyzed (Chi S et al, Nature, 2009, 460 (7254): 479-86).

As a result, the inventors identified that miRNA may bind to target mRNA although not binding with exact complementarity with a miRNA seed region. More specifically, binding of the miRNA seed region defined by a base sequence in positions 1 to 8 from the 5′ end of miRNA with perfect pairing with at least 6 consecutive bases, and particularly, most significantly by base pairing in position 2 to 7 from the 5′ end, with mRNA is referred to as canonical target recognition. Although not with consecutive and exact complementarity with the miRNA seed region, which deviates from the above-described rule, recognition as a target of miRNA and binding to miRNA is referred to as non-canonical target recognition. According to the result of Ago HITS-CLIP assay, it can be seen that the frequency of non-canonical target recognition through a seed region by miRNA is approximately 50% of a canonical recognition frequency.

Accordingly, it can be seen that the conventional RNA interference material (e.g., siRNA or shRNA) designed to include the sequence of the miRNA seed region has a biological function by suppressing several non-canonical target genes in addition to a canonical target gene.

DISCLOSURE
Technical Problem

Therefore, the present invention is directed to solving a limitation of the conventional RNA interference material designed to include the intact sequence of miRNA seed region and improving is efficiency.

More specifically, the conventional RNA interference material designed to include the intact sequence of the miRNA seed region suppresses both a canonical target gene and a non-canonical target gene, and the canonical target gene is strongly suppressed, but the non-canonical target gene is very weakly suppressed. Therefore, the present invention is directed to providing an RNA interference-inducing nucleic acid having an effect of efficiently improving biological functions exhibited by suppressing a non-canonical target gene by conventional miRNA, or selectively exhibiting one of the biological functions, that is, only biological functions exhibited by suppressing a non-canonical target gene by conventional miRNA.

Technical Solution

To solve the above-described problems, the present invention provides an RNA interference-inducing nucleic acid which suppresses a non-canonical target gene of miRNA by modifying a partial sequence of specific miRNA.

More specifically,

the present invention provides an RNA interference-inducing nucleic acid, which suppresses a non-canonical target gene of miRNA by modifying a partial sequence of specific miRNA in one or more single strands of the double strands of the nucleic acid inducing RNA interference,

wherein the RNA interference-inducing nucleic acid has a base sequence in positions 2 to 7 from the 5′ end of specific miRNA, which is the most significant site involving in pairing with target mRNA, the base sequence having the sequence of four bases in positions 2 to 5 from the 5′ end and the 6^thand 7^thbases that are the same and complementary to a base capable of pairing with the 6^thbase of the specific miRNA, including all complementary bases including G:A and G:U wobble pairs, to allow non-canonical target base pairs bound to a bulge generated in the target gene between positions 5 and 6 of the miRNA to be a consecutive base pair by the disappearance of the bulge, or

the RNA interference-inducing nucleic acid has a modified base sequence in which at least one guanine base is substituted with uracil or adenine, preferably, in a sequence in positions 2 to 7 based on the 5′ end of a base sequence between 1 to 9 bases from the 5′ end of miRNA, such that a G:A or G:U wobble pair at the corresponding site becomes a canonical base pair of U:A or A:U.

Preferably, the specific miRNA has the same seed sequence selected from the group consisting of miR-124, miR-155, miR-122, miR-1, let-7, miR-133, miR-302 and miR-372, and consists of 18 to 24 bases.

Preferably, the RNA interference-inducing nucleic acid has any one or more of the sequences of bases in positions 2 to 7 from the 5′ end:

an RNA interference-inducing nucleic acid having a base sequence of 5′-AA GGC C-3′ (miR-124-BS) as an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-124 (SEQ ID NO: 1);

an RNA interference-inducing nucleic acid having a base sequence of 5′-GG AGU U-3′ (miR-122-BS) as an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-122 (SEQ ID NO: 2);

an RNA interference-inducing nucleic acid having a base sequence of 5′-UA AUG G-3′ (miR-155-BS) as an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-155 (SEQ ID NO: 3); or

an RNA interference-inducing nucleic acid having a base sequence of 5′-GG AAU U-3′ (miR-1-BS) as an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-1 (SEQ ID NO: 4).

Preferably, the base sequence of the RNA interference-inducing nucleic acid is represented by any one or more as follows:

an RNA interference-inducing nucleic acid having a base sequence of 5′-UAA GGC CAC GCG GUG AAU GCC-3′ (miR-124-BS) as an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-124 (SEQ ID NO: 5);

an RNA interference-inducing nucleic acid having a base sequence of 5′-UGG AGU UGU GAC AAU GGU GUU-3′ (miR-122-BS) as an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-122 (SEQ ID NO: 6);

an RNA interference-inducing nucleic acid having a base sequence of 5′-UUA AUG GCUAAU CGU GAU AGG-3′ (miR-155-BS) as an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-155 (SEQ ID NO: 7); or

an RNA interference-inducing nucleic acid having a base sequence of 5′-UGG AAU UGU AAA GAA GUA UGU-3′ (miR-1-BS) as an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-1 (SEQ ID NO: 8).

Preferably, the RNA interference-inducing nucleic acid is any one or more of the base sequences between the 1^stto 9^thbases from the 5′ end:

an RNA interference-inducing nucleic acid preferably having a modified base sequence in which at least one guanine is substituted with uracil among the sequence in positions 2 to 7 from the 5′ end, such as a base sequence of 5′-UAA UGC ACG-3′ (miR-124-G4U) (SEQ ID NO: 9), 5′-UAA GUC ACG-3′ (miR-124-G5U) (SEQ ID NO: 10) or 5′-UAA UUC ACG-3′ (miR-124-G4,5U) (SEQ ID NO: 11), as the RNA interference-inducing nucleic acid suppressing the non-canonical target gene of miR-124;

an RNA interference-inducing nucleic acid preferably having a modified base sequence in which at least one guanine is substituted with uracil among the sequence in positions 2 to 7 from the 5′ end, such as a base sequence of 5′-UUG AAU GUA-3′ (miR-1-G2U) (SEQ ID NO: 12), 5′-UGU AAU GUA-3′ (miR-1-G3U) (SEQ ID NO: 13), 5′-UGG AAU UUA-3′ (miR-1-G7U) (SEQ ID NO: 14), 5′-UUU AAU GUA-3′ (miR-1-G2,3U) (SEQ ID NO: 15), 5′-UGU AAU UUA-3′ (miR-1-G3,7U) (SEQ ID NO: 16), 5′-UUG AAU UUA-3′ (miR-1-G2,7U) (SEQ ID NO: 17) or 5′-UUU AAU UUA-3′ (miR-1-G2,3,7U) (SEQ ID NO: 18), as the RNA interference-inducing nucleic acid suppressing the non-canonical target gene of miR-1;

as an RNA interference-inducing nucleic acid suppressing the non-canonical target gene of miR-122,

an RNA interference-inducing nucleic acid having a base sequence of 5′-UUG AGU GUG-3′ (miR-122-G2U) (SEQ ID NO: 19), 5′-UGU AGU GUG-3′ (miR-122-G3U) (SEQ ID NO: 20), 5′-UGG AUU GUG-3′ (miR-122-G5U) (SEQ ID NO: 21), 5′-UGG AGU UUG-3′ (miR-122-G7U) (SEQ ID NO: 22), 5′-UGG AGU GUU-3′ (miR-122-G9U) (SEQ ID NO: 23), 5′-UUU AGU GUG-3′ (miR-122-G2,3U) (SEQ ID NO: 24), 5′-UUG AUU GUG-3′ (miR-122-G2,5U) (SEQ ID NO: 25), 5′-UUG AGUUUG-3′ (miR-122-G2,7U) (SEQ ID NO: 26), 5′-UUG AGU GUU-3′ (miR-122-G2,9U) (SEQ ID NO: 27), 5′-UGU AUU GUG-3′ (miR-122-G3,5U) (SEQ ID NO: 28), 5′-UGU AGU UUG-3′ (miR-122-G3,7U) (SEQ ID NO: 29), 5′-UGU AGU GUU-3′ (miR-122-G3,9U) (SEQ ID NO: 30), 5′-UGG AUU UUG-3′ (miR-122-G5,7U) (SEQ ID NO: 31), 5′-UGG AUU GUU-3′ (miR-122-G5,9U) (SEQ ID NO: 32), or 5′-UGG AGU UUU-3 (miR-122-G7,9U) (SEQ ID NO: 33), and preferably, having a modified base sequence in which at least one guanine is substituted with uracil among the sequence in positions 2 to 7 from the 5′ end, such as 5′-UUG AGU GUG-3′ (miR-122-G2U) (SEQ ID NO: 19), 5′-UGU AGU GUG-3′ (miR-122-G3U) (SEQ ID NO: 20), 5′-UGG AUU GUG-3′ (miR-122-G5U) (SEQ ID NO: 21), 5′-UGG AGU UUG-3′ (miR-122-G7U) (SEQ ID NO: 22), 5′-UUUAGU GUG-3′ (miR-122-G2,3U) (SEQ ID NO: 24), 5′-UUG AUU GUG-3′ (miR-122-G2,5U) (SEQ ID NO: 25), 5′-UUG AGUUUG-3′ (miR-122-G2,7U) (SEQ ID NO: 26), 5′-UGU AUU GUG-3′ (miR-122-G3,5U) (SEQ ID NO: 28), 5′-UGU AGU UUG-3′ (miR-122-G3,7U) (SEQ ID NO: 29) or 5′-UGG AUU UUG-3′ (miR-122-G5,7U) (SEQ ID NO: 31);

an RNA interference-inducing nucleic acid having a modified base sequence in which at least one guanine is substituted with uracil in the sequence in positions 2 to 7 from the 5′ end, such as a base sequence of 5′-UUU UGU CCC-3′ (miR-133-G4U) (SEQ ID NO: 34), 5′-UUU GUU CCC-3′ (miR-133-G5U) (SEQ ID NO: 35) or 5′-UUU UUU CCC-3′(miR-133-G4,5U) (SEQ ID NO: 36) as an RNA interference-inducing nucleic acid suppressing the non-canonical target gene of miR-133;

an RNA interference-inducing nucleic acid having a base sequence of 5′-UUA GGU AGU-3′ (let-7-G2U) (SEQ ID NO: 37), 5′-UGA UGU AGU-3′ (let-7-G4U) (SEQ ID NO: 38), 5′-UGA GUU AGU-3′ (let-7-G5U) (SEQ ID NO: 39), 5′-UGA GGU AUU-3′ (let-7-G8U) (SEQ ID NO: 40), 5′-UUA UGU AGU-3′ (let-7-G2,4U) (SEQ ID NO: 41), 5′-UUA GUU AGU-3′ (let-7-G2,5U) (SEQ ID NO: 42), 5′-UUA GGU AUU-3′ (let-7-G2,8U) (SEQ ID NO: 43), 5′-UGA UUU AGU-3′ (let-7-G4,5U) (SEQ ID NO: 44), 5′-UGA UGU AUU-3′ (let-7-G4,8U) (SEQ ID NO: 45), or 5′-UGA GUUAUU-3′ (let-7-G5,8U) (SEQ ID NO: 46), and preferably, having a modified base sequence in which at least one guanine is substituted with uracil in the sequence in positions 2 to 7 based on the 5′ end, such as 5′-UUA GGU AGU-3′ (let-7-G2U) (SEQ ID NO: 37), 5′-UGA UGU AGU-3′ (let-7-G4U) (SEQ ID NO: 38), 5′-UGA GUU AGU-3′ (let-7-G5U) (SEQ ID NO: 39), 5′-UUA UGU AGU-3′ (let-7-G2,4U) (SEQ ID NO: 41), 5′-UUA GUU AGU-3′ (let-7-G2,5U) (SEQ ID NO: 42) or 5′-UGAUUU AGU-3′ (let-7-G4,5U) (SEQ ID NO: 44) as an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of let-7;

an RNA interference-inducing nucleic acid preferably having a modified base sequence in which at least one guanine is substituted with uracil in the sequence in positions 2 to 7 based on the 5′ end, such as 5′-UAA UUG CUU-3′ (miR-302a-G4U) (SEQ ID NO: 47), 5′-UAA GUU CUU-3′ (miR-302a-G6U) (SEQ ID NO: 48), or 5′-UAA UUU CUU-3′ (miR-302a-G4,6U) (SEQ ID NO: 49), as an RNA interference-inducing nucleic acid suppressing the non-canonical target gene of miR-302a; or

an RNA interference-inducing nucleic acid preferably having a modified base sequence in which at least one guanine is substituted with uracil in the sequence in positions 2 to 7 from the 5′ end, such as 5′-AAAUUG CUG-3′ (miR-372-G4U) (SEQ ID NO: 50), 5′-AAA GUU CUG-3′ (miR-372-G6U) (SEQ ID NO: 51), 5′-AAA GUG CUU-3′ (miR-372-G9U) (SEQ ID NO: 52), 5′-AAA UUU CUG-3′ (miR-372-G4,6U) (SEQ ID NO: 53), 5′-AAA UUG CUU-3′ (miR-372-G4,9U) (SEQ ID NO: 54), or 5′-AAA GUU CUU-3′ (miR-372-G6,9U) (SEQ ID NO: 55), as an RNA interference-inducing nucleic acid suppressing the non-canonical target gene of miR-372.

Preferably, the RNA interference-inducing nucleic acid has one or more of the following base sequences:

as an RNA interference-inducing nucleic acid suppressing the non-canonical target gene of miR-124,

an RNA interference-inducing nucleic acid having a base sequence of 5′-UAA UGC ACG CGG UGAAUG CCA A-3′ (miR-124-G4U) (SEQ ID NO: 56), 5′-UAA GUC ACG CGG UGAAUG CCA A-3′ (miR-124-G5U) (SEQ ID NO: 57) or 5′-UAA UUC ACG CGG UGAAUG CCAA-3′(miR-124-G4,5U) (SEQ ID NO: 58);

as an RNA interference-inducing nucleic acid suppressing the non-canonical target gene of miR-1,

an RNA interference-inducing nucleic acid having a base sequence of 5′-UUG AAU GUA AAG AAG UAU GUA U-3′ (miR-1-G2U) (SEQ ID NO: 59), 5′-UGU AAU GUA AAG AAG UAU GUA U-3′ (miR-1-G3U) (SEQ ID NO: 60), 5′-UGG AAU UUA AAG AAG UAU GUA U-3′ (miR-1-G7U) (SEQ ID NO: 61), 5′-UUU AAU GUA AAG AAG UAU GUA U-3′ (miR-1-G2,3U) (SEQ ID NO: 62), 5′-UGU AAU UUA AAG AAG UAU GUA U-3′ (miR-1-G3,7U) (SEQ ID NO: 63), 5′-UUG AAU UUAAAG AAG UAU GUA U-3′ (miR-1-G2,7U) (SEQ ID NO: 64) or 5′-UUU AAU UUAAAG AAG UAU GUA U-3′ (miR-1-G2,3,7U) (SEQ ID NO: 65);

as an RNA interference-inducing nucleic acid suppressing the non-canonical target gene of miR-122,

an RNA interference-inducing nucleic acid having a base sequence of 5′-UUG AGU GUG ACA AUG GUG UUU G-3′ (miR-122-G2U) (SEQ ID NO: 66), 5′-UGU AGU GUG ACA AUG GUG UUU G-3 (miR-122-G3U) (SEQ ID NO: 67), 5′-UGG AUU GUG ACA AUG GUG UUU G-3′ (miR-122-G5U) (SEQ ID NO: 68), 5′-UGG AGU UUG ACA AUG GUG UUU G-3′ (miR-122-G7U) (SEQ ID NO: 69), 5′-UGG AGU GUU ACA AUG GUG UUU G-3′ (miR-122-G9U) (SEQ ID NO: 70), 5′-UUU AGU GUG ACA AUG GUG UUU G-3′ (miR-122-G2,3U) (SEQ ID NO: 71), 5′-UUG AUU GUG ACA AUG GUG UUU G-3′ (miR-122-G2,5U) (SEQ ID NO: 72), 5′-UUG AGU UUG ACA AUG GUG UUU G-3′ (miR-122-G2,7U) (SEQ ID NO: 73), 5′-UUG AGU GUU ACA AUG GUG UUU G-3′ (miR-122-G2,9U) (SEQ ID NO: 74), 5′-UGU AUU GUG ACA AUG GUG UUU G-3 (miR-122-G3,5U) (SEQ ID NO: 75), 5′-UGU AGU UUG ACA AUG GUG UUU G-3 (miR-122-G3,7U) (SEQ ID NO: 76), 5′-UGU AGU GUU ACA AUG GUG UUU G-3 (miR-122-G3,9U) (SEQ ID NO: 77), 5′-UGG AUU UUG ACA AUG GUG UUU G-3′ (miR-122-G5,7U) (SEQ ID NO: 78), 5′-UGG AUU GUU ACA AUG GUG UUU G-3′ (miR-122-G5,9U) (SEQ ID NO: 79) or 5′-UGG AGU UUU ACA AUG GUG UUU G-3′ (miR-122-G7,9U) (SEQ ID NO: 80);

as an RNA interference-inducing nucleic acid suppressing the non-canonical target gene of miR-133, an RNA interference-inducing nucleic acid having the base sequence of 5′-UUU UGU CCC CUU CAA CCA GCU G -3′ (miR-133-G4U) (SEQ ID NO: 81), 5′-UUU GUU CCC CUU CAA CCA GCU G-3′ (miR-133-G5U) (SEQ ID NO: 82) or 5′-UUU UUU CCC CUU CAA CCA GCU G-3′(miR-133-G4,5U) (SEQ ID NO: 83);

as an RNA interference-inducing nucleic acid suppressing the non-canonical target gene of let-7, an RNA interference-inducing nucleic acid having a base sequence of 5′-UUA GGU AGU AGG UUG UAU AGU U-3′ (let-7-G2U) (SEQ ID NO: 84), 5′-UGA UGU AGU AGG UUG UAU AGU U-3′ (let-7-G4U) (SEQ ID NO: 85), 5′-UGA GUU AGU AGG UUG UAU AGU U-3′ (let-7-G5U) (SEQ ID NO: 86), 5′-UGA GGU AUU AGG UUG UAU AGU U-3′ (let-7-G8U) (SEQ ID NO: 87), 5′-UUA UGU AGU AGG UUG UAU AGU U-3′ (let-7-G2,4U) (SEQ ID NO: 88), 5′-UUA GUU AGU AGG UUG UAU AGU U-3′ (let-7-G2,5U) (SEQ ID NO: 89), 5′-UUA GGU AUU AGG UUG UAU AGU U-3′ (let-7-G2,8U) (SEQ ID NO: 90), 5′-UGA UUU AGU AGG UUG UAU AGU U-3′ (let-7-G4,5U) (SEQ ID NO: 91), 5′-UGA UGU AUU AGG UUG UAU AGU U-3′ (let-7-G4,8U) (SEQ ID NO: 92) or 5′-UGA GUU AUU AGG UUG UAU AGU U-3′ (let-7-G5,8U) (SEQ ID NO: 93);

as an RNA interference-inducing nucleic acid suppressing the non-canonical target gene of miR-302a, an RNA interference-inducing nucleic acid having a base sequence of 5′-UAA UUG CUU CCA UGU UUU GGU GA-3′ (miR-302a-G4U) (SEQ ID NO: 94), 5′-UAA GUU CUU CCA UGU UUU GGU GA-3′ (miR-302a-G6U) (SEQ ID NO: 95), or 5′-UAA UUU CUU CCA UGU UUU GGU GA-3′ (miR-302a-G4,6U) (SEQ ID NO: 96); or

as an RNA interference-inducing nucleic acid suppressing the non-canonical target gene of miR-372, an RNA interference-inducing nucleic acid having a base sequence of 5′-AAA UUG CUG CGA CAU UUG AGC GU-3′ (miR-372-G4U) (SEQ ID NO: 97), 5′-AAA GUU CUG CGA CAU UUG AGC GU-3′ (miR-372-G6U) (SEQ ID NO: 98), 5′-AAA GUG CUU CGA CAU UUG AGC GU-3′ (miR-372-G9U) (SEQ ID NO: 99), 5′-AAA UUU CUG CGA CAU UUG AGC GU-3′ (miR-372-G4,6U) (SEQ ID NO: 100), 5′-AAA UUG CUU CGA CAU UUG AGC GU-3′ (miR-372-G4,9U) (SEQ ID NO: 101) or 5′-AAA GUU CUU CGA CAU UUG AGC GU-3′ (miR-372-G6,9U) (SEQ ID NO: 102).

The present invention provides a composition for inhibiting the expression of a non-canonical target gene of miRNA, which includes an RNA interference-inducing nucleic acid.

The present invention provides a method of inhibiting the expression of a non-canonical target gene of miRNA, which includes administering the composition including an RNA interference-inducing nucleic acid into a subject.

The present invention provides the use of an RNA interference-inducing nucleic acid for inhibiting the expression of a non-canonical target gene of miRNA.

The present invention provides a composition for regulating cell cycling, differentiation, dedifferentiation, morphology, migration, division, proliferation or apoptosis, which includes an RNA interference-inducing nucleic acid.

The present invention provides a method of regulating cell cycling, differentiation, dedifferentiation, morphology, migration, division, proliferation or apoptosis, which includes administering the composition including an RNA interference-inducing nucleic acid into a subject.

The present invention provides the use of an RNA interference-inducing nucleic acid for regulating cell cycling, differentiation, dedifferentiation, morphology, migration, division, proliferation or apoptosis.

Preferably, the composition is any one or more of the compositions selected from:

a composition for inducing cancer cell death, which includes an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-124;

a composition for inducing neurite differentiation, which includes an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-124;

a composition for inducing cell cycle arrest in liver cancer cells, which includes an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-122;

a composition for promoting differentiation of muscle cells or muscle fibrosis, which includes an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-1;

a composition for inducing muscle cell death, which includes an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-155;

a composition for inducing cell death of neuroblastomas, which includes an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-124;

a composition for promoting cell division or proliferation of neuroblastomas, which includes an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-124;

a composition for inducing myocardial hypertrophy, which includes an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-1;

a composition for inducing myocardial hypertrophy, which includes an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-133;

a composition for inducing cell cycle arrest in cancer cells, which includes an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of let-7;

a composition for inducing the cell cycle progressing activity of hepatocytes, which includes an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of let-7;

a composition for promoting dedifferentiation, which includes an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-302a;

a composition for promoting dedifferentiation, which includes an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-372; or

a composition for inhibiting cell migration of liver cancer cells, which includes an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-122.

The present invention provides a method of preparing an RNA interference-inducing nucleic acid, which inhibits the expression of a non-canonical target gene of the miRNA by modifying a partial sequence of specific miRNA in one or more single strands of the double strands of the nucleic acid inducing RNA interference, the method including the following steps:

constructing an RNA interference-inducing nucleic acid having a sequence of four bases in positions 2 to 5 from the 5′ end of the specific miRNA, and bases in positions 6 and 7, which are the same and complementary to a base capable of pairing with the 6^thbase of the specific miRNA, including all complementary bases including G:A and G:U wobble pairs, to allow non-canonical target base pairs bound to a bulge generated in the target gene between positions 5 and 6 of the miRNA to be a consecutive base pair by the disappearance of the bulge, or

constructing an RNA interference-inducing nucleic acid having a modified base sequence in which at least one guanine is substituted with uracil in a base sequence between the first to ninth bases from the 5′ end of specific miRNA, in which a G:A or G:U wobble pair at the corresponding site becomes a canonical base sequence of U:A or A:U.

In addition, the present invention provides a method of screening a test material for regulating cell cycling, differentiation, dedifferentiation, morphology, migration, division, proliferation or death, which includes the following steps:

transfecting an RNA interference-inducing nucleic acid into a target cell;

treating the target cell with a test material; and

confirming the expression level or expression of a non-canonical target gene of miRNA suppressing an RNA interference-inducing nucleic acid in target cells.

In addition, the present invention provides an RNA interference-inducing nucleic acid as follows:

1. 2′OMe-Modified RNA Interference-Inducing Nucleic Acid

The present invention provides an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miRNA by modifying a partial sequence of specific miRNA in one or more single strands of the double strands of the nucleic acid inducing RNA interference, wherein

the RNA interference-inducing nucleic acid has a sequence of four bases in positions 2 to 5 from the 5′ end of the specific miRNA, and bases in positions 6 and 7, which are the same and complementary to a base capable of pairing with the 6^thbase of the specific miRNA, including all complementary bases including G:A and G:U wobble pairs, to allow non-canonical target base pairs bound to a bulge generated in the target gene between positions 5 and 6 of the miRNA to be a consecutive base pair by the disappearance of the bulge, and

the specific miRNA is characterized by adding a methyl group (2′OMe) to the 2′ position of the ribosyl ring of the 6^thnucleotide from the 5′ end.

Preferably, the RNA interference-inducing nucleic acid inhibits only the expression of a canonical seed target gene of the corresponding RNA interference-inducing nucleic acid.

Preferably, the RNA interference-inducing nucleic acid is characterized by specifically suppressing a non-canonical nucleation bulge site of the specific miRNA, and removing a non-canonical nucleation bulge site which may be newly generated.

In addition, the present invention provides a composition for inhibiting the expression of a non-canonical target gene of miRNA, which includes an RNA interference-inducing nucleic acid.

In addition, the present invention provides a method of inhibiting the expression of a non-canonical target gene of miRNA, which includes an RNA interference-inducing nucleic acid.

In addition, the present invention provides the use of an RNA interference-inducing nucleic acid for inhibiting the expression of a non-canonical target gene of miRNA.

In addition, the present invention provides a method of preparing an RNA interference-inducing nucleic acid, which inhibits the expression of a non-canonical target gene of the miRNA by modifying a partial sequence of specific miRNA in one or more single strands of the double strands of the nucleic acid inducing RNA interference, the method including the following steps:

adding a methyl group (2′OMe) to the 2′ position of the ribosyl ring of the 6^thnucleotide from the 5′ end of the specific miRNA.

2. RNA Interference-Inducing Nucleic Acids Suppressing Non-Canonical Nucleation Bulge Target Site of miRNA (SEQ ID NOs: 103 to 528)

The present invention provides an RNA interference-inducing nucleic acid, which suppresses a non-canonical target gene of miRNA by modifying a partial sequence of specific miRNA in one or more single strands of the double strands of the nucleic acid inducing RNA interference, wherein

Preferably, the RNA interference-inducing nucleic acid is characterized by selectively suppressing only a non-canonical nucleation bulge site and not suppressing a canonical target gene of miRNA.

Preferably, the specific miRNA consists of 6 to 24 bases, which includes the sequence of four bases in positions 2 to 5 from the 5′ end of the same seed sequence of one selected from the group consisting of let-7/98/4458/4500, miR-125a-5p/125b-5p/351/670/4319, miR-124/124ab/506, miR-9/9ab, miR-29abcd, miR-103a/107/107ab, miR-221/222/222ab/1928, miR-26ab/1297/4465, miR-15abc/16/16abc/195/322/424/497/1907, miR-126-3p, miR-30abcdef/30abe-5p/384-5p, miR-33ab/33-5p, miR-34ac/34bc-5p/449abc/449c-5p, miR-19ab, miR-99ab/100, miR-17/17-5p/20ab/20b-5p/93/106ab/427/518a-3p/519d, miR-27abc/27a-3p, miR-218/218a, miR-22/22-3p, miR-185/882/3473/4306/4644, miR-181abcd/4262, miR-338/338-3p, miR-127/127-3p, miR-101/101ab, miR-149, miR-324-5p, miR-24/24ab/24-3p, miR-33a-3p/365/365-3p, miR-139-5p, miR-138/138ab, miR-143/1721/4770, miR-25/32/92abc/363/363-3p/367, miR-574-5p, miR-7/7ab, miR-145, miR-135ab/135a-5p, miR-148ab-3p/152, miR-28-5p/708/1407/1653/3139, miR-130ac/301ab/301b/301b-3p/454/721/4295/3666, miR-3132, miR-155, miR-485-3p, miR-132/212/212-3p, hsa-miR-9-3p, miR-374ab, miR-129-3p/129ab-3p/129-1-3p/129-2-3p, hsa-miR-126-5p, miR-425/425-5p/489, miR-423-3p, miR-21/590-5p, miR-31, hsa-miR-20b-3p, hsa-let-7d-3p, miR-191, miR-18ab/4735-3p, miR-369-3p, hsa-miR-5187-5p, miR-382, miR-485-5p/1698/1703/1962, hsa-miR-136-3p, miR-576-3p, miR-204/204b/211, miR-769-5p, miR-342-5p/4664-5p, miR-361-5p, miR-199ab-3p/3129-5p, miR-142-3p, miR-299-5p/3563-5p, miR-193/193b/193a-3p, hsa-miR-1277-5p, miR-140/140-5p/876-3p/1244, hsa-miR-30a/d/e-3p, hsa-let-7i-3p, miR-409-5p/409a, miR-379/1193-5p/3529, miR-136, miR-154/872, miR-4684-3p, miR-361-3p, miR-335/335-5p, miR-423a/423-5p/3184/3573-5p, miR-371/373/371b-5p, miR-1185/3679-5p, miR-3613-3p, miR-93/93a/105/106a/291a-3p/294/295/302abcde/372/373/428/519a/520be/520acd-3p/1378/1420ac, miR-876-5p/3167, miR-329/329ab/362-3p, miR-582-5p, miR-146ac/146b-5p, miR-380/380-3p, miR-499-3p/499a-3p, miR-551a, miR-142-5p, hsa-miR-17-3p, miR-199ab-5p, miR-542-3p, miR-1277, hsa-miR-29c-5p, miR-3145-3p, hsa-miR-106b-3p, hsa-miR-22-5p, miR-744/1716, hsa-miR-132-5p, miR-488, miR-501-3p/502-3p/500/502a, miR-486-5p/3107, miR-450a/451a, hsa-miR-30c-3p, miR-499-5p, miR-421, miR-197, miR-296-5p, miR-326/330/330-5p, miR-214/761/3619-5p, miR-612/1285/3187-5p, miR-409-3p, miR-378/422a/378bcdefhi, miR-342-3p, miR-338-5p, miR-625, miR-200bc/429/548a, hsa-miR-376a-5p, miR-584, miR-411, miR-573/3533/3616-5p/3647-5p, miR-885-5p, hsa-miR-99-3p, miR-876-3p, miR-654-3p, hsa-miR-340-3p, miR-3614-5p, hsa-miR-124-5p, miR-491-5p, miR-96/507/1271, miR-548a-3p/548ef/2285a, hsa-miR-32-3p, miR-3942-5p/4703-5p, miR-34b/449c/1360/2682, hsa-miR-23a/b-5p, miR-362-5p/500b, miR-677/4420, miR-577, miR-3613-5p, miR-369-5p, miR-150/5127, miR-544/544ab/544-3p, hsa-miR-29a-5p, miR-873, miR-3614-3p, miR-186, miR-483-3p, hsa-miR-374a-3p, miR-196abc, hsa-miR-145-3p, hsa-miR-29b-2-5p, hsa-miR-221-5p, miR-323b-3p, miR-616, miR-330-3p, hsa-miR-7-3p, miR-187, hsa-miR-26a-3p, miR-452/4676-3p, miR-129-5p/129ab-5p, miR-223, miR-4755-3p, miR-1247, miR-3129-3p, hsa-miR-335-3p, miR-542-5p, hsa-miR-181a-3p, hsa-miR-186-3p, hsa-miR-27b-5p, miR-491-3p, miR-4687-3p, hsa-miR-101-5p, miR-4772-5p, miR-337-3p, hsa-miR-223-5p, hsa-miR-16/195-3p, miR-3677-3p, hsa-miR-766-5p, miR-299/299-3p/3563-3p, miR-3140-3p, miR-532-5p/511, hsa-miR-24-5p, miR-4778-5p, miR-642b, miR-483-5p, miR-767-5p, hsa-miR-31-3p, miR-574-3p, miR-3173-3p, miR-2127/4728-5p, hsa-miR-103a-2-5p, miR-3591-3p, hsa-miR-625-3p, hsa-miR-15b-3p, miR-522/518e/1422p, miR-548d-3p/548acbz, hsa-miR-452-3p, miR-192/215, miR-1551/4524, hsa-miR-425-3p, miR-3126-3p, hsa-miR-125b-2-3p, miR-324-3p/1913, hsa-miR-141-5p, hsa-miR-365a/b-5p, hsa-miR-29b-1-5p, miR-563/380-5p, miR-1304, miR-216c/1461/4684-5p, hsa-miR-2681-5p, miR-194, miR-296-3p, hsa-miR-205-3p, miR-888, miR-4802-3p, hsa-let-7a/g-3p, miR-762/4492/4498, hsa-miR-744-3p, hsa-miR-148b-5p, miR-514/514b-3p, miR-28-3p, miR-550a, hsa-miR-125b-1-3p, hsa-miR-506-5p, hsa-miR-1306-5p, miR-3189-3p, miR-675-5p/4466, hsa-miR-34a-3p, hsa-miR-454-5p, miR-509-5p/509-3-5p/4418, hsa-miR-19a/b-5p, miR-4755-5p, hsa-miR-93-3p, miR-3130-5p/4482, hsa-miR-488-5p, hsa-miR-378a-5p, miR-575/4676-5p, miR-1307, miR-3942-3p, miR-4677-5p, miR-339-3p, miR-548b-3p, hsa-miR-642b-5p, miR-188-5p, hsa-miR-652-5p, miR-2114, miR-3688-5p, hsa-miR-15a-3p, hsa-miR-181c-3p, miR-122/122a/1352, miR-556-3p, hsa-miR-218-2-3p, miR-643, miR-140-3p, miR-1245, hsa-miR-2115-3p, miR-518bcf/518a-3p/518d-3p, miR-3200-3p, miR-545/3065/3065-5p, miR-1903/4778-3p, hsa-miR-302a-5p, hsa-miR-183-3p, miR-3144-5p, miR-582-3p, miR-4662a-3p, miR-3140-5p, hsa-miR-106a-3p, hsa-miR-135a-3p, miR-345/345-5p, miR-125a-3p/1554, miR-3145-5p, miR-676, miR-3173-5p, hsa-miR-5586-3p, miR-615-3p, miR-3688-3p, miR-4662a-5p, miR-4659ab-5p, hsa-miR-5586-5p, hsa-miR-514a-5p, miR-10abc/10a-5p, hsa-miR-888-3p, miR-3127-5p, miR-508-3p, hsa-miR-185-3p, hsa-miR-200c-5p,hsa-miR-550a-3p, miR-513c/514b-5p, miR-490-3p, hsa-miR-5187-3p, miR-3664-3p, miR-3189-5p, miR-4670-3p, miR-105/105ab, hsa-miR-135b-3p, hsa-miR-5010-3p, miR-493/493b, miR-3605-3p, miR-188-3p, hsa-miR-449c-3p, miR-4761-5p, miR-224, miR-4796-5p, hsa-miR-551b-5p, miR-556-5p, hsa-miR-122-3p, miR-4677-3p, miR-877, miR-576-5p, miR-490-5p, hsa-miR-589-3p, miR-4786-3p, hsa-miR-374b-3p, hsa-miR-26b-3p, miR-3158-3p, miR-4423-3p, miR-518d-5p/519bc-5p520c-5p/523b/526a, miR-4707-3p, hsa-miR-10a-3p, miR-526b, hsa-miR-676-5p, hsa-miR-660-3p, hsa-miR-5004-3p, miR-193a-5p, hsa-miR-222-5p, miR-4661-3p, hsa-miR-25-5p, miR-4670-5p, miR-659, miR-1745/3194-3p, hsa-miR-182-3p, miR-298/2347/2467-3p, hsa-miR-130b-5p, miR-4746-3p, miR-1893/2277-5p, miR-3619-3p, hsa-miR-138-1-3p, miR-4728-3p, miR-3127-3p, miR-671-3p, hsa-miR-211-3p, hsa-miR-2114-3p, hsa-miR-877-3p, miR-3157-5p, miR-502-5p, miR-500a, miR-548g, miR-523, hsa-miR-584-3p, miR-205/205ab, miR-4793-5p, hsa-miR-363-5p, hsa-miR-214-5p, miR-3180-5p, miR-1404/2110, miR-3157-3p, hsa-miR-191-3p, miR-1346/3940-5p/4507, miR-4746-5p, miR-3939, hsa-miR-181a-2-3p, hsa-miR-500a-3p, hsa-miR-196b-3p, hsa-miR-675-3p, hsa-miR-548aj/g/x-5p, miR-4659ab-3p, hsa-miR-5001-3p, hsa-miR-1247-3p, miR-2890/4707-5p, hsa-miR-150-3p, hsa-miR-629-3p, miR-2277-3p, miR-3547/3663-3p, miR-34bc-3p, miR-518ef, miR-3187-3p, miR-1306/1306-3p, miR-3177-3p, miR-lab/206/613, miR-128/128ab, miR-1296, miR-598/598-3p, miR-887, miR-1-5p, miR-376c/741-5p, miR-374c/655, miR-494, miR-651, miR-1301/5047, miR-381-5p, miR-216a, miR-300/381/539-3p, miR-1249, miR-579, miR-656, miR-433, miR-1180, miR-597/1970, miR-190a-3p, miR-1537, miR-874-5p, miR-410/344de/344b-1-3p, miR-370, miR-219-2-3p/219-3p, miR-3620, miR-504/4725-5p, miR-2964/2964a-5p, miR-450a-2-3p, miR-511, miR-6505-3p, miR-433-5p, miR-6741-3p, miR-370-5p, miR-579-5p, miR-376c-5p,miR-376b-5p, miR-552/3097-5p, miR-1910, miR-758, miR-6735-3p, miR-376a-2-5p, miR-585, miR-451 and miR-137/137ab, and bases in position 6 and 7 are the same and complementary to a base capable of pairing with the 6^thbase of the specific miRNA.

Preferably, the RNA interference-inducing nucleic acid includes a base sequence represented by any one or more of SEQ ID NOs: 103 to 528 (see Table 3).

In addition, the present invention provides a composition for inhibiting the expression of a non-canonical target gene of miRNA, which includes an RNA interference-inducing nucleic acid.

In addition, the present invention provides a method of inhibiting the expression of a non-canonical target gene of miRNA, which includes administering a composition including an RNA interference-inducing nucleic acid, into a subject.

In addition, the present invention provides the use of an RNA interference-inducing nucleic acid for suppressing a non-canonical target gene of miRNA.

3. RNA Interference-Inducing Nucleic Acids suppressing Non-Canonical G:A Wobble Target Site of miRNA (SEQ ID NOs: 529 to 863)

the RNA interference-inducing nucleic acid has a modified base sequence in which at least one guanine is substituted with uracil in a base sequence between the second to ninth bases from the 5′ end of specific miRNA, and the G:A wobble at the corresponding site becomes the canonical base pair of U:A.

Preferably, the RNA interference-inducing nucleic acid includes a sequence of 6 to 8 consecutive bases, starting from the second base from the 5′ end of specific miRNA, in which

at least one guanine base is substituted with an uracil base.

Preferably, the RNA interference-inducing nucleic acid selectively suppresses only a non-canonical target gene of miRNA binding to a G:A wobble pair, and does not suppress a canonical target gene of miRNA.

Preferably, the specific miRNA consists of 6 to 24 bases, which includes one selected from the group consisting of hsa-miR-1-3p, hsa-miR-194-5p, hsa-miR-193a-5p, hsa-miR-15b-3p, hsa-miR-200c-5p, hsa-miR-214-5p, hsa-miR-134-5p, hsa-miR-145-3p, hsa-miR-22-5p, hsa-miR-423-3p, hsa-miR-873-3p, hsa-miR-122-5p, hsa-miR-143-3p, hsa-miR-485-5p, hsa-miR-409-5p, hsa-miR-24-3p, hsa-miR-223-3p, hsa-miR-144-5p, hsa-miR-379-5p, hsa-miR-146b-5p/hsa-miR-146a-5p, hsa-miR-539-5p, hsa-miR-296-5p, hsa-miR-767-5p, hsa-miR-34a-5p/hsa-miR-34c-5p, hsa-let-7f-5p/hsa-let-7d-5p/hsa-let-7b-5p/hsa-let-7a-5p/hsa-let-7e-5p/hsa-miR-202-3p/hsa-let-7i-5p/hsa-miR-98-5p/hsa-let-7c-5p/hsa-let-7g-5p, hsa-miR-1271-3p, hsa-miR-138-5p, hsa-miR-19b-3p/hsa-miR-19a-3p, hsa-miR-27a-5p, hsa-miR-146b-3p, hsa-miR-7-5p, hsa-miR-423-5p, hsa-miR-324-5p, hsa-miR-629-5p, hsa-miR-139-3p, hsa-miR-30d-5p/hsa-miR-30e-5p/hsa-miR-30a-5p/hsa-miR-30c-5p/hsa-miR-30b-5p, hsa-miR-221-3p/hsa-miR-222-3p, hsa-miR-509-3p, hsa-miR-769-5p, hsa-miR-142-3p, hsa-miR-185-5p, hsa-miR-508-3p/hsa-miR-219a-5p, hsa-miR-31-5p, hsa-miR-103a-3p/hsa-miR-107, hsa-miR-542-3p, hsa-miR-219a-2-3p, hsa-miR-29c-3p/hsa-miR-29a-3p/hsa-miR-29b-3p, hsa-miR-125b-1-3p, hsa-miR-411-5p, hsa-miR-196a-5p/hsa-miR-196b-5p, hsa-miR-3622a-5p, hsa-miR-127-5p, hsa-miR-22-3p, hsa-miR-153-3p, hsa-miR-15b-5p/hsa-miR-16-5p/hsa-miR-424-5p, hsa-let-7g-3p/hsa-miR-493-5p/hsa-let-7c-3p, hsa-let-7i-3p, hsa-miR-218-5p, hsa-miR-1307-5p, hsa-miR-127-3p, hsa-miR-210-3p, hsa-miR-187-3p, hsa-miR-192-3p, hsa-miR-192-5p, hsa-miR-21-5p, hsa-miR-500a-3p, hsa-miR-203a-3p, hsa-miR-30c-2-3p, hsa-miR-488-3p, hsa-miR-301a-3p/hsa-miR-30 lb-3p, hsa-miR-126-3p, hsa-miR-26b-3p, hsa-miR-324-3p, hsa-miR-3065-3p, hsa-miR-124-5p, hsa-miR-345-5p, hsa-miR-615-3p, hsa-miR-889-5p/hsa-miR-135a-5p/hsa-miR-135b-5p, hsa-miR-18a-5p, hsa-miR-708-5p/hsa-miR-28-5p, hsa-miR-224-5p, hsa-miR-100-3p, hsa-miR-873-5p, hsa-miR-4662a-5p, hsa-miR-99b-3p/hsa-miR-99a-3p, hsa-miR-433-5p, hsa-miR-3605-5p, hsa-miR-744-5p, hsa-miR-1296-5p, hsa-miR-133a-3p, hsa-miR-382-5p, hsa-miR-425-5p, hsa-miR-377-5p, hsa-miR-3180-3p, hsa-miR-758-3p, hsa-miR-93-3p, hsa-miR-154-5p, hsa-miR-124-3p, hsa-miR-194-3p, hsa-miR-375, hsa-miR-148a-5p, hsa-miR-2277-5p, hsa-miR-17-3p, hsa-miR-4772-3p, hsa-miR-329-5p, hsa-miR-182-5p/hsa-miR-96-5p, hsa-miR-2467-5p/hsa-miR-485-5p, hsa-miR-149-5p, hsa-miR-29b-2-5p, hsa-miR-122-3p, hsa-miR-302a-3p/hsa-miR-520a-3p/hsa-miR-519b-3p/hsa-miR-520b/hsa-miR-519c-3p/hsa-miR-520c-3p/hsa-miR-519a-3p, hsa-miR-532-5p, hsa-miR-132-5p, hsa-miR-541-5p, hsa-miR-671-3p, hsa-miR-518e-3p, hsa-miR-487a-5p, hsa-miR-589-5p/hsa-miR-146b-5p/hsa-miR-146a-5p, hsa-miR-196b-5p/hsa-miR-196a-5p, hsa-miR-486-3p, hsa-miR-378a-3p, hsa-miR-27b-5p, hsa-miR-6720-3p, hsa-miR-574-3p, hsa-miR-29a-5p, hsa-miR-30c-2-3p/hsa-miR-30c-1-3p, hsa-miR-199b-3p, hsa-miR-574-5p, hsa-miR-4677-3p, hsa-miR-654-3p, hsa-miR-652-3p, hsa-miR-19a-3p/hsa-miR-19b-3p, hsa-let-7c-5p/hsa-miR-98-5p/hsa-let-7g-5p/hsa-let-7f-5p/hsa-miR-202-3p/hsa-let-7b-5p/hsa-let-7e-5p/hsa-let-7a-5p/hsa-let-7d-5p/hsa-let-7i-5p, hsa-miR-3663-3p, hsa-miR-152-3p/hsa-miR-148b-3p/hsa-miR-148a-3p, hsa-miR-193b-5p, hsa-miR-502-3p/hsa-miR-501-3p, hsa-miR-299-3p, hsa-miR-140-5p, hsa-miR-96-5p/hsa-miR-182-5p, hsa-miR-193b-3p, hsa-miR-365a-3p, hsa-miR-486-5p, hsa-miR-493-3p, hsa-miR-548am-5p, hsa-miR-20b-5p/hsa-miR-20a-5p/hsa-miR-93-5p/hsa-miR-17-5p/hsa-miR-106b-5p, hsa-miR-541-3p, hsa-miR-452-5p, hsa-miR-221-5p, hsa-miR-518f-3p, hsa-miR-370-3p, hsa-miR-107/hsa-miR-103a-3p, hsa-miR-338-3p, hsa-miR-409-3p, hsa-let-7d-5p/hsa-let-7g-5p/hsa-let-7i-5p/hsa-let-7f-5p/hsa-let-7e-5p/hsa-let-7a-5p/hsa-let-7b-5p/hsa-let-7c-5p, hsa-miR-130b-3p/hsa-miR-301a-3p/hsa-miR-130a-3p/hsa-miR-301b-3p, hsa-miR-512-3p, hsa-miR-191-5p, hsa-miR-509-3-5p, hsa-miR-92a-3p/hsa-miR-92b-3p/hsa-miR-363-3p/hsa-miR-25-3p/hsa-miR-32-5p, hsa-miR-183-5p, hsa-miR-1307-3p, hsa-miR-499a-5p/hsa-miR-208a-3p, hsa-miR-186-5p, hsa-miR-450b-5p, hsa-miR-450a-5p, hsa-miR-101-3p/hsa-miR-144-3p, hsa-miR-320a, hsa-miR-199b-5p/hsa-miR-199a-5p, hsa-miR-135a-5p, hsa-miR-145-5p, hsa-miR-26a-5p, hsa-miR-34c-5p, hsa-miR-125b-5p, hsa-miR-526b-5p, hsa-miR-16-5p/hsa-miR-15b-5p/hsa-miR-424-5p/hsa-miR-15 a-5p, hsa-miR-9-3p, hsa-miR-363-5p, hsa-miR-1298-3p, hsa-miR-148a-3p, hsa-miR-302a-3p, hsa-miR-9-5p, hsa-miR-28-3p, hsa-miR-508-3p, hsa-miR-137, hsa-miR-5010-5p, hsa-miR-523-5p, hsa-miR-128-3p, hsa-miR-199a-5p/hsa-miR-199b-5p, hsa-miR-181a-2-3p, hsa-miR-27a-3p/hsa-miR-27b-3p, hsa-let-7d-3p, hsa-miR-129-5p, hsa-miR-424-3p, hsa-miR-181a-3p, hsa-miR-10a-5p, hsa-miR-196b-5p, hsa-miR-92a-1-5p, hsa-miR-483-5p, hsa-miR-1537-3p, hsa-miR-106b-5p/hsa-miR-20a-5p/hsa-miR-17-5p/hsa-miR-93-5p, hsa-miR-30a-3p/hsa-miR-30e-3p, hsa-miR-374a-3p, hsa-miR-675-5p, hsa-miR-503-5p, hsa-miR-340-5p, hsa-miR-208a-3p, hsa-miR-200b-3p/hsa-miR-200c-3p, hsa-miR-518f-5p/hsa-miR-523-5p, hsa-miR-625-3p, hsa-miR-194-5p, hsa-let-7g-3p, hsa-miR-514a-5p, hsa-miR-381-3p, hsa-miR-513c-5p/hsa-miR-514b-5p, hsa-miR-520a-5p, hsa-miR-125b-5p/hsa-miR-125a-5p, hsa-miR-141-3p, hsa-miR-874-3p, hsa-miR-202-5p, hsa-miR-140-3p, hsa-miR-361-3p, hsa-miR-513b-5p, hsa-miR-33a-5p, hsa-let-7a-5p/hsa-let-7c-5p/hsa-let-7b-5p/hsa-let-7d-5p/hsa-let-7f-5p/hsa-let-7e-5p/hsa-let-7i-5p/hsa-let-7g-5p, hsa-miR-136-3p, hsa-miR-508-5p, hsa-miR-204-5p/hsa-miR-211-5p, hsa-miR-146a-5p/hsa-miR-146b-5p, hsa-miR-23a-3p, hsa-miR-21-3p, hsa-miR-877-5p, hsa-miR-302a-5p, hsa-miR-139-5p, hsa-miR-99a-5p/hsa-miR-100-5p/hsa-miR-99b-5p, hsa-miR-216a-5p and hsa-miR-3157-3p, and preferably, a modified base sequence in which at least one guanine base is substituted with uracil in the sequence in positions 2 to 7 or 2 to 9 based on the 5′ end, to allow the G:A wobble pair at the corresponding site to be a canonical base pair of U:A.

Preferably, the RNA interference-inducing nucleic acid has a sequence of 6 to 8 consecutive bases containing 2 to 7 or 2 to 9 bases from the 5′ end, represented by any one or more of SEQ ID NOs: 529 to 863 (see Table 4).

In addition, the present invention provides a composition for inhibiting the expression of a non-canonical target gene of miRNA, which includes the RNA interference-inducing nucleic acid.

In addition, the present invention provides the use of an RNA interference-inducing nucleic acid for inhibiting the expression of a non-canonical target gene of miRNA.

In addition, the present invention provides a method of preparing an RNA interference-inducing nucleic acid, which inhibits the expression of a non-canonical target gene of miRNA by modifying a partial sequence of specific miRNA in one or more single strands of the double strands of the nucleic acid inducing RNA interference, the method including the following step: constituting an RNA interference-inducing nucleic acid to have a modified base sequence in which at least one guanine base is substituted with a uracil base in the sequence of 6 to 8 consecutive bases, starting from the second base from the 5′ end of specific miRNA.

Hereinafter, the present invention will be described in detail.

When miRNA binds with target mRNA, even if not exactly complementary to a miRNA seed region, it is recognized as a miRNA target and binds to mRNA, and this is called non-canonical target recognition. The inventors focused on the non-canonical target recognition of miRNA to develop an RNA interference-inducing nucleic acid selectively suppressing the non-canonical target gene of miRNA by modifying a partial sequence of miRNA and elucidated its efficiency, and thus the present invention was completed.

the RNA interference-inducing nucleic acid has a sequence of four bases in positions 2 to 5 from the 5′ end of the specific miRNA, and bases in positions 6 and 7, which are the same and complementary to a base capable of pairing with the 6^thbase of specific miRNA, including all complementary bases including a G:A or G:U wobble pair, to allow non-canonical target base pairs bound to a bulge generated in the target gene between positions 5 and 6 of the miRNA to be a consecutive base pair by the disappearance of the bulge, or

the RNA interference-inducing nucleic acid has a modified base sequence in which at least one guanine base is substituted with uracil or adenine in the base sequence between the first to 9^thbases from the 5′ end of specific miRNA, and particularly, in the sequence from the 2^ndto 7^thbases based on the 5′ end to allow a G:A or G:U wobble pair at the corresponding site to be a canonical base pair of U:A or A:U.

The RNA interference-inducing nucleic acid according to the present invention may be one or more single strands of the double strands of the nucleic acid inducing RNA interference, preferably including miRNA, small hairpin RNA (shRNA) and small interfering RNA (siRNA), DsiRNA, lsiRNA, ss-siRNA, asiRNA, piRNA or endo-siRNA.

The RNA interference-inducing nucleic acid according to the present invention is based on two types of non-canonical target recognition of miRNA.

More specifically, the miRNA recognizes a normal target gene as well as a non-canonical target gene. The non-canonical target gene recognized by the miRNA does not have a complementary relationship exactly corresponding to the miRNA.

Aspects of a non-canonical target gene recognized by the miRNA and an RNA interference-inducing nucleic acid suppressing the non-canonical target gene are as follows.

One type of recognition of miRNA-binding non-canonical target genes has a consecutive complementary relationship with all of the bases in positions 1 to 5 from the 5′ end of miRNA. However, in a gene recognized by the base in position 6 from the 5′ end of miRNA (paired with the base in position 6 from the 5′ end of miRNA), the base recognized by the base in position 6 from the 5′ end of miRNA is a base that has a complementary relationship with miRNA after pushing an immediately consecutive base in a bulge form, rather than a base that is immediately contiguous to a base having a complementary relationship with the base in position 5 from the 5′ end of the miRNA.

The RNA interference-inducing nucleic acid suppressing the non-canonical target gene recognized by miRNA of one aspect has a four-base sequence in positions 2 to 5 from the 5′ end of specific miRNA. In addition, two consecutive bases in the four-base sequence are the same and have a base sequence complementary to a base that can be paired with the 6^thbase of the specific miRNA, and the base that can be paired with the 6^thbase of the specific miRNA includes all complementary bases including G:A and G:U wobble pairs.

The RNA interference-inducing nucleic acid suppressing a non-canonical target gene recognized by the specific miRNA commonly includes a sequence of at least four bases in positions 2 to 5 from the 5′ end of the specific miRNA, compared with the base sequence of the miRNA.

In addition, the 6^thand 7^thbases from the 5′ end of the RNA interference-inducing nucleic acid may be the same, and these two bases may be complementary to a base that can be paired with the 6^thbase of specific miRNA, which includes all complementary bases including G:A and G:U wobble pairs. For example, an arrangement of the 6^thbase of the specific miRNA: a base that is able to be paired with the 6^thbase: a base complementary to the base that is able to be paired with the 6^thbase may be (A:U, G:A,C), (G:A,U,C:U,A,G), (U:G,A:C,U) or (C:G:C). For example, when the 6^thbase of the specific miRNA is A, a sequence of the 6^thand 7^thbases from the 5′ end of the RNA interference-inducing nucleic acid may be AA or CA; when the 6^thbase of the specific miRNA is G, a sequence of the 6^thand 7^thbases from the 5′ end of the RNA interference-inducing nucleic acid may be UG, AG or GG; when the 6^thbase of the specific miRNA is U, a sequence of the 6^thand 7^thbases from the 5′ end of the RNA interference-inducing nucleic acid may be CU or UU; or when the 6^thbase of the specific miRNA is C, a sequence of the 6^thand 7^thbases from the 5′ end of the RNA interference-inducing nucleic acid may be CC.

Preferably, the 6^thbase from the 5′ end of the RNA interference-inducing nucleic acid and the 6^thbase from the 5′ end of the specific miRNA are the same, and the 7^thbase from the 5′ end of the RNA interference-inducing nucleic acid and the 6^thbase from the 5′ end of the specific miRNA are the same.

In addition, the RNA interference-inducing nucleic acid preferably includes the 7^thbase from the 5′ end of the specific miRNA as the 8^thbase from the 5′ end thereof.

In still another aspect, aspects of a non-canonical target gene recognized by. miRNA and an RNA interference-inducing nucleic acid suppressing the non-canonical target gene are as follows.

In the case of the non-canonical target gene recognized by miRNA, bases of the miRNA and the target gene recognized by the miRNA have a G:A or G:U wobble pair relationship, in addition to complementary pairing of A:U, G:C, C:G or U:A.

When the miRNA and the target gene recognized by the miRNA are in a G:A wobble relationship, an arrangement of the base of the specific miRNA: the base of the specific gene recognized by the specific miRNA: the base of the RNA interference-inducing nucleic acid suppressing the target gene recognized by the specific miRNA is G:A:U. In addition, when the miRNA and the target gene recognized by the miRNA have a G:U wobble relationship, an arrangement of the base of the specific miRNA: the base of the specific gene recognized by the specific miRNA: the base of the RNA interference-inducing nucleic acid suppressing the target gene recognized by the specific miRNA is G:U:A.

The RNA interference-inducing nucleic acid according to the present invention has a modified base sequence in which one or more guanine (G) bases of the base sequence from the 1^stto 9^thbases from the 5′ end of the specific miRNA are substituted with uracil (U) bases. In addition, the RNA interference-inducing nucleic acid according to the present invention has a modified base sequence in which one or more guanine (G) bases are substituted with adenine (A) bases in the sequence from the 1^stto 9^thbases, and preferably, the 2^ndto 7^thbases from the 5′ end of the specific miRNA. When one or more guanine (G) bases are included in the base sequence from the 1^stto 9^thbases from the 5′ end of the specific miRNA, all of the included guanine (G) bases may be substituted with uracil (U) or adenine (A), and at least one guanine base is substituted with uracil (U) or adenine (A). When at least one guanine (G) is included in the base sequence from the 1^stto 9^thbases, and preferably, the 2^ndto 7^thbases from the 5′ end of the specific miRNA, the other bases except the base substituted with uracil or adenine may be the same as the bases of the specific miRNA.

The RNA interference-inducing nucleic acid according to the present invention selectively suppresses a non-canonical target gene of the miRNA, and does not suppress a canonical target gene of the miRNA.

According to an exemplary embodiment of the present invention (see FIG. 2), the function of regulating the miRNA expression of a target gene of the RNA interference-inducing nucleic acid according to the present invention is specific for a non-canonical target gene, and an inhibitory effect is not shown for a canonical target gene.

Therefore, when the RNA interference-inducing nucleic acid according to the present invention is used, only the expression of a non-canonical target gene may be selectively inhibited, thereby selectively inducing only an effect expected by the expression inhibition of the RNA interference-inducing nucleic acid.

In the present invention, the specific miRNA may be one or more selected from the group consisting of miR-124, miR-155, miR-122, miR-1, miR-133, let-7, mR-302a and miR-372.

In the present invention, the base sequence of each of human miR-124, miR-155, miR-122, miR-1, miR-133, let-7, miR-302a and miR-372 is described in MIMAT0000422, MIMAT0000646, MIMAT0000421, MIMAT0000416, MIMAT0000427, MIMAT0000062, MIMAT0000684 or MIMAT0000724, respectively, in the miRNA sequence database, miRBase (http://www.mirbase.org/).

However, the specific miRNA according to the present invention is not limited to human miRNA, and includes miRNAs derived from animals including mammals.

As the RNA interference-inducing nucleic acid suppressing a non-canonical target gene recognized by the specific miRNA according to the present invention, the RNA interference-inducing nucleic acid which has a sequence of four bases in positions 2 to 5 from the 5′ end of the specific miRNA, and bases in positions 6 and 7, which are e the same and complementary to a base capable of being paired with the 6^thbase of the specific miRNA, including all complementary bases including G:A and G:U wobble pairs, has a length of 6 or more bases, but the present invention is not limited thereto. The RNA interference-inducing nucleic acid normally has a length of approximately 21 to 24 bases.

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-124 when the specific miRNA is miR-124. The RNA interference-inducing nucleic acid has a sequence of four bases in positions 2 to 5 from the 5′ end of the miR-124, and bases in positions 6 and 7, which are the same and complementary to a base capable of being paired with the 6^thbase of miR-124, including all complementary bases including G:A and G:U wobble pairs.

For example, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid (miR-124BS) in which the sequence of the 2^ndto 7^thbases from the 5′ end is 5′-AA GGC C-3′. More preferably, the RNA interference-inducing nucleic acid is an RNA interference-inducing nucleic acid (miR-124BS) having the base sequence of 5′-UAA GGC CAC GCG GUG AAU GCC-3′.

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-122 when the specific miRNA is miR-122, which has a sequence of four bases in positions 2 to 5 from the 5′ end of miR-122, and bases in positions 6 and 7, which are the same and complementary to a base capable of being paired with the 6^thbase of miR-122, including all complementary bases having a G:A or G:U wobble pair.

For example, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid (miR-122BS) in which the sequence of the 2^ndto 7^thbases from the 5′ end is 5′-GG AGU U-3. More preferably, the RNA interference-inducing nucleic acid is an RNA interference-inducing nucleic acid (miR-122BS) having a base sequence of 5′-UGG AGU UGU GAC AAU GGU GUU-3′.

The RNA interference-inducing nucleic acid according to the present invention may be an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-155 when the specific miRNA is miR-155, which has a sequence of four bases in positions 2 to 5 from the 5′ end of miR-155, and bases in positions 6 and 7, which are the same and complementary to a base capable of being paired with the 6^thbase of miR-155, including all complementary bases including G:A and G:U wobble pairs.

For example, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid (miR-155BS) in which the base sequence in positions 2 to 7 from the 5′ end is 5′-UA AUG G-3′. More preferably, the RNA interference-inducing nucleic acid is an RNA interference-inducing nucleic acid (miR-155BS) having a base sequence of 5′-UUA AUG GC UAA U CGU GAU AGG-3′.

The RNA interference-inducing nucleic acid according to the present invention may be an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-1 when the specific miRNA is miR-1, which has a sequence of four bases in positions 2 to 5 from the 5′ end of the miR-1, and bases in positions 6 and 7, which are the same and complementary to a base capable of being paired with the 6^thbase of the miR-1, including all complementary bases including G:A and G:U wobble pairs.

For example, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid (miR-1BS) in which the base sequence in positions 2 to 7 from the 5′ end is 5′-GG AAU U-3′. More preferably, the RNA interference-inducing nucleic acid is an RNA interference-inducing nucleic acid (miR-1BS) having a base sequence of 5′-UGG AAU UGU AAA GAA GUA UGU-3′.

As the RNA interference-inducing nucleic acid suppressing a non-canonical target gene recognized by specific miRNA according to the present invention, the RNA interference-inducing nucleic acid having a modified base sequence in which at least one guanine (G) base is substituted with uracil (U) or adenine (A) in the base sequence in positions 1 to 9 from the 5′ end of the specific miRNA, preferably, the sequence from the 2^ndto 7^thbases from the 5′ end, has a length of 6 or more bases, but the present invention is not limited thereto. The RNA interference-inducing nucleic acid may normally have approximately 21 to 24 bases in length.

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-124 when the specific miRNA is miR-124, which has a modified base sequence in which at least one guanine (G) base is substituted with uracil (U) or adenine (A) in the base sequence in positions 1 to 9 from the 5′ end of the miR-124, preferably, the sequence from the 2^ndto 7^thbases from the 5′ end.

For example, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid in which the base sequence in positions 1 to 9 from the 5′ end is 5′-UAA UGC AC-3′ (miR-124-G4U), 5′-UAA GUC AC-3′ (miR-124-G5U) or 5′-UAA UUC AC-3′ (miR-124-G4,5U). More preferably, the RNA interference-inducing nucleic acid is an RNA interference-inducing nucleic acid having a base sequence of 5′-UAA UGC ACG CGG UGA AUG CCA A-3′ (miR-124-G4U), 5′-UAA GUC ACG CGG UGA AUG CCA A-3′ (miR-124-G5U) or 5′-UAA UUC ACG CGG UGA AUG CCA A-3′ (miR-124-G4,5U).

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-1 when the specific miRNA is miR-1, which has a modified base sequence in which at least one guanine (G) base is substituted with uracil (U) or adenine (A) in the base sequence in positions 1 to 9 from the 5′ end of the miR-1.

For example, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid in which the base sequence in positions 1 to 9 from the 5′ end is 5′-UUG AAU GUA-3′ (miR-1-G2U), 5′-UGU AAU GUA-3′ (miR-1-G3U), 5′-UGG AAU UUA-3′ (miR-1-G7U), 5′-UUU AAU GUA-3′ (miR-1-G2,3U), 5′-UGU AAU UUA-3′ (miR-1-G3,7U), 5′-UUG AAU UUA-3′ (miR-1-G2,7U) or 5′-UUU AAU UUA-3′ (miR-1-G2,3,7U). More preferably, the RNA interference-inducing nucleic acid is an RNA interference-inducing nucleic acid having a base sequence of 5′-UUG AAU GUA AAG AAG UAU GUA U-3′ (miR-1-G2U), 5′-UGU AAU GUA AAG AAG UAU GUA U-3′ (miR-1-G3U), 5′-UGG AAU UUA AAG AAG UAU GUA U-3′ (miR-1-G7U), 5′-UUU AAU GUA AAG AAG UAU GUA U-3′ (miR-1-G2,3U), 5′-UGU AAU UUA AAG AAG UAU GUA U-3′ (miR-1-G3,7U), 5′-UUG AAU UUA AAG AAG UAU GUA U-3′ (miR-1-G2,7U) or 5′-UUU AAU UUA AAG AAG UAU GUA U-3′ (miR-1-G2,3,7U).

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-122 when the specific miRNA is miR-122, which has a modified base sequence in which at least one guanine (G) base is substituted with uracil (U) or adenine (A) in the base sequence in positions 1 to 9 from the 5′ end of the miR-122, preferably, the sequence from the 2^ndto 7^thbases from the 5′ end.

For example, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid in which the base sequence in positions 1 to 9 from the 5′ end is 5′-UUG AGU GUG-3′ (miR-122-G2U), 5′-UGU AGU GUG-3′ (miR-122-G3U), 5′-UGG AUU GUG-3′ (miR-122-G5U), 5′-UGG AGU UUG-3′ (miR-122-G7U), 5′-UGG AGU GUU-3′ (miR-122-G9U), 5′-UUU AGU GUG-3′ (miR-122-G2,3U), 5′-UUG AUU GUG-3′ (miR-122-G2,5U), 5′-UUG AGU UUG-3′ (miR-122-G2,7U), 5′-UUG AGU GUU-3′ (miR-122-G2,9U), 5′-UGU AUU GUG (miR-122-G3,5U), 5 ′-UGU AGU UUG-3′ (miR-122-G3,7U), 5′-UGU AGU GUU-3′ (miR-122-G3,9U), 5 ′-UGG AUU UUG-3′ (miR-122-G5,7U), 5′-UGG AUU GUU-3′ (miR-122-G5,9U) or 5′-UGG AGU UUU-3 (miR-122-G7,9U). More preferably, the RNA interference-inducing nucleic acid is an RNA interference-inducing nucleic acid having a base sequence of 5′-UUG AGU GUG ACA AUG GUG UUU G-3′ (miR-122-G2U), 5′-UGU AGU GUG ACA AUG GUG UUU G-3 (miR-122-G3U), 5′-UGG AUU GUG ACA AUG GUG UUU G-3′ (miR-122-G5U), 5′-UGG AGU UUG ACA AUG GUG UUU G-3′ (miR-122-G7U), 5′-UGG AGU GUU ACA AUG GUG UUU G-3′ (miR-122-G9U), 5′-UUU AGU GUG ACA AUG GUG UUU G-3′ (miR-122-G2,3U), 5′-UUG AUU GUG ACA AUG GUG UUU G-3′ (miR-122-G2,5U), 5′-UUG AGU UUG ACA AUG GUG UUU G-3′ (miR-122-G2,7U), 5′-UUG AGU GUU ACA AUG GUG UUU G-3′ (miR-122-G2,9U), 5′-UGU AUU GUG ACA AUG GUG UUU G-3 (miR-122-G3,5U), 5′-UGU AGU UUG ACA AUG GUG UUU G-3 (miR-122-G3,7U), 5′-UGU AGU GUU ACA AUG GUG UUU G-3 (miR-122-G3,9U), 5′-UGG AUU UUG ACA AUG GUG UUU G-3′ (miR-122-G5,7U), 5′-UGG AUU GUU ACA AUG GUG UUU G-3′ (miR-122-G5,9U) or 5′-UGG AGU UUU ACA AUG GUG UUU G-3′ (miR-122-G7,9U).

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-133 when the specific miRNA is miR-133, which has a modified base sequence in which at least one guanine (G) base is substituted with uracil (U) or adenine (A) in the base sequence in positions 1 to 9 from the 5′ end of the miR-133, preferably, the sequence from the 2^ndto 7^thbases from the 5′ end.

For example, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid in which the base sequence in positions 1 to 9 from the 5′ end is 5′-UUU UGU CCC-3′ (miR-133-G4U), 5′-UUU GUU CCC-3′ (miR-133-G5U) or 5′-UUU UUU CCC-3′(miR-133-G4,5U). More preferably, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid having a base sequence of 5′-UUU UGU CCC CUU CAA CCA GCU G -3′ (miR-133-G4U), 5′-UUU GUU CCC CUU CAA CCA GCU G-3′ (miR-133-G5U) or 5′-UUU UUU CCC CUU CAA CCA GCU G-3′(miR-133-G4,5U).

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of let-7 when the specific miRNA is let-7, which has a modified base sequence in which at least one guanine (G) base is substituted with uracil (U) or adenine (A) in the base sequence in positions 1 to 9 from the 5′ end of the let-7, preferably, the sequence from the 2^ndto 7^thbases from the 5′ end.

For example, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid in which the base sequence in positions 1 to 9 from the 5′ end is 5′-UUA GGU AGU-3′ (let-7-G2U), 5′-UGA UGU AGU-3′ (let-7-G4U), 5′-UGA GUU AGU-3′ (let-7-G5U), 5′-UGA GGU AUU-3′ (let-7-G8U), 5′-UUA UGU AGU-3′ (let-7-G2,4U), 5′-UUA GUU AGU-3′ (let-7-G2,SU), 5′-UUA GGU AUU-3′ (let-7-G2,8U), 5′-UGA UUU AGU-3′ (let-7-G4,SU), 5′-UGA UGU AUU-3′ (let-7-G4,8U) or 5′-UGA GUU AUU-3′ (let-7-G5,8U). More preferably, the RNA interference-inducing nucleic acid is an RNA interference-inducing nucleic acid having a base sequence of 5′-UUA GGU AGU AGG UUG UAU AGU U-3′ (let-7-G2U), 5′-UGA UGU AGU AGG UUG UAU AGU U-3′ (let-7-G4U), 5′-UGA GUU AGU AGG UUG UAU AGU U-3′ (let-7-G5U), 5′-UGA GGU AUU AGG UUG UAU AGU U-3′ (let-7-G8U), 5′-UUA UGU AGU AGG UUG UAU AGU U-3′ (let-7-G2,4U), 5′-UUA GUU AGU AGG UUG UAU AGU U-3′ (let-7-G2,5U), 5′-UUA GGU AUU AGG UUG UAU AGU U-3′ (let-7-G2,8U), 5′-UGA UUU AGU AGG UUG UAU AGU U-3′ (let-7-G4,5U), 5′-UGA UGU AUU AGG UUG UAU AGU U-3′ (let-7-G4,8U) or 5′-UGA GUU AUU AGG UUG UAU AGU U-3′ (let-7-G5,8U).

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-302a when the specific miRNA is miR-302a, which has a modified base sequence in which at least one guanine (G) base is substituted with uracil (U) or adenine (A) in the base sequence in positions 1 to 9 from the 5′ end of the miR-302a, preferably, the sequence from the 2^ndto 7^thbases from the 5′ end.

For example, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid in which the base sequence in positions 1 to 9 from the 5′ end is 5′-UAA UUG CUU-3′ (miR-302a-G4U), 5′-UAA GUU CUU-3′ (miR-302a-G6U) or 5′-UAA UUU CUU-3′ (miR-302a-G4,6U). More preferably, the RNA interference-inducing nucleic acid is an RNA interference-inducing nucleic acid having a base sequence of 5′-UAA UUG CUU CCA UGU UUU GGU GA-3′ (miR-302a-G4U), 5′-UAA GUU CUU CCA UGU UUU GGU GA-3′ (miR-302a-G6U) or 5′-UAA UUU CUU CCA UGU UUU GGU GA-3′ (miR-302a-G4,6U).

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-372 when the specific miRNA is miR-372, which has a modified base sequence in which at least one guanine (G) base is substituted with uracil (U) or adenine (A) in the base sequence in positions 1 to 9 from the 5′ end of the miR-372, preferably, the sequence from the 2^ndto 7^thbases from the 5′ end.

For example, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid in which the base sequence in positions 1 to 9 from the 5′ end is 5′-AAA UUG CUG-3′ (miR-372-G4U), 5′-AAA GUU CUG-3′ (miR-372-G6U), 5′-AAA GUG CUU-3′ (miR-372-G9U), 5′-AAA UUU CUG-3′ (miR-372-G4,6U), 5′-AAA UUG CUU-3′ (miR-372-G4,9U), or 5′-AAA GUU CUU-3′ (miR-372-G6,9U). More preferably, the RNA interference-inducing nucleic acid is an RNA interference-inducing nucleic acid suppressing a non-canonical target gene of miR-372, having a base sequence of 5′-AAA UUG CUG CGA CAU UUG AGC GU-3′ (miR-372-G4U), 5′-AAA GUU CUG CGA CAU UUG AGC GU -3′ (miR-372-G6U), 5′-AAA GUG CUU CGA CAU UUG AGC GU -3′ (miR-372-G9U), 5′-AAA UUU CUG CGA CAU UUG AGC GU -3′ (miR-372-G4,6U), 5′-AAA UUG CUU CGA CAU UUG AGC GU-3′ (miR-372-G4,9U) or 5′-AAA GUU CUU CGA CAU UUG AGC GU-3′ (miR-372-G6,9U).

In addition, the present invention provides an RNA interference-inducing nucleic acid, which suppresses a non-canonical target gene of miRNA by modifying a partial sequence of specific miRNA in one or more single strands of the double strands of the nucleic acid inducing RNA interference,

the specific miRNA is characterized by adding a methyl group (2′OMe) to the 2′ position of the ribosyl ring of the 6^thnucleotide from the 5′ end.

The RNA interference-inducing nucleic acid according to the present invention has a sequence of four bases in positions 2 to 5 from the 5′ end of the specific miRNA, and bases in positions 6 and 7, which are the same and complementary to a base capable of pairing with the 6^thbase of the specific miRNA, including all complementary bases including G:A and G:U wobble pairs, and when a methyl group (2′OMe) is added to the 2′ position of the ribosyl ring of the 6^thnucleotide from the 5′ end of the specific miRNA, there is no limitation on the rest of the base sequence except this, and any base sequence may be included. Here, the RNA interference-inducing nucleic acid may include a sequence of 6 to 24 bases, preferably, 6 to 15 bases, and more preferably, 6 to 8 bases, but there is no particular limitation on the length of the nucleic acid.

The RNA interference-inducing nucleic acid according to the present invention includes a base sequence chemically modified by adding a methyl group (2′OMe) to the 2′ position of the ribosyl ring of the 6^thnucleotide from the 5′ end of the specific miRNA, the RNA interference-inducing nucleic acid in which the 2′OMe modification occurs may selectively inhibit only the expression of a canonical seed target gene thereof, but not inhibit the expression of a non-canonical seed target gene. Therefore, the RNA interference-inducing nucleic acid in which the 2′OMe modification occurs may maintain the purpose of the present invention for specifically suppressing only a non-canonical nucleation bulge site of the specific miRNA, and may have an effect of completely removing a newly generated non-canonical nucleation bulge site.

In the RNA interference-inducing nucleic acid according to the present invention, there is no limitation on the type of miRNA to which the methyl group (2′OMe) can be added as long as the 2′OMe is capable of being added to the 2′ position of the ribosyl ring of the 6^thnucleotide from the 5′ end. However, according to an exemplary embodiment of the present invention, the miRNA to which the 2′OMe may be added may be miR-124 or miR-1.

In addition, the present invention provides an RNA interference-inducing nucleic acid which suppress a non-canonical target gene of miRNA by modifying a partial sequence of specific miRNA in one or more single strands of the double strands of a nucleic acid inducing RNA interference, wherein

As long as the RNA interference-inducing nucleic acid according to the present invention has a sequence of four bases in positions 2 to 5 from the 5′ end of the specific miRNA, and bases in positions 6 and 7, which are the same and complementary to a base capable of pairing with the 6^thbase of the specific miRNA, including all complementary bases including G:A and G:U wobble pairs, it may have any base sequence without limitation on the rest of the base sequence except the above-described characteristics. Here, the RNA interference-inducing nucleic acid may include a sequence of 6 to 24 bases, preferably, 6 to 15 bases, and more preferably, 6 to 8 bases, but there is not specific limitation on the length of the nucleic acid.

The RNA interference-inducing nucleic acid according to the present invention has a sequence of four bases in positions 2 to 5 from the 5′ end of the specific miRNA, and bases in positions 6 and 7, which are the same and complementary to a base capable of pairing with the 6^thbase of the specific miRNA, including all complementary bases including G:A and G:U wobble pairs, to allow non-canonical target base pairs bound to a bulge generated in the target gene between positions 5 and 6 of the miRNA to be a consecutive base pair by the disappearance of the bulge, thereby selectively suppressing only a non-canonical nucleation bulge site and not suppressing a canonical target gene of miRNA.

The RNA interference-inducing nucleic acid according to the present invention may have a base sequence represented by any one or more of SEQ ID NOs: 103 to 528 (see Table 3).

In addition, the present invention provides an RNA interference-inducing nucleic acid which suppresses only the expression of a non-canonical target gene of miRNA by modifying a partial sequence of specific miRNA in one or more single strands of the double strands of the nucleic acid inducing RNA interference according to the present invention, wherein

the RNA interference-inducing nucleic acid has a modified base sequence in which at least one guanine (G) base is substituted with an uracil base in the sequence between the 2^ndto 9^thbases from the 5′ end of the specific miRNA or the sequence in positions 2 to 7 from the 5′ end to allow a G:A wobble pair at the corresponding site to be a canonical base pair of U:A.

The RNA interference-inducing nucleic acid according to the present invention may have at least one guanine (G) base substituted with an uracil base in the sequence between the 2^ndto 9^thbases from the 5′ end of the specific miRNA or the sequence between the 2^ndto 7^thbases from the 5′ end, and as long as it has a modified base sequence in which at least one guanine base between the 2^ndto 9^thbases from the 5′ end of the specific miRNA is substituted with an uracil base, the RNA interference-inducing nucleic acid may have any base sequence without limitation on the rest of the sequence except the above-described characteristics. Here, in the RNA interference-inducing nucleic acid according to the present invention, all bases except the base substituted with an uracil (U) base in the sequence between the 2^ndto 9^thbases from the 5′ end of the specific miRNA or between the 2^ndto 7^thbases from the 5′ end may be the same as the bases of the specific miRNA.

As the RNA interference-inducing nucleic acid suppressing a non-canonical target gene recognized by specific miRNA according to the present invention, an RNA interference-inducing nucleic acid having a modified base sequence in which at least one guanine base is substituted with an uracil base in the base sequence between the 2^ndto 9^thbases from the 5′ end of the specific miRNA or the sequence in positions 2 to 7 from the 5′ end, may have a sequence of 6 to 24 bases, preferably, 6 to 15 bases, and more preferably, 6 to 8 bases, but there is no specific limitation on the length of the nucleic acid as long as it is an RNA interference-inducing nucleic acid having a modified base sequence in which at least one guanine base is substituted with an uracil base in the sequence of 6 to 8 consecutive bases, starting with the 2^ndbase from the 5′ end of the specific miRNA. Here, as long as the RNA interference-inducing nucleic acid has a modified base sequence in which at least one guanine base is substituted with an uracil base in the sequence of 6 to 8 consecutive bases, starting with the 2^ndbase from the 5′ end of the specific miRNA, there is no limitation on the rest of the base sequence except the above-mentioned characteristics, and any base sequence may be included.

The RNA interference-inducing nucleic acid according to the present invention has a modified base sequence in which at least one guanine (G) base is substituted with an uracil (U) base in the sequence of 6 to 8 consecutive bases, starting with the 2^ndbase from the 5′ end to allow a G:A wobble pair at the corresponding site to be a canonical base pair of U:A, thereby selectively suppressing only a non-canonical target gene of miRNA binding as a G:A wobble pair and not suppressing a canonical target gene of miRNA.

The RNA interference-inducing nucleic acid according to the present invention may include a base sequence represented by any one or more of SEQ ID NOs: 529 to 863 (see Table 4).

When the above-described RNA interference-inducing nucleic acid according to the present invention is used, a non-canonical target gene of miRNA may be specifically suppressed.

Therefore, the present invention provides a composition for inhibiting the expression of a non-canonical target gene of miRNA, which includes an RNA interference-inducing nucleic acid, or a kit for inhibiting the expression of a non-canonical target gene of miRNA, which includes an RNA interference-inducing nucleic acid.

In addition, when the above-described RNA interference-inducing nucleic acid according to the present invention is used, an action caused by the inhibition of the expression of a non-canonical target gene of miRNA by specifically suppressing the non-canonical target gene of miRNA may be selectively regulated.

More specifically, when the RNA interference-inducing nucleic acid according to the present invention is used, the non-canonical target gene of miRNA is specifically suppressed, and thus the action (e.g., cell cycle, differentiation, dedifferentiation, morphology, migration, division, proliferation or death) caused by the expression of a non-canonical target gene of miRNA may be specifically regulated by using the above-described RNA interference-inducing nucleic acid according to the present invention.

Accordingly, the present invention provides a composition or kit for regulating cell cycling, differentiation, dedifferentiation, morphology, migration, division, proliferation or death, which includes an RNA interference-inducing nucleic acid.

The “cell cycle” used herein is a continuous process of growing, dividing and growing cells again, and refers that cells repeatedly go through four phases of an M phase (mitosis), a G1 phase (first division preparation phase), an S phase (DNA synthesis phase) and a G2 phase (second division preparation phase). The G1 phase is for preparing for DNA synthesis from immediately after cell division to the initiation of DNA synthesis, and the G2 phase is for preparing for cell division from the termination of DNA synthesis to the initiation of the cell division.

The “regulation of a cell cycle” used herein includes induction of cell transition between the four phases, or promotion or arrest of the transition, for example, the induction of cells in the G2/M phase to transition to the G1/G2 phase.

The “cell differentiation” used herein is a process by which cells acquire morphological and functional specificities, in which the cells are changed in size or shape, membrane potential, metabolic activity, and response to a signal through differentiation.

The “regulation of cell differentiation” used herein refers to the proliferation or delay of a rate of cell differentiation, induction to initiate differentiation, or inhibition not to initiate differentiation, and for example, includes induction of nerve cell differentiation to acquire morphological specificity (e.g., neurite differentiation). In addition, for example, the induction of myocyte differentiation to acquire morphological specificity (e.g., the fibrosis of myocytes) may be included as an example of the regulation of cell differentiation.

The “dedifferentiation” used herein refers to a phenomenon in which differentiation reverses or completely-differentiated cells acquire pluripotency in the undifferentiated period before differentiation and then are changed like cells in mesophase.

The “regulation of dedifferentiation” used herein includes induction or promotion of cell dedifferentiation, or inhibition or delay of the progression of dedifferentiation, and the result of regulating dedifferentiation may be determined by confirming, for example, the form of cell growth (e.g., formation of a population, etc.), the activity of dedifferentiation factors (Oct3/4, Sox2, c-Myc, Klf4), etc.

The “cell morphology” used herein refers to phenotypic features including a cell shape, structure, size and the like, and varies depending on the type of organism, and the type of tissue or organ even in the same organism.

The “regulation of cell morphology” used herein refers to a change in phenotypic features including the shape, structure and size of cells, and includes the promotion, delay, induction or inhibition of the natural change in cell morphology and the promotion or induction of a non-natural change in cell morphology. For example, the induction of a myocardial hypertrophy of myocardial cells may be included as an example of the regulation of cell morphology.

The “cell migration” used herein refers to the mobility of cells, which is an important process for the growth and physiological activity of an animal, and cells usually respond rapidly to a given stimulus and normally migrate in a non-collective shape, whereas other types of cells may migrate only during a specific growth period or in special circumstances. Cell migration mainly occurs during the development of neural and vascular tubes or the wound healing of epithelial tissue, and the migration of cell populations contributes to the metastasis of various types of tumors.

The “regulation of cell migration” used herein refers to the delay, promotion, induction or inhibition (suppression) of cell migration.

The “cell division and proliferation” used herein is a process by which a parent cell is divided into two daughter cells to increase a cell number.

The “regulation of cell division and proliferation” used herein includes the delay, promotion, inhibition or induction of cell division and proliferation. When cells are induced to the M phase (mitosis) in the cell cycle, it may include cell division and proliferation. For example, an increase in the number of cells distributed in the G0/G1 phase and a decrease in the number of cells distributed in the G2/M phase may be an example of the regulation of cell division and proliferation.

The “apoptosis” used herein refers to a stage leading to death due to genetic properties while cells maintain their functional role, and includes early apoptosis and late apoptosis. According to apoptosis, a new protein is synthesized as the entire cell atrophies, leading to death by a suicide gene of the cells.

The “regulation of apoptosis” used herein includes the induction, delay, promotion or inhibition of apoptosis.

More specifically, the present invention provides a composition for inducing cancer cell death, which includes an RNA interference-inducing nucleic acid (preferably, miR-124BS) suppressing a non-canonical target gene of miR-124;

a composition for inducing neurite differentiation, which includes an RNA interference-inducing nucleic acid (preferably, miR-124BS) suppressing a non-canonical target gene of miR-124;

a composition for inducing cell cycle arrest in liver cancer cells, which includes an RNA interference-inducing nucleic acid (preferably, miR-122BS) suppressing a non-canonical target gene of miR-122;

a composition for promoting differentiation of muscle cells or muscle fibrosis, which includes an RNA interference-inducing nucleic acid (preferably, miR-1BS) suppressing a non-canonical target gene of miR-1;

a composition for inducing muscle cell death, which includes an RNA interference-inducing nucleic acid (preferably, miR-155BS) suppressing a non-canonical target gene of miR-155;

a composition for inducing cell death of neuroblastomas, which includes an RNA interference-inducing nucleic acid (preferably, miR-124-G5U) suppressing a non-canonical target gene of miR-124;

a composition for promoting cell division or proliferation of neuroblastomas, which includes an RNA interference-inducing nucleic acid (preferably, miR-124-G4U) suppressing a non-canonical target gene of miR-124;

a composition for inducing myocardial hypertrophy, which includes an RNA interference-inducing nucleic acid (preferably, miR-1-G2U, miR-1-G3U or miR-1-G7U) suppressing a non-canonical target gene of miR-1;

a composition for inducing myocardial hypertrophy, which includes an RNA interference-inducing nucleic acid (preferably, miR-133-G4U) suppressing a non-canonical target gene of miR-133;

a composition for inducing cell cycle arrest in cancer cells, which includes an RNA interference-inducing nucleic acid (preferably, let-7-G2U, let-7-G2,4U) suppressing a non-canonical target gene of let-7;

a composition for inducing the cell cycle progressing activity of hepatocytes, which includes an RNA interference-inducing nucleic acid (preferably, let-7-G4U) suppressing a non-canonical target gene of let-7;

a composition for promoting dedifferentiation, which includes an RNA interference-inducing nucleic acid (preferably, miR-302a-G4U) suppressing a non-canonical target gene of miR-302a;

a composition for promoting dedifferentiation, which includes an RNA interference-inducing nucleic acid (preferably, miR-372-G4U) suppressing a non-canonical target gene of miR-372; or

a composition for inhibiting cell migration of liver cancer cells, which includes an RNA interference-inducing nucleic acid (preferably, miR-122-G2U or miR-122-G2,3U) suppressing a non-canonical target gene of miR-122.

In addition, when the RNA interference-inducing nucleic acid according to the present invention is used, the action of the non-canonical target gene of miRNA may be selectively regulated by specifically suppressing the non-canonical target gene of miRNA, and therefore may be applied in various fields (e.g., pharmaceuticals, cosmetics, cytology, etc.).

In addition, the RNA interference-inducing nucleic acid according to the present invention may be applied in a method of screening a test material for regulating cell cycling, differentiation, dedifferentiation, morphology, migration, division, proliferation or apoptosis.

More specifically, the present invention provides a method of screening a test material for regulating cell cycling, differentiation, dedifferentiation, morphology, migration, division, proliferation or apoptosis, which includes:

transfecting the RNA interference-inducing nucleic acid according to the present invention into target cells;

treating the target cells with a test material; and

checking an expression level or expression of the non-canonical target gene of miRNA, which is suppressed by the RNA interference-inducing nucleic acid, in the target cells.

In the present invention, the target cells are derived from mammals including a human without limitation, and tissue from which the target cells are extracted or the type of cells is not limited. The target cells may include, for example, neural cells, cardiomyocytes, cancer cells and muscle cells.

In the present invention, the introduction of the RNA interference-inducing nucleic acid into target cells may be conducted properly according to various methods known in the art, and for example, the induction may be performed by methods well known to those of ordinary skill in the art to which the present invention belongs, for example, transfection using calcium phosphate (Graham, F L et al., Virology, 52:456 (1973)), transfection by DEAE dextrin, transfection by microinjection (Capecchi, M R, Cell, 22:479 (1980)), transfection by a cationic lipid (Wong, T K et al., Gene, 10:87 (1980)), electroporation (Neumann E et al., EMBO J, 1:841 (1982)), transduction or transfection, as described in the documents (Basic methods in molecular biology, Davis et al., 1986 and Molecular cloning: A laboratory manual, Davis et al., 1986). The introduction is preferably performed by transfection. Transfection efficiency may vary greatly depending on the type of cells, as well as cell culture conditions or a transfection reagent.

In the present invention, the test material refers to an unknown material used in screening to examine whether cell cycling, differentiation, dedifferentiation, morphology, division, proliferation or apoptosis is affected by regulating the expression of the non-canonical target gene of miRNA, and includes a compound, an extract, a protein or a nucleic acid, but the present invention is not limited thereto.

In the present invention, the expression level or expression of the non-canonical target gene of miRNA may be confirmed by various expression analysis methods, for example, RT-PCR, ELISA (see Sambrook, J et al, Molecular Cloning A Laboratory Manual, 3rd ed Cold Spring Harbor Press (2001)), western blotting or FACS analysis, but the present invention is not limited thereto.

The expression level or expression of the non-canonical target gene of miRNA in the present invention is confirmed, and as a result of comparison with the case of only treatment with the RNA interference-inducing nucleic acid without a test material, when there is a difference, it can be determined that the test material affects the expression of the non-canonical target gene of miRNA. Further, it can be determined that the test material has a function of regulating cell cycling, differentiation, dedifferentiation, morphology, migration, division, proliferation or apoptosis.

In addition, the present invention provides a method of preparing the above-described RNA interference-inducing nucleic acid.

More specifically, the present invention provides a method of preparing an RNA interference-inducing nucleic acid which suppresses a non-canonical target gene of miRNA by modifying a partial sequence of specific miRNA in one or more single strands of the double strands of a nucleic acid inducing RNA interference, which includes the following steps:

constructing an RNA interference-inducing nucleic acid to have a sequence of four bases in positions 2 to 5 from the 5′ end of the specific miRNA, and bases in positions 6 and 7, which are the same and complementary with a base capable of being paired with the 6^thbase of specific miRNA, including all complementary bases including G:A and G:U wobble pairs, or

constructing an RNA interference-inducing nucleic acid to have a modified base sequence in which at least one guanine base is substituted with uracil or adenine in the base sequence between the 1^stto 9^thbases from the 5′ end of specific miRNA.

adding a methyl group (2′OMe) to the 2′ position of the ribosyl ring of the 6^thnucleotide from the 5′ end of the specific miRNA.

constituting an RNA interference-inducing nucleic acid to have a modified base sequence in which at least one guanine base is substituted with an uracil base in the sequence of 6 to 8 consecutive bases, starting from the second base from the 5′ end of specific miRNA.

The RNA interference-inducing nucleic acid has already been described, and thus to avoid duplication, the description will be omitted.

The construction of the RNA interference-inducing nucleic acid is performed according to various methods known in the art, and the methods are not limited to a specific method.

In addition, the present invention provides an interface-induced nucleic acid modified by adding a methyl group (2′OME) to a 2′ site of the ribosyl group of the 6^thnucleotide from the 5′ end of specific miRNA.

The modified interface-induced nucleic acid maintains the purpose of the present invention for specifically suppressing only a non-canonical nucleation bulge site of the corresponding miRNA, and has an effect of completely removing non-canonical nucleation bulge binding which may newly occur.

Advantageous Effects

When an interference-inducing nucleic acid according to the present invention is used, a biological function exhibited by suppressing a non-canonical target gene of conventional miRNA can be effectively improved, or a part of the functions of the conventional miRNA, that is, only a biological function exhibited by suppressing a non-canonical target gene, can be selectively exhibited. In addition, a cell cycle, differentiation, dedifferentiation, morphology, migration, proliferation or apoptosis can be regulated by the interference-inducing nucleic acid, and thus it is expected to be used in various fields such as pharmaceuticals, cosmetics, etc.

DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the action mechanism of an interference-inducing nucleic acid suppressing a non-canonical target of miRNA.

FIGS. 2A to 2C show the non-canonical target-specific expression inhibitory effect of miR-124-BS by measuring the suppression of target genes of a canonical target (seed) of miR-124 and a non-canonical nucleation bulge target using a luciferase reporter enzyme.

FIGS. 3A and 3B show the result of fluorescence-activated cell sorting (FACS) performed on miR-124-BS to confirm the apoptosis induced by the canonical target (seed) and non-canonical nucleation bulge target of miR-124 in cervical cancer cells (HeLa).

FIGS. 4A and 4B show the result of observing cell morphology and a marker expression pattern through expression of miR-124-BS in human neural tumor blastoma (Sh-Sy-5y), confirming neurite differentiation induced by a non-canonical nucleation bulge target of miR-124.

FIGS. 5A and 5B show the result of observing cell morphological and molecular biological characteristics, confirming neurite branch differentiation induced by miR-124-BS(4753), which is a natural form of miR-124-BS regulating a nucleation bulge of miR-124 with a canonical target (seed) and miR-124-(3714) having the same seed sequence as miR-124.

FIGS. 6A to 6C show the result of observing cell morphological and molecular biological characteristics, confirming that neurite branch differentiation induced by a non-canonical nucleation bulge target of miR-124 in a mouse neuroblastoma (N2a) is a mechanism caused by the regulation of the expression of an MAPRE1 gene.

FIGS. 7A to 7C show the RNA-Seq analysis result of confirming the inhibition of the expression of a non-canonical nucleation bulge target gene induced by miR-124-BS in mouse neuroblastoma (N2a) at the transcriptome level.

FIGS. 8A to 8C show the result of observing cell cycle arrest induced by a non-canonical nucleation bulge target of miR-122 in a human liver cancer cell line (HepG2) using miR-122-BS and flow cytometry.

FIGS. 9A to 9C show the cell morphological and molecular biological features and flow cytometry of miR-1-BS and miR-155-BS, confirming the increase in thickness of myocytes induced by a non-canonical nucleation bulge target of miR-1 and the inhibition of myocyte differentiation and induction of apoptosis, which are induced by a non-canonical nucleation bulge target of miR-155, in a muscle cell line.

FIGS. 10A to 10D show the bioinformatics analysis results of Ago HITS-CLIP, which are the RNA data of Argonaute protein-bound miRNA and a target in human brain tissue, confirming that there are a canonical seed target as well as a non-canonical G:A wobble seed target, which naturally occur.

FIGS. 11A to 11D show the cell morphology and flow cytometry result for observing apoptosis of miR-124-G4U and miR-124-G5U, confirming that the non-canonical 4G:A wobble seed target and the non-canonical 5G:A wobble seed target of miR-124 in mouse neuroblastomas (N2a) show totally different functions from miR-124 in brain cell differentiation.

FIGS. 12A and 12B show the flow cytometry (fluorescence-activated cell sorting: FACS) result for analyzing cell proliferation and cell cycling with miR-124-G4U and miR-124-G5U to confirm the biological functions of suppressing the non-caconical 4G:A wobble seed target and the non-canonical 5G:A wobble seed target of miR-124 in human neuroblastomas (SH-sy-5y).

FIGS. 13A to 13D show the flow cytometry (fluorescence-activated cell sorting; FACS) result for a cardiomyocyte cell line (h9c2) with a fluorescent protein reporter, confirming that a non-canonical 7G:A wobble seed target of miR-1 has a gene-suppressing function.

FIGS. 14A to 14C show the result of observing cell morphological and molecular biological features of a myocardial hypertrophy-inducing effect after miR-1-G2U, miR-1-G3U and miR-1-G7U are expressed in rat neonatal cardiomyocytes, confirming the functions of the non-canonical 2G:A, 3G:A and 7G:A wobble seed targets of miR-1.

FIGS. 15A and 15B show the result of observing cell morphological features for a myocardial hypertrophy-inducing effect after miR-133-G4U is expressed in myocardial cells (h9c2) to confirm the function of a non-canonical 4G:A wobble seed target of miR-133.

FIGS. 16A to 16B show the results of measuring the enzyme activity of a luciferase reporter in a human liver cancer cell line (HepG2), confirming that gene expression is inhibited through non-canonical 2G:A and 3G:A wobble seed targets of miR-122.

FIGS. 17A to 17D show the result of observing cell morphological features of the expression of miR-122-G2U and miR-122-G2,3,U to confirm a function in which the inhibition of the expression of a non-canonical 2G:A wobble seed target and a non-canonical 2,3G:A wobble seed target suppresses the migration in a human liver cancer cell line (HepG2) by miR-122.

FIGS. 18A and 18B show the results of flow cytometry for the expression of miR-122-G2,3U to confirm that the inhibition of the expression of a non-canonical 2,3G:A wobble seed target by miR-122 induces cell cycle arrest in a human liver cancer cell line (HepG2).

FIGS. 19A and 19B show the result of flow cytometry for the expression of miR-122-G2,3U and miR-122-G2,7U to confirm that the inhibition of the expression of a non-canonical 2,3G:A wobble seed target and a non-canonical 2,7G:A wobble seed target by miR-122 induces cell cycle arrest of a human liver cancer cell line (HepG2).

FIGS. 20A and 20B show the result of flow cytometry for the expression of let-7a-G2U, let-7a-G2,4U and let-7-G4U to confirm that the inhibition of the expression of non-canonical 2G:A and 2,4G:A wobble seed targets by let-7 induces cell cycle arrest of a human liver cancer cell line (HepG2) and the inhibition of the expression of a non-canonical 4G:A wobble seed target promotes a cell cycle.

FIGS. 21A to 21C show that miR-372-G4U or miR-302a-G4U inducing the inhibition of the expression of a non-canonical 4G:A wobble seed target by miR-372 or miR-302a promotes dedifferentiation with miR-372 or miR-302a, confirmed with an Oct4 gene expression reporter.

FIGS. 22A and 22B show the results of confirming the phenomenon in which a 2′OMe modification in position 6 from the 5′ end does not suppress a non-canonical nucleation bulge target of a corresponding RNA interference derivative.

FIGS. 23A and 23B show the results of confirming that a 2′OMe modification in position 6 from the 5′ end does not suppress total non-canonical nucleation bulge target mRNAs in a transcriptome.

FIGS. 24A and 24B show the results of analyzing miRNAs binding to non-canonical nucleation bulge sites through Ago HITS CLIP experiments.

FIGS. 25A to 25F show the results of identifying a G-to-U modification recognizing non-canonical GA wobble seed target as a canonical target in tumor miRNA by analyzing sequence variations in a miRNA sequencing database (TCGA) of cancer patients.

MODES OF THE INVENTION

Hereinafter, the present invention will be described in further detail with reference to the following examples. The following examples are merely provided to exemplify the present invention, and the present invention are not limited to the following examples.

EXAMPLE 1
Action Mechanism of Interference-Inducing Nucleic Acid Suppressing Non-Canonical Target of miRNA

Through Ago HITS CLIP assay, the inventors confirmed from an Argonaute protein-binding RNA complex sequence that, when miRNA is base-paired with a target site in Argonaute protein, the miRNA binds with target messenger RNA (mRNA) without making a complete complementary pairing with the miRNA seed, in addition to a canonical seed target having a complete complementary pairing with the miRNA seed. Moreover, as a result of sequencing of RNA having the above-described characteristics, it was confirmed that there are a target base-paired by the induction of a nucleation bulge to the target by the 6^thbase of the miRNA (non-canonical nucleation bulge target site of miRNA) and a target site allowing G:A wobble pairing in a miRNA seed region (non-canonical G:A wobble seed target site of miRNA), which naturally occur (FIG. 1).

As illustrated in FIG. 1, miR-124 has at least 6 consecutive complementary base pairings to the base sequence of a target site, which is 5′-GUGCCUUA-3′ with a seed base sequence (5′-UAAGGCAC-3′) by means of the Argonaute protein, and the target site having such base pairings has been reported as a canonical seed target site (canonical seed site) of miRNA (Nature, 2009, 460 (7254): 479-86).

In addition, in the Argonaute-bound RNA complex, a target in which the miR-124 seed base sequence (5′-UAAGGCAC-3′) makes complementary base pairing with a non-canonical nucleation bulge target site (5′-GUGGCCUU-3′) of miRNA to allow G of target mRNA in positions 5 and 6 from the 5′ end of miR-124 to be arranged in a bulge was identified, and based on this, the seed base sequence of miR-124 enabling complementary base pairing with the non-canonical nucleation bulge target site of miRNA has a base sequence (5-UAAGGCCA-3) in which the 6^thbase is repeated once more between positions 6 and 7 of the miR-124 seed. The consecutive and perfectly complementary sequence with respect to the above-described sequence was used as miR-124BS (BS: bulge site) siRNA suppressing a non-canonical nucleation bulge target of miR-124.

When miRNA-bound target mRNA sequence in the Argonaute-bound RNA complex was analyzed, other than a conventional complementary base sequence, it was confirmed that G:A wobble base pairing occurs in the binding of the target mRNA with the seed sequence of miRNA. Therefore, in the case of miR-124, the 4^thG base from the 5′ end of the seed base sequence (5′-UAAGGCAC-3′) makes a G:A wobble pair, thereby recognizing a non-canonical G:A wobble seed target site of miRNA (5′-UGGCAUU-3′). Based on this, siRNA was designed with a consecutively and perfectly complementary sequence with the non-canonical G:A wobble seed target site of miR-124 to invent miR-124-GU, and siRNA was used as siRNA inhibiting the expression of the non-canonical G:A wobble seed target of miR-124.

EXAMPLE 2
Confirmation of Inhibitory Ability of miRNA-BS in Which Non-Canonical Nucleation Bulge Target Site and Consecutively and Perfectly Complementary Base Sequence are Included in Seed Region

In a canonical seed target site of natural miR-124, the sequence complementary to the 2^ndto 7^thbases from the 5′ end of miR-124 is 5′-GUGCCUU-3′, and the canonical seed target site makes perfectly complementary base pairing with at least 6 consecutive bases with the seed of miR-124 (5′-UAAGGCAC-3′) (FIG. 2A).

A non-canonical nucleation bulge target site (Nuc site; nucleation bulge site) of miR-124 found by Ago HITS CLIP assay is 5′-GUGGCCUU-3′, the 6^thbase (C) of miR-124 is base-paired with the target by forming a nucleation bulge, and therefore, G of target RNA paired between positions 5 and 6 from the 5′ end of miR-124 is formed in a bulge. Based on this, siRNA was designed using the base sequence having consecutively and perfectly complementary binding with the non-canonical nucleation bulge target site (nucleation bulge site) (5′-UAAGGCCA-3′) as a miRNA seed and named miR-124-BS siRNA (FIG. 2A). It is considered that miR-124-BS recognizes a non-canonical target of miR-124, which is a nucleation bulge target site (Nuc site), as a canonical seed target, and specifically and more strongly inhibits gene expression of the non-canonical target, which was confirmed through luciferase reporter assay (FIGS. 2B and C).

First, to confirm how much miR-124 can suppress a non-canonical target, which is a nucleation bulge target site, compared with a conventional canonical target, which is a seed target, a corresponding site for miR-124 was inserted into a luciferase reporter, and various concentrations (0, 0.01, 0.1, 0.5, 2.5 and 15 nM) of miR-124 were also transfected into cells, followed by measuring inhibitory concentration 50 (IC₅₀) with luciferase activity. As a result, it can be confirmed that the IC₅₀of the canonical seed site was approximately 0.5 nM, which was similar to the IC₅₀of the non-canonical nucleation bulge site of approximately 0.2 nM (FIG. 2B). However, the highest inhibitory ratio for the canonical seed site was approximately 50%, and the highest inhibitory ratio for the non-canonical nucleation bulge site was approximately 80%, indicating that the inhibition through the non-canonical nucleation bulge site is slightly weak. Particularly, through the measurement of luciferase activity, it was confirmed that gene suppression is initiated at a concentration of approximately 0.01 nM or more for the canonical seed target and 0.1 nM or more for the non-canonical nucleation bulge target (FIG. 2B). Accordingly, it was able to be seen that miR-124 regulates the expression of the canonical target and the non-canonical nucleation bulge target, and has high canonical target expression regulatory efficiency.

The luciferase reporter assay performed herein was conducted by cloning the sequence of the corresponding miR-124 target site into a Renilla luciferase gene (3′ untranslated region (3′UTR)) site of a psi-check2 vector (Promega) twice in a row. The sequence of the non-canonical bulge site uses a naturally-occurring non-canonical nucleation bulge target site in the 3′ UTR site of MINK1. The miR-124, as a human-derived sequence, was prepared as a duplex of a guide strand and a passenger strand, which are chemically synthesized by Bioneer according to a miRBase database, separated by HPLC, according to by the method provided by the manufacturer. The miR-124 and the luciferase reporter vector were co-transfected into approximately 10,000 cervical cancer cells (HeLa: ATCC CCL-2) using a Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. The HeLa cells were incubated in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin, and during transfection, the cells were incubated in an antibiotic-free complete medium. Twenty-four hours after transfection, luciferase activity was measured using a dual-luciferase reporter assay system (Promega) according to the manufacturer's protocol, and here, the measurement of Renilla luciferase activity was repeated at least three times using a Glomax Luminometer (Promega), and calculated by normalization by firefly luciferase activity.

To confirm the functions of miR-124-BS invented to specifically suppress only the non-canonical nucleation bulge target of the miR-124, an experiment was conducted with a luciferase reporter in the same manner as described above. As a result, the miR-124-BS did not inhibit luciferase reporter enzyme activity with respect to the canonical seed target of miR-124 at any concentration (0 to 10 nM), but an effect of inhibiting luciferase reporter enzyme activity with respect to the non-canonical nucleation bulge target reporter of miR-124 was possibly confirmed at a concentration of 0.1 nM or more (FIG. 2C). Here, the gene inhibitory efficiency was measured by IC50, showing that the inhibition of the non-canonical nucleation bulge target is approximately 0.5 nM. In terms of this, the function of regulating the expression of the miR-124 non-canonical nucleation bulge target of miR-124-BS may be interpreted as being specific for the non-canonical nucleation bulge target site (FIG. 2C). The luciferase reporter assay used herein was performed by synthesizing miR-124-BS by Bioneer in the same manner as described above, co-transfecting HeLa cells (ATCC CCL-2) with a corresponding luciferase reporter vector, and measuring luciferase enzyme activity for 24 hours.

In this example, the inventors invented miRNA-BS having a consecutive and perfectly complementary seed site sequence with respect to a corresponding site sequence in order to suppress only a non-canonical organic target of miRNA, and when a luciferase reporter assay was performed using miR-124 to verify the effect of miRNA-BS, it was able to be demonstrated that the miR-124-BS does not inhibit the expression of a canonical seed target, and is able to effectively inhibit the expression of a non-canonical bulge target.

EXAMPLE 3
Observation of Apoptosis of Cervical Cancer Cells Induced by Inhibition of Expression of Non-Canonical Nucleation Bulge Target of miR-124

After confirming that the miR-124-BS suppresses only a non-canonical bulge target among several targets suppressed by miR-124 in the above-described example, through this, it was intended to confirm whether only a specific function of the miR-124 can be exhibited. Since specifically expressed only in nerve cells, the miR-124 is known to mainly play a role related to nerve cells. However, miR-124 expression decreases in gliomas generated by tumors, with regard to this, when the miR-124 expression is artificially induced in tumor cells, it was observed that the apoptosis of tumor cells may occur. Therefore, to confirm whether various and different functions of miR-124 vary according to the type of target, the miR-124-BS was transfected into cervical cancer cells (HeLa), and the apoptosis of the cells was observed through flow cytometry (FIG. 3A).

The experiment was performed by transfecting 75 nM of the miR-124 or miR-124-BS duplex into cervical cancer cells (HeLa: ATCC CCL-2) using an RNAiMAX reagent (Invitrogen) according to the manufacturer's protocol, treating the cells with trypsin 72 hours after transfection, detaching the cells from the culture dish, treating the cells using an Annexin V: FITC Apoptosis Detection Kit II (BD Pharmingen) according to the method provided by the manufacturer to detect annexin V for apoptosis and propidium iodide (PI) for necrosis by BD Aria I (BD Biosciences). Here, as a control, a sequence was synthesized to be the same as the sequence of cel-miR-67, which is miRNA of C. elegans.

As a result of comparing these experimental results for the control, miR-124 expression and miR-124-BS expression, when miR-124 was expressed, it was observed that, in cervical cancer (HeLa) cells, compared with the control, a ratio of progressing cell death by apoptosis (the number of cells stained with annexin V) increases from approximately 30% to approximately 78%, and it was confirmed that the miR-124-BS expression also increased to approximately 73%, which is 2-fold higher than the control (FIG. 3A). According to the result of Example 2 in that the miR-124-BS does not suppress a canonical seed site of the miR-124 and suppresses only a non-canonical bulge site, it can be seen that the apoptosis of cervical cancer cells occurs by the inhibition of the expression of the non-canonical nucleation bulge target.

It has been reported that the miR-124 is a brain tissue-specific miRNA, and does not have a brain tissue-specific function in other cells not showing tissue cell-specific transcriptome expression (Farh K et al, 2005, Science, 310 (5755) 1817-21). Therefore, to observe a function of the miR-124 that is caused by suppressing a non-canonical bulge target in nerve cells, the miR-124-BS was transfected into a cerebellar neural stem cell line, C17.2, and then cell differentiation was observed in cell morphology (FIG. 3B, upper image). As a result, when the miR-124 was transfected, the neurite differentiation of C17.2 cells was extended and promoted, but when the miR-124-BS was transfected, it was observed that there was differentiation, and branches were short but different. Therefore, it can be seen that general neurite differentiation induced by the miR-124 occurs by suppressing a canonical seed site target. Here, the miRNA transfection was conducted with an RNAiMax reagent by synthesizing 50 nM miRNA in the same manner as in Example 2.

In addition, since the miR-124-BS previously induced apoptosis in HeLa cells, whether such a function is also exhibited in nerve cells was observed through flow cytometry for C17.2 cells in the same manner as in Example 2. As a result, in the case of the miR-124 expression, an apoptosis ratio was 22.4%, almost similar to the control which is 21%, and in the case of the miR-124-BS expression, an apoptosis ratio was 33.4%, which is slightly increased (FIG. 3B, middle image). This shows the same tendency as in cervical cancer cells in that apoptosis occurs through the suppression of a non-canonical bulge target of the miR-124, but the increase rate is much smaller than that in the cervical cancer cells. The miR-124 mainly plays a differentiation-involved role in neural stem cells, and here, the regulation of a cell cycle may play a critical role. Therefore, after miR-124 and miR-124-BS were transfected into C17.4 cells, a cell cycle was observed by propidium iodide (PI) staining through flow cytometry (FIG. 3B, lower image). Here, it was seen that the miR-124 induces cell cycle arrest in the G0/G1 phase to 83%, which is slightly higher than the control, which is 69%. In comparison, it was possible to observe that miR-124-BS expression does not significantly induce cell cycle arrest. This experiment was performed by transfecting corresponding miRNA in the same manner as in the above-described example, incubating the transfected cells for 48 hours, detaching the cells, fixing them with ethanol, reacting the cells with 1 mg/ml of PI (Sigma-Aldrich) and 0.2 mg/ml of RnaseA at 37° C. for 30 minutes, and analyzing them through flow cytometry using BD FACSCalibur (BD Biosciences).

Summarizing the results in the above examples, only by inhibition of the expression of a non-canonical nucleation bulge target of miR-124 by the miR-124-BS, the death of cervical cancer cells (Hela) was induced like the miR-124, and therefore, it can be seen that the inhibitory function of the non-canonical bulge target of the miR-124 is the death of cancer cells. In addition, in neural stem cells, as neurite differentiation occurs due to miR-124 expression, little apoptosis and cell cycle arrest occur, and in the miR-124-BS suppressing only a non-canonical bulge target of miR-124, such a function is exhibited in a slightly different form. Accordingly, it can be seen that the nerve cell differentiation function of miR-124 is usually caused by the suppression of a canonical seed target gene which is conventionally known, or in a different form, it was able to be considered that the nerve cell differentiation function of miR-124 may occur by a non-canonical bulge target.

EXAMPLE 4
Confirmation of Neuronal Differentiation in Different Form With Many Branches and Short Neurites (Branched Neurite Outgrowth) by Inhibiting Expression of miR-124 Non-Canonical Nucleation Bulge Target by miR-124-BS

After confirming the fact that the miR-124-BS induces a different type of neurite differentiation from miR-124 in the previous example, to further examine this, a human neuroblastomas (sh-sy-5y) cell line (CRL-2266) was used for the experiment. Here, in addition to the miR-124-BS, miR-124-BS(4753) designed to have the same miRNA sequence having the same miR-124 seed sequence which is able to recognize and suppress a non-canonical nucleation bulge site of the miR-124, but the other part of which is different was synthesized by the method described in the previous example and used for the experiment. The sequence of the miR-124-BS(4753) used herein is 5′-CAAGGCCAAAGGAAGAGAACAG-3′, and a control was used by being synthesized to be the same as the miRNA of C. elegans, cel-miR-67 (NT; non-targeting). The corresponding miRNA was transfected by the same method as described in the previous example, the cells were cultured for 60 hours, and then the morphological change of the cells was observed using an optical microscope (FIG. 4A).

As a result, the miR-124 expression was possible to observe by the morphological change which makes a neurite of the human neuroblastoma (Sh-sy-5y) cell differentiate longer, whereas in the miR-124-BS(4753) having the same seed sequence that can suppress only a non-canonical bulge target of miR-124 as the miR-124-BS, a highly branched neurite outgrowth was observed in one cell (FIG. 4A). Based on this, the same experiment was additionally performed on catecholamine-containing nerve cells (CAD, CRL-11179). Here, corresponding miRNA was transfected, the cells were incubated for 52 hours, and then the cells were observed (FIG. 4B). As a result, like the result shown from the human neuroblastoma (Sh-sy-5y, CRL-2266) cells, the miR-124 is small in number but forms a longer neurite, whereas it was possible to observe that the miR-124-BS(4753) shows a differentiation phenomenon in which many short neurite branches overgrow (FIG. 4B). Additionally, miR-124 and miR-124-BS(4753) are transferred into cells at the same ratio, cells with long neurites and cells with many neurite branches were observed simultaneously. This shows the possibility that, when both of the invented miR-124-BS and the conventional miR-124 were expressed, it can artificially promote various types of neuronal differentiation similar to brain cells shown in a more complicated neural network.

Additionally, the neuronal differentiation of the CAD cell line promoted by the miRNA was confirmed by immunostaining for a nerve cell-specific marker, Tuj1 (FIG. 4B, lower image). The experiment performed herein was performed by transfecting corresponding miRNA, fixing the CAD cells with 4% paraformaldehyde 52 hours after transfection, immuno-staining the cells with a primary antibody, Tuj1 (MRB-435P, Covance Antibody Products) at 1:1000 and an Alexa 594 fluorescent material-conjugated secondary antibody (Abcam: ab150076) at 1:1000, and staining the nucleus with DAPI.

From the result of the above-described example, it can be seen that miR-124 has a function of forming many short neurite branches, which is different from the function of the conventional miR-124 by suppressing only a non-canonical nucleation bulge target. Particularly, there are a different sequence from the miR-124-BS and the same seed sequence, which was observed using the miR-124-BS(4753) capable of suppressing only the non-canonical nucleation bulge target, elucidating the fact that an RNA interference derivative including the seed sequence capable of recognizing the non-canonical nucleation bulge target exhibits the same effect as the miR-124-BS. In addition, it can be shown that complex neurite branches generated by the miR-124-BS can be applied as a method of promoting various types of neuronal differentiation similar to human brain cells showing a complicated neural network.

EXAMPLE 5
Confirmation of Morphological and Molecular Biological Features of Branched Neurite Outgrowth Induced Thereby After miR-124-BS is Expressed Using siRNA and shRNA Vector

To additionally confirm the specific branched neurite outgrowth phenomenon induced by the miR-124-BS expression in mouse neuroblastomas N2a (CCL-131) cells, as in Example 4, the miR-124 and the miR-124-BS(4753) were transfected into the cells, and then the morphological change of the cells was observed while the cells were incubated for 72 hours (FIG. 5A). In addition, simultaneously, the miR-124(3714) which has the same miR-124 seed sequence, but is different in the other part of the sequence was synthesized as the sequence of 5′-GAAGGCAGCAGUGCUCCCCUGU-3′ to form an siRNA-type duplex and then transfected into cells for an experiment. Here, a control was used by being synthesized to be the same as the sequence of cel-miR-67 (NT; non-targeting), which is miRNA of C. elegans.

As a result, in the experiment group into which the miR-124 is delivered, nerve cells having a long neurite similar to that in catecholamine-containing nerve cells, CAD, were able to be observed, and in cells to which miR-124-BS(4753) was transfected, the morphological feature of a cell having a short neurite branched in various directions was possible to confirm (FIG. 5A, upper). In addition, in an experimental group into which miR-124(3714) was transfected, like the miR-124, it was possible to observe that there are many cells having a relatively longer neurite (FIG. 5A, upper). Such morphological changes in cells were able to be confirmed by detecting an increase in expression levels of an antibody against a corresponding protein expressed in differentiated nerve cells, Tuj1 (Covance Antibody Products, MRB-435P), vGLUT1 (Synaptic Systems, 135-303) and MAP2 (Millipore, MAB3418) through immunoblotting, compared with the expression of four types of control proteins (ACT-B, TUB-A, GAPDH and H3B) (FIG. 5A, lower). Accordingly, it was possible to confirm that the neuronal differentiation caused by miR-124-BS(4753) that is similar to but different from that by miR-124 is slightly different in a morphological aspect, but induced in the same manner in terms of a gene expression marker, compared with the case caused by miR-124, and it was able to seen that the miR-124(3714) shown to mainly suppress a canonical seed target in the same manner as miR-124 because it has the same seed sequence exhibits a characteristic of differentiating the branch of a nerve cell longer than expected. In addition, when both of the miR-124-BS(4753) and miR-124(3714) are expressed, the expression levels of proteins shown in the differentiated nerve cells increased, confirming that these have molecular characteristics of differentiated forms of nerve cells.

In all of the examples described above, the miRNA expression was performed by transfecting a synthesized duplex into cells, or also by transfecting a vector for expressing the form of shRNA, which is a hairpin-shaped RNA capable of generating the miRNA. Therefore, a vector for expressing miR-124, miR-124-BS(4753) or miR-124(3714) in the form of shRNA was formed using a pCAG-miR30 plasmid (Addgene #14758), which is a vector for expressing pre-miRNA. The pCAG-miR30 vector used herein used a CAG promoter to strongly express miRNA, was designed to express a backbone of miRNA-30, thereby having an advantage of maximizing miRNA expression (Paddison P et al, Nature methods, 2004, 1(2): 163-7).

The pCAG vector formed to express each of the miR-124, miR-124-BS(4753) and miR-124(3714) was transfected into N2a cells, using a pCAG vector not expressing anything, and then cell morphology was observed (FIG. 5B). In an experiment group in which a hair-pin structure was transfected into neuroblastoma (N2a) cells, similar changes to the changes in cell morphology shown by the miR-124, miR-124-BS(4753) and miR-124(3714) duplexes were shown. When incubated for 72 hours after transfection, in the experimental groups in which the miR-124 and the miR-124(3714) are expressed, respectively, neuroblastoma cells with a long neurite were able to be observed. In contrast, in the experimental group in which the miR-124-BS(4753) is expressed, neuroblastoma cells with a short neurite branching in various directions were able to be observed (FIG. 5B, upper). Such changes in cell morphology were confirmed from the increase in expression of Tuj1 (O. von Bohlen et al. 2007 Cell and Tissue Research, 329, 3, 409-20), which is a protein expressed in differentiated nerve cells, or expression of synaptophysin (SYP) (FIG. 5B, lower). Therefore, it was possible to confirm that the cells into which the miR-124, miR-124-BS(4753), and miR-124(3714) hair-pin vectors are introduced have the characteristics of differentiated nerve cells. The increase in TUJ1 was observed by immunoblotting in the same manner as in the above-described example, and synaptophysin was detected using the primer sequences disclosed in the previous document (S. E. Kwon et al. 2011 Neuron 70, 847-54) through quantitative polymerase chain reaction (qPCR). This assay was conducted by transfecting a corresponding vector into N2a cells using a Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer's protocol, 24 hours after transfection, extracting total RNA using an RNeasy kit (Qiagen), performing reverse transcription with Superscript III RT (Invitrogen) according to the corresponding protocol, performing qPCR with an SYBR Green PCR Master Mix (Applied Biosystems) to measure an mRNA level of SYP as a corresponding primer, and then normalizing it to a GAPDH mRNA level.

From the results in the example, it can be seen that, even when a vector expressing the miRNA transfection in the form of shRNA, if only having the same seed sequence of the miR-124 (miR-124(3714)), like the miR-124, generally long neurite differentiation occurs by the inhibition of the expression of a canonical target gene. In addition, when the non-canonical bulge target of miR-124 is also expressed in the same manner as the miR-124-BS through the seed region in the form of shRNA (miR-124-BS(4753)), branched neurite outgrowth may occur. It was possible to confirm that both of the canonical target suppression and non-canonical bulge target suppression by the miR-124 show molecular gene marker expression in the form of neurite differentiation of nerve cells, and both show differentiation into nerve cells.

EXAMPLE 6
Confirmation of Induction of Neurite Differentiation of Mouse Neuroblastoma (N2a) Cells by miR-124-BS Through Regulation of MAPRE1 Expression

Additionally, the miR-124, miR-124-B S(4753) and miR-124(3714)-induced neurite differentiation in neuroblastoma (N2a) cells was observed in terms of cell morphology, which was quantitatively analyzed (FIG. 6A). During quantitative analysis, to distinguish a neurite branch derived from each nerve cell, a pUltra (Addgene #24129) plasmid expressing a green fluorescent protein (GFP) and a corresponding miRNA expression vector were co-transfected in the same manner as in the above-described example, cell culture was conducted for 72 hours, neurites were observed using a fluorescent microscope (Leica) to count the number of neurite branches, and the length of the neurite generated per cell was analyzed using the ImageJ program.

As a result, with respect to 100 cells, the number of neurite branches expressed per cell was increased in the miR-124, miR-124-BS(4753) and miR-124(3714) experimental groups approximately 3- to 5-fold compared to the control (FIG. 6A, lower left). However, when the miR-124-BS(4753) is expressed, compared with the miR-124 or miR-124(3714) is expressed, significantly more branches were observed. In addition, when it was confirmed that, in the miR-124-BS(4753) and miR-124(3714) experimental groups, the length of a neurite generated per cell was increased 4- to 6-fold compared to the control, and in further detail, it was observed that, when the miR-124 or miR-124(3714) was expressed, compared with when the miR-124-BS(4753) was expressed, a longer neurite branch is produced (FIG. 6A, lower right). This is the same result as observed in cell morphology in the previous examples (FIGS. 4, 5 and 6).

To investigate whether the specific neuronal differentiation phenomenon caused by the miR-124-BS is induced by a non-canonical bulge site of any gene, the non-canonical bulge target sequence of miR-124 was searched for in the sequence of the 3′ UTR region of the gene related to neuronal differentiation. As a result, a microtubule-associated protein RP/EB family member 1 (MAPRE1: Elena Tortosa et al. 2013 EMBO32,1293-1306) gene reported to regulate the differentiation of nerve cells was found. MAPRE1 may be a protein attached to the end of a microtubule constituting a neurite in the generation of the neurite, and may determine the shape of a neurite through the regulation of the formation of the corresponding microtubule. Accordingly, to confirm that the MAPRE1 gene is recognized as the non-canonical bulge target of miR-124 and inhibits the expression thereof, after miR-124-BS was transfected into N2a cells, the mRNA level of MAPRE1 was measured by qPCR assay according to the method performed in Example 5 (FIG. 6B). When qPCR was conducted with two different primers, it was possible to confirm that both of the two results were able to show that MAPRE1 expression decreased to approximately 40 to 70% in the miR-124-BS-transfected experimental group, compared with the control. Through this, it was possible to confirm that the expression of MAPRE1 is inhibited by the non-canonical bulge site of the miR-124.

MAPRE1 is the non-canonical bulge target of the miR-124, and if it is a very important target, the type of neuronal differentiation caused by the suppression of the non-canonical bulge target of the miR-124-BS is further promoted by the reduction in MAPRE-1 expression. Accordingly, to confirm this, two types of siRNA capable of suppressing MAPRE1 were prepared and transfected into N2a cells with miR-124, followed by performing an experiment of confirming a change in cell morphology (FIG. 6C). As a result, compared with the control expressing only miR-124, in the experimental group expressing miR-124 with the siRNA for MAPRE1, it was possible to observe that the number of neurite branches is highly increased, which was observed to be the same as in the experiment using two types of MAPRE1 siRNA having a different sequence (FIG. 6C). In this experiment, when RNA is transfected into cells in the same manner as in the method used in the previous example, the morphology was observed while the cells were incubated for 48 hours thereafter.

Therefore, it was confirmed that the short, branched neurite outgrowth induced by the miR-124-BS is a biological function that can be shown by at least partially inhibiting the expression of a MAPRE1 gene by the non-canonical bulge target site of miR-124.

EXAMPLE 7
Analysis of Tendency of Suppressing Non-Canonical Bulge Target Gene of miR-124 When miR-124-BS is Transfected Into Cells at Transcriptome Level

It was confirmed that a gene suppressed by miR-124-BS in mouse neuroblastoma (N2a) cells eventually induces the nerve cell differentiation which produces many neurite branches of neuroblastoma (FIG. 5). This phenomenon is considered to occur by a mechanism of recognizing and suppressing the non-canonical bulge target of miR-124, and such possibility was confirmed with MAPRE1 found as a target gene (FIG. 6). However, it may be actually a phenomenon caused by suppression of hundreds of non-canonical bulge target genes of miR-124.

After miR-124 and miR-124-BS were transfected into N2a cells, RNA-Seq assay was performed on the miR-124-BS invented by the inventors to confirm only the non-canonical bulge target gene of miR-124 at the transcriptome level expressing all genes. Here, as a control, a duplex having the same base sequence as miR-124 and an abasic substitution (pi) in position 6 from the 5′ end (miR-124-6pi) was used, and it was reported that such substitution does not make pairing with target mRNA at all since there is no base in position 6 from the 5′ end of miRNA (Lee H S et. Al. 2015, Nat Commun. 6:10154).

The RNA-Seq assay was performed by delivering each of the miR-124, the miR-124-BS and the miR-124-6pi duplex into N2a cells at 75 nM using a modification of the sequence of cel-miR-67, which is miRNA of C. elegans as an experimental control, in which the position 6 from the 5′ end is substituted with an abasic spacer (NT-6pi), extracting total RNA with an RNAeasy (Qiagen) 24 hours after incubation, constructing a library at Otogenetics, and subjecting it to next-generation sequencing. Afterward, a FASTAQ file, which is the sequence data obtained from the assay, was mapped to a mouse genomic sequence (mm9) with the TopHat2 program, expression values (FPKM) were obtained with Cufflink and Cuffdiff programs and normalized to a result in mouse neuroblastomas (N2a) cells into which a control NT-6pi was transfected to perform analysis with a fold change, (log2 ratio).

To analyze whether the mRNA level of a gene having a target site of miRNA is reduced by the expression of the corresponding miRNA from the RNA-Seq result, genes with a phastCons score of 0.9 or more were screened to select those in which a canonical seed site (7mer paired with bases between positions 2 to 8 from the 5′ end) of the miR-124 has 3′ UTR and the sequence of the corresponding site is conserved in various species, and these profiling results were compared and analyzed with a cumulative fraction in the order of inhibition depending on the expression of the corresponding miRNA. In addition, the non-canonical bulge site (nuc, 7mer) of miR-124 was also identified in the same manner as described above, and the suppressive fraction of the corresponding gene was analyzed with a cumulative fraction in the same manner as described above. Here, since a gene having the non-canonical bulge site of the miR-124 also has a canonical seed site, it is difficult to determine the suppressive effect thereof, in contrast to when there is no canonical seed sequence of miR-124 in total mRNA (nuc only), the case where there is only a canonical seed site and no non-canonical bulge site (seed only) was also analyzed.

In the RNA-Seq sequencing for the miR-124-expressed experimental group, when a fold change according to the miR-124 expression was analyzed according to a cumulative fraction, a phenomenon in which a gene (miR-124 seed) having the canonical seed site conserved in the miR-124 in the 3′ UTR is highly suppressed compared with the distribution of total mRNA was confirmed (FIG. 7A, upper), and compared with this, it was possible to observe that a gene having the non-canonical bulge site of the miR-124 is significantly suppressed and then very weakly suppressed (FIG. 7A, lower). However, such a suppressive phenomenon was not observed in miR-124-6pi-transfected cells as a control (FIG. 7B).

When the miR-124-BS developed by the inventors was expressed, in RNA-Seq sequencing, when a fold change according to the miR-124-BS expression was analyzed according to a cumulative fraction, it was confirmed that a weak inhibitory effect was shown in a gene (miR-124 seed) having the canonical seed site conserved in the miR-124 in the 3′ UTR, but there is no change in a gene (miR-124 seed only) having the corresponding canonical seed site without a non-canonical bulge site of the miR-124 (FIG. 7C, upper). However, it was able to seen that the suppression of both of the gene having the non-canonical bulge target of the miR-124 (miR-124 nuc) and the gene having a corresponding target (miR-124 nuc only) occurs strongly and effectively (FIG. 7C, lower). In FIG. 7C, when the miR-124-BS was expressed, the gene having only the canonical seed site of miR-124 (miR-124 seed only) was not suppressed, demonstrating that the canonical seed site of miR-124 was not suppressed at all, but it is predicted that the weak suppression of the seed site target of miR-124 by the miR-124-BS is probably caused by the co-existence of the canonical site and the non-canonical bulge site of the miR-124.

From the result of the example, it was able to be seen that miRNA very strongly and effectively suppresses a conventional canonical seed target gene, but very weakly suppresses the non-canonical bulge target at the transcriptome level, and it was possible to confirm that miRNA-BS inhibits the expression of the non-canonical bulge target of miRNA, but does not suppress the conventional canonical seed sequence at the transcriptome level.

EXAMPLE 8
Confirmation of Cell Cycle Arrest Induced by miR-122-BS in Human Liver Cancer Cell Line (HepG2) Through Flow Cytometry

Based on the result of observing the induction of nerve cell differentiation of miR-124 by the non-canonical nucleation bulge target of miR-124, an experiment for the biological function of the non-canonical nucleation bulge target of another miRNA was performed. MiR-122 has 5′-UGG AGU GU-3′ as a seed base sequence, and miR-122-BS, which is the base sequence of siRNA base-paired with the non-canonical nucleation bulge target site thereof, may have one more U in position 6, and in this case, thus have 19 bases represented by 5′-UGG AGU U GUG ACA AUG GUG-3′ at the 5′ end thereof and deoxy thymine nucleotides (dts) as bases in positions 20 and 21. Particularly, in a duplex structure, the corresponding dt parts may consist of a guide strand and a passenger strand to form a two-nucleotide overhang at the 3′ end. Here, the passenger strand made perfectly complementary base pairing with the guide strand, and included two 2′OMes at both of the positions 1 and 2 from the 5′ end and two dts at the end of the base sequence (FIG. 8A). The guide strand and the passenger strand of the miR-122-BS were chemically synthesized by Bionia, separated by HPLC, and were prepared in a duplex according to the method provided by the manufacturer.

The miR-122-BS prepared as described above (FIG. 8A) was transfected into a human liver cancer cell line (HepG2), and the change in cell cycle was then observed through flow cytometry. In this experiment, each of NT-6pi as a control and the miR-122 and miR-122-BS duplexes was transfected into HepG2 cells at 50 nM using an RNAiMAX (Invitrogen) reagent, and the cells were incubated for 24 hours, treated with 100 ng/ml of nocodazole for 16 hours and synchronized to the G2/M phase (division preparation phase/division phase), followed by analyzing a cell cycle with a Muse™ Cell Cycle Kit (Catalog No. MCH100106, Millipore) and a Muse Cell Analyzer (Millipore) according to the experimental method provided by the manufacturer.

As a result, compared with the cell cycle analysis for the NT-6pi-transfected control, in the miR-122-BS experimental group, the G0/G1-phase cells increased approximately 2-fold from 10.7% to 24.3%, confirming that the cell cycle arrest in the liver cancer (HepG2) cells increased (FIGS. 8B and 8C). An increase in the G0/G1-phase cells was not observed in the miR-122-transfected cells. Subsequently, the G2/M-phase cell distribution of the miR-122-BS-transfected human liver cancer cell line (HepG2) was reduced from 83.4% to 67.3% compared with the control, and this result was not observed in the miR-122-transfected cells (FIGS. 8B and 8C).

Accordingly, it was possible to observe that the miR-122-BS shows a biological function of inducing cell cycle arrest in a human liver cancer cell line through regulation of the expression of the non-canonical nucleation bulge target of the miR-122, which is a completely different function from the biological function of the miR-122.

EXAMPLE 9
Confirmation of Muscle Fibrosis Function in Skeletal Muscle Cells (C2C12), Induced by miR-1-BS, and Apoptosis Mediated by miR-155-BS

Since a novel biological function different from the function shown by the miRNA-BS-mediated inhibition of the expression of the canonical seed target of miRNA has been observed, to observe how such a function is exhibited in muscle cells, miRNA-BS was applied to miR-1 and miR-155, which are expressed in muscle cells and play a critical role.

First, since the miR-1 is a muscle tissue-specific miRNA, and has been reported to function in muscle cell differentiation (Chen J et.al, Nature genetics, 2006, 38(2): 228-233), miR-1-BS was expressed in the skeletal muscle cell line, C2C12, and then a cell morphological change (FIG. 9A) and the expression of a gene marker expressed in differentiation (FIG. 9B) were investigated. Here, miR-1-BS was synthesized in the same manner as described in the previous example and transfected into the cells, and the cells were incubated in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml of streptomycin for 48 hours and then observed after being divided under a growth media (GM) condition (FIG. 9A, upper) and further incubated for 48 hours under a FBS-removed deprivation media condition (DM) (FIG. 9A, lower). Afterward, the cells were treated with 4% para-formaldehyde (PFA) to fix the cells, a myosin 2 heavy chain protein expressed in differentiated muscle cells was reacted with a primary antibody (MF20: Developmental Studies Hybridoma Bank) and then stained with an Alexa 488 fluorescent material-attached mouse secondary antibody (ab150105) to observe cell morphology and differentiation (FIG. 9A).

As a result, when miR-1-BS was expressed under the growth media (GM) condition in the mouse skeletal muscle cells (C2C12), compared with the NT (control) or miR-1 duplex-transfected cells, a larger number of differentiated muscle cells was possible to confirm by MF20 staining. Under the deprivation media (DM) condition that reduces serum during incubation, the progression of the differentiation into a muscle fiber was observed, and it was possible to observe a fiber in a thicker form compared with the skeletal muscle cells expressing NT as a control (FIG. 9A). This is caused by faster induction of muscle cell differentiation by miR-1-BS under the GM condition. The differentiation of muscle cells can be confirmed by the expression level of a gene whose expression is increased during differentiation as a marker at the molecular level, and therefore, sk-actin and myogenin expression levels were detected through reverse transcription (RT)-PCR using a primer specific for the corresponding mRNA, compared with the mRNA level of b-actin, which has no difference in expression level in differentiation (FIG. 9B). As a result, when both of the miR-1(1) and miR-1-BS(1BS) are expressed, it was possible to confirm that the transcription levels of the sk-actin and the myogenin increase, and the transcription level of the myogenin is increased more by the miR-1-BS than the miR-1.

The miR-155 is miRNA inhibiting muscle cell differentiation (Seok H et. Al, JBC, 286(41):35339-46, 2011), the effect of miR-155-BS was observed through the differentiation of skeletal muscle cells (C2C12) in the same manner as performed for the miR-1 above (FIG. 9C). As a result, after NT as a control, miR-155 and miR-155-BS were expressed, it was observed that no significant morphological differences were observed under the GM condition, whereas under the DM condition inducing muscle cell differentiation, the control was differentiated, when the miR-155 was expressed, the differentiation was inhibited, and when the miR-155-BS was expressed, it was confirmed that differentiation disappeared through immunostaining with a myosin 2 heavy chain protein (FIG. 9C). Particularly, when closely examining the C2C12 cells expressing miR-155-BS, it was observed that the miR-155-BS induces apoptosis. In contrast, in the miR-155-transfected muscle cells, apoptosis was not observed at all (FIG. 9C, lower). For further investigation, the C2C12 cells expressing the corresponding miRNA were grown under the GM condition for 48 hours, and then subjected to flow cytometry in the same manner as in Example 3 to examine apoptosis (FIG. 9D). As a result, it was confirmed that, in the miR-155-BS-transfected experimental group, apoptosis increased approximately 1.5 to 2.5-fold compared with the control (FIGS. 9D and 9E).

In this example, it was possible to confirm that the miR-1-BS promotes the differentiation of skeletal muscle cells and induces the differentiation of a thick muscle fiber, the miR-155-BS induces the death of skeletal muscle cells, and these functions are different from the function of the conventional miR-1 for contracting muscle cells and the function of the miR-155 for inhibiting muscle cell differentiation.

EXAMPLE 10
Discovery of Non-Canonical Target Site Allowing Wobble Base Pairing at Seed Position of miRNA Through Ago HITS CLIP Assay

As a method of analyzing a miRNA target at the transcriptome level, an experimental method called Ago HITS CLIP has been developed and is widely used (Nature, 2009, 460 (7254): 479-86). The Ago HITS CLIP assay is a method of inducing covalent bonding between RNA and Argonaute protein in cells by UV irradiation of cells or a tissue sample, isolating the generated RNA-Argonaute complex through immunoprecipitation using an antibody specifically recognizing Argonaute, and analyzing the isolated RNA through next-generation sequencing. The base sequence data obtained thereby may not only identify target mRNA of miRNA through bioinformatics analysis, but also precisely analyze its binding site and sequence (Nature, 2009, 460 (7254): 479-86). Ago HITS-CLIP was first applied to mouse cerebral cortex tissue to perform mapping for the miRNA target in the brain tissue. Since then, this method has been applied to various tissues. Through numerous Ago HITS-CLIP assays, miRNA binding sites in various tissues were identified, and it was found that there are many non-canonical binding sites which make similar miRNA seed binding particularly in the above-mentioned target sequence, but different binding in the sequence of target mRNA (Nat Struct Mol Biol. 2012 Feb. 12; 19(3):321-7).

It was observed that some of the known non-canonical binding rules are present as wobbles through G:U base pairing, other than the conventional known base pairings. The wobble base pairing is found in specific RNA, different from the conventional base pairing, and it was shown that the well-known G:U wobble pair may also be paired to a non-canonical target of miRNA. However, compared with this, a G:A wobble pair which has weak binding and is not often found may form base pairing in specific RNA, but other than this, has not been investigated at all. However, in the case of the seed sequence of miRNA, since the free energy of base pairing is structurally stabilized by the Argonaute protein, generally weak G:A base pairing may be relatively significant, and based on fact described above, the inventors conducted analysis as follows.

First, to confirm whether wobble base pairing including a G:A pair is present in the miRNA target of human brain tissue, Ago HITS-CLIP data from the gray matter of the brain tissue after death was analyzed (Boudreau R L et al, Neuron, Vol. 81 (2), 2014, 294-305) (FIG. 10A). This analysis was performed by pairing an RNA sequence that had been bound with Argonaute-miRNA according to the method used in Ago HITS-CLIP development (Nature, 2009, 460 (7254): 479-86) to a human genomic sequence by Bowtie2 using a FASTQ file. In addition, the corresponding sequencing result paired to a human miRNA sequence at the same time so as to also analyze 20 types of miRNAs (top 20 miRNAs; FIG. 10B) frequently binding to the Argonaute protein in the corresponding brain tissue was analyzed (FIG. 10B). Here, it was observed that a considerably large number of canonical seed target sites are found at miRNA binding sites (FIG. 3B, median: 1292.5 sites, error range: +/−706.62). Afterward, from the Ago HITS-CLIP data, whether non-canonical target sites that are bound with G:U or G:A wobble pairing are present in such a miRNA seed base sequence was examined. Here, for analysis, a target sequence which cannot achieve complementary pairing of bases due to a base sequence identical to the miRNA seed base sequence, was used as a control.

As a result of the analysis, all of G:A wobble pairs pair-allowed target sites (median: 1119.5 sites, error range: +/−526.48) and G:U wobble pair-allowed target sites (median: 995 sites, error range: +/−523.22) showed higher distribution than the control (median 891 sites, error range: +/−410.71), indicating that, particularly, there are approximately 1.1 times more miRNA target sites having a G:A wobble pair than those having a G:U wobble pair (FIG. 10B). MiR-124 specifically expressed in brain tissue has two G bases in positions 4 and 5 from the 5′ end in the seed region, and thus can achieve G:U and G:A wobble base pairing (FIG. 10C). Accordingly, as a result of distinguishing and analyzing the G:A and G:U wobble base pairing by the G bases at the specific sites (FIG. 10D), the largest number (1,992) of canonical seed target sites (seed sites) of miR-124 were found. However, as compared with the above, 1,542 sites with a G:U wobble pair at the base in position 4 were found, and then 1,542 sites with a G:A wobble pair were observed. In addition, 1,800 target sites with a G:U wobble pair and 1,196 target sites with a G:A wobble pair were observed, and it was possible to confirm that all of the investigated sites are present in a number greater than the number of the control (647 sites) (FIG. 10D). Accordingly, when target mRNA is recognized with the base sequence of the seed region, a non-canonical G:U or G:A wobble base pair as well as a canonical base sequence with respect to the seed region is allowed, and it was able to be seen that through these types of pairing, miRNA can bind with target mRNA through such G:A wobble base pairing. Such miRNA target recognition through G:A wobble base pairing is a novel result that has not been reported yet, and as observed above, it was possible to confirm that non-canonical targets are recognized in many miRNAs.

In this example, as it was found that the recognition of the non-canonical target of miRNA allows G:A wobble pairing to the seed sequence, it is considered that many miRNAs may bind to the mRNA of a target gene, thereby inhibiting the expression of the gene.

EXAMPLE 11
Confirmation of Development of mIRNA-GU Recognizing G:A Seed Site, Which is Non-Canonical Target of miRNA, and Effect of Increasing Nerve Cell Death of miR-124-G5U When miR-124 is Applied

In Example 10, it was found that the non-canonical target recognition of miRNA may allow a G:A wobble pair in the seeding sequence, which is referred to as a non-canonical G:A seed site, and in contrast, miRNA-GU, which is the sequence of a new RNA interference-inducing material capable of complementarily pairing was invented. When applying such sequencing technology to miR-124, the miR-124 is the base sequence in which a seed sequence is 5′-AAGGCAC-3′ and a non-canonical G:A wobble seed target is capable of having G:A wobble base pairing at the 4^thand 5^thG bases, and therefore, when such a G:A wobble pair is changed into the miRNA-GU sequence, which is a format for changing the wobble pair into a conventional complementary base pair, the sequence may be changed into miR-124-G4U (5′-AAUGCAC-3′) or miR-124-G5U (5′-AAGUCAC-3′). Since the miR-124-G4U and miR-124-G5U changed as described above may be complementarily paired with a non-canonical target site weakly paired in a G:A wobble pair and recognized by the conventional miR-124, the function of the non-canonical G:A seed target in the miR-124 may be exhibited.

Accordingly, an experiment was performed by applying a non-canonical G:A wobble seed target to the miR-124 to examine the biological function mediated by the non-canonical G:A wobble seed target of the miRNA. To this end, NT (referred to as N2a of FIG. 11B) as a control, the miR-124, the miR-124-G4U or the miR-124-G5U were transfected into neuroblastomas N2a cells according to the same manner as described in the previous example, and incubated for 72 hours to observe cell morphology (FIG. 11B). As a result, when the miR-124 is expressed, the differentiation phenomenon in which neurites are formed occurs, but in the case of the miR-124-G4U or miR-124-G5U-transfected experimental group, a phenomenon in which differentiation does not occur is observed (FIG. 11B). Since neuronal differentiation ability, which is the conventional miR-124 function, is changed by the miRNA-GU, a change in apoptosis was observed in a human neuroblastomas (Sh-sy-5y) cell line. Here, the flow cytometry was performed with a Muse Annexin V and Dead Cell Assay Kit (Millipore), similar to the method used in Example 9, using a Muse™ Cell Analyzer (Millipore). As a result, it was confirmed that the miR-124-G-5U expression increases 2- or more fold compared with the control (FIG. 11C), and when such increase was analyzed by dividing into early apoptosis and late apoptosis, it was analyzed that, compared with the miR-124-transfected experimental group, in the miR-124-G-5U-transfected experimental group, both of early apoptosis and late apoptosis significantly increase (FIG. 11D). In the miR-124-G-4U-transfected experimental group, compared with the miR-124-transfected experimental group, it was possible to observe that late apoptosis was significantly inhibited (FIGS. 11C and 11D), and the number of living cells is also high (FIG. 11B).

Based on this, it was possible to confirm that the miR-124-GU-mediated suppression of the non-canonical G:A wobble seed target eliminates the neuronal differentiation ability induced by miR-124, and exhibits an apoptosis regulating function, which is another biological function, instead of the neuronal differentiation ability.

EXAMPLE 12
Promotion of Cell Division of Neuroblasts Induced by miR-124-G4U

In the previous example, since the miR-124-G4U lost neural differentiation ability and did not have a specific function in apoptosis, cell division was examined. That is, each of the control NT, the miR-124, the miR-124-G4U and the miR-124-G5U was transfected into neuroblastoma (N2a) cells in the same manner as described in the previous example, and flow cytometry for the cell division and proliferation phenomenon was performed (FIG. 12A). The experiment used herein was performed by treating cells using a Muse Ki67 Proliferation Kit (Millipore) according to the experimental method provided by the manufacturer, and analyzing cell division using a muse cell analyzer (Millipore). This method is for quantitatively analyzing the number of cells increased by cell division and proliferation by measuring the number of Ki67-stained cells. When miR-124 is expressed in N2a cells, compared with the control NT, as the cells are differentiated, the number of cells with reduced Ki67 staining intensity increases. However, in the miR-124-G4U-transfected experimental group, compared with the control (NT), the number of proliferated cells, Ki67-stained cells, slightly increased from 61% to 65%. In contrast, it was possible to observe that the miR-14-G5U has no difference from the control.

Additionally, to investigate an effect of miR-124-G4U on a cell cycle, flow cytometry was performed using a human neuroblastomas cell line (sh-sy-5y) (FIG. 12B). The analysis was performed using a Muse™ Cell Cycle Kit (Catalog No. MCH100106, Millipore) and a Muse Cell Analyzer (Millipore) to examine the functions of the miR-124-G4U and the miR-124-G5U by cell cycle. As a result, in the case of miR-124, cell cycle arrest was observed by an increase in the number of cells at the G0/G1 phase increases, but in the cases of the miR-124-G4U and the miR-124-G5U, the cell cycle arrest mediated by miR-124 disappears and the cell cycle goes back to the same as the control.

According to the above-described example, it was able to be seen that the miR-124-G4U of the RNA interference-inducing modified sequences suppressing the non-canonical G:A wobble seed target of miR-124 has a function of increasing cell division and proliferation of neuroblasts.

EXAMPLE 13
Confirmation that miR-1 can Inhibit the Expression of a Non-Canonical G:A Wobble Seed Target Gene in a Cardiomyocyte Cell Line Using a Fluorescent Protein Reporter

In the above-described example, the miRNA was able to non-canonically bind with target mRNA by allowing a G:A wobble pair in a seed sequence, and it was found that the resulting complex was able to have a novel function, and then a reporter assay for confirming whether such binding can actually induce the suppression of a target gene was conducted at the cellular level. Here, this assay was conducted on miR-1 known to be highly expressed in muscle-like cells such as cardiomyocytes h9c2, and particularly, on a G:A wobble base pair, focusing on G present in position 7 from the 5′ end of miR-1 (FIG. 13). To measure the gene suppression mediated by miRNA more precisely at the individual cell level, a fluorescent protein expression reporter system was constructed and used (FIG. 13). The fluorescent protein expression reporter system was formed to express fluorescent proteins with two colors in one vector. Here, a green fluorescent protein (GFP) was expressed in a SV40 promoter, several miRNA target sites were consecutively arranged at the 3′untranslated region (3′ UTR), a miRNA-mediated gene inhibitory effect was measured by the intensity of the GFP, and the red fluorescent protein (RFP) was expressed in a TK promoter so that a GF signal was normalized to the RFP intensity and used (FIG. 13A, upper).

A reporter was formed by inserting a non-canonical target site (7G:A-site) which is complementary to the bases in positions 2 to 8 from the 5′ end of miR-1 and has a G:A wobble at the 7^thbase (G) into the fluorescent protein expression reporter vector constructed as described above, and then whether the reporter is suppressed by miR-1 was confirmed through an experiment. Here, in this experiment, 500 ng of the GFP-7G:A site reporter vector and 25 μM of miR-1 were co-transfected into a cardiomyocyte cell line (H9c2) using a Lipofectamine 2000 (Invitrogen) reagent according to the manufacturer's protocol, and 24 hours after transfection, each fluorescent signal was measured through flow cytometry using Attune NxT (Life Technology). As a result, it was confirmed that the 7G:A site of the miR-1 was very effectively suppressed by miR-1 expression (FIG. 13A, middle) as compared with cells into which the control nucleic acid (cont; NT) was transfected (FIG. 13A, left). That is, as a result of analyzing the degrees of the expression of two types of fluorescent proteins in the cardiomyocyte cell line (h9c2) in four parts (Q5-1, Q5-2, Q5-3 and Q5-4), in the miR-1-transfected cells, compared with the control NT-transfected cells, the distribution of cells showing the RFP at an intensity of 103 and the GFP at an intensity of 103 was reduced from 59.51% (control: Q5-3) to 41.80% (miR-1: Q5-3).

In the control, it was observed that the GFP-7G:A site reporter vector was inhibited to some extent even in the h9c2 cells without miR-1 transfection. It may be predicted that the inhibition is mediated by miR-1 previously expressed in the h9c2 cells through G:A wobble base pairing with the reporter target mRNA. To confirm this, a miRIDIAN microRNA hairpin inhibitor (miR-1 inhibitor, FIG. 13A, right) which can inhibit the miR-1 expression in the h9c2 cells was purchased from Dharmacon, and transfected into cells at 50 μM. As a result, compared with the control (FIG. 13A, left), when the miR-1 inhibitor was transfected (FIG. 13A, right), the distribution of cells showing the RFP at an intensity of 103 and the GFP at an intensity of 103 was increased to 81.56% (miR-1 inhibitor: Q5-3), confirming that the miR-1 present in the cells can inhibit gene expression by the 7G:A site of miR-1 (FIG. 13A).

This was confirmed by additionally transfecting each of a control (GFP-no site) in which a miRNA target site was not added to a fluorescent protein expression reporter and a miR-1 7G:A site-inserted reporter, that is, a GFP-7G:A site into the H9c2 cells and comparing their activities, and the result was quantitatively compared with the case of co-transfection with miR-1 as a positive control (FIG. 13B). For the comparative analysis, the RFP values measured by a flow cytometer were divided by section, and the GFP values were averaged and converted into a log ratio, and then the log ratio was relatively calculated as the GFP level of the control, that is, the GFP-no site, was set as 1 (relative log GFP). Here, when there is a site complementarily paired with the 7^thG base of the miR-1 through G:A wobble pairing (GFP-7G:A site), particularly, in the section showing RFP expression (log RFP) from 1.5 to 3, it was confirmed that GFP expression (relative log GFP) caused by a G:A wobble is reduced by approximately 10% from 1. Such inhibition was observed in the same manner as the pattern in which GFP expression (relative log GFP) was inhibited by approximately 40% to 10% in the positive control, particularly, in the section of RFP expression (log RFP) from 1.5 to 4.

To confirm that the result observed in the above example was not influenced by the intensities of different promoters used in the corresponding fluorescent protein reporter and the difference in fluorescent activity between two different fluorescent proteins, a fluorescent protein reporter, that is, p.UTA.3.0 (Lemus-Diaz N et al, Scientific Reports, (7), 2017), in which two fluorescent proteins were interchanged was purchased from Addgene (plasmid #82447). Here, a reporter assay was conducted after constructing a reporter vector (RFP-7G:A site) by repeatedly inserting the miR-1-7G:A sequence five times into the 3′ UTR of the RFP regulated by a SV40 promoter in a p.UTA.3.0 vector, and the RFP expression level was used by modifying a GFP value normalized to a degree of transfection into cells (FIG. 13C, upper). As a result, like the previous example, the suppression of miR-1 7G; A site by miR-1 expressed in h9c2 cells was confirmed by comparing the results with the control of a reporter having no miRNA target site (RFP-no site) and a miR-1 7G:A site-inserted reporter (RFP-7G:A site), and the same phenomenon as in the previous example was also observed by the co-transfection of the positive control and the miR-1 (FIGS. 13C and 13D).

From the above, it was confirmed that the non-canonical target of miRNA found by Ago HITS-CLIP assay cannot only actually bind to the seed sequence of miRNA through G:A wobble pairing, but also inhibits the expression of the corresponding target gene. According to this, it is considered that the non-canonical target of miRNA is able to easily bind to the target site of miRNA through G:A wobble pairing, and if this can be similarly induced by miRNA-GU, only the biological function of miRNA mediated by the non-canonical target suppression will be used.

EXAMPLE 14
Confirmation of Function of Inducing the Hypertrophy of Cardiomyocytes Through Regulation of Non-Canonical G:A Wobble Seed Target Gene at G2, G3 and G7 of miR-1

To confirm the possibility of regulating a biological function through the above-described non-canonical gene suppression by the G:A wobble base pair acting as a non-canonical target of miRNA by using miRNA-GU, an experiment was conducted with the miR-1 of a cardiomyocyte, which has been described in the previous example.

To investigate whether a non-canonical G:A wobble target has a specific biological function, miR-1 should recognize only a non-canonical G:A wobble pair target site, rather than a canonical target, and thus miRNA-GU prepared by substituting the corresponding G with U in the seed region of the miR-1 to be paired with A, not C, was used for the experiment. In this experiment, for physiological conditions more similar to the heart, primary rat neonatal cardiomyocytes derived from rat cardiac tissue were used. Here, for cardiomyocyte culture, cardiomyocytes were isolated from cardiac tissue of a neonatal rat (1 day after birth) through an enzyme reaction (Neomyt kit, Cellutron), and the cardiomyocytes were cultured in a 4:1 mixture of Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% FBS (fetal bovine serum), 5% HS (horse serum), 100 U/ml penicillin and 100 μg/ml streptomycin and M199 (WellGene). By adding brdU to the prepared media, the cells were cultured differently from other cells with cell cycles in cardiac tissue.

It was first confirmed whether myocardial hypertrophy is induced in the primarily cultured cardiomyocyte obtained as described above (FIG. 14A). Cardiomyocytes have a characteristic of being able to increase their size after cell cycle arrest, which is called myocardial hypertrophy. Myocardial hypertrophy is a heart disease model system whose cell morphology and gene expression profile change are well known, which plays a role in compensating for the deterioration in function of cardiomyocytes and is known as the intermediate physiological stage of a heart disease. In the myocardial hypertrophy cell model, when the cardiomyocytes were treated with an adrenergic receptor agonist such as phenylephrine (PE), myocardial hypertrophy may be induced (Molkentin, J D et al, 1998, Cell 93(2), 215-228). Accordingly, myocardial hypertrophy was induced by treating the cultured cardiomyocytes with 100 μM phenylephrine, and then compared with a control which is not treated at all (FIG. 14A, rCMC), thus the increase in size of the cardiomyocyte was morphologically confirmed (FIG. 14A, +PE). In addition, when miR-1 was transfected into the primary cardiomyocyte, a decrease in size of the cardiomyocyte was observed (FIG. 14A, upper right). This is concurrent with the well-known myocardial hypertrophy and the miR-1 function in cardiomyocytes (Molkentin, J D et al, 1998, Cell 93(2), 215-228, Ikeda S et al, Mol cell boil, 2009, vo129, 2193-2204).

MiR-1 contains three Gs in the seed region, and therefore, to recognize only a non-canonical G:A wobble pair target site, not a canonical target, miR-1 in which G is substituted with U was designed and applied to the 2^ndposition (miR-1-G2U), the 3^rdposition (miR-1-G3U) and the 7^thposition (miR-1-G7U) from the 5′ end depending on where G is located. Here, the substituted base sequences of the miR-1 used in FIG. 14 are as follows (miR-1-G2U: 5′p-UUGAAUGUAAAGAAGUAUGUAU-3′; miR-1-G3U: 5′p-UGUAAUGUAAAGAAGUAUGUAU-3′; and miR-1-G7U: 5′p-UGGAAUUUAAAGAAGUAUGUAU-3′). These sequences were synthesized as a guide strand, and a passenger strand was prepared by being designed with reference to that of the conventional miR-1, chemically synthesized by Bioneer and separated by HPLC. The sequences were prepared in a duplex of a guide strand and a passenger strand according to the method provided by the manufacturer. The transfection of the corresponding miRNA into the primary cardiomyocyte cell line was performed using an RNAiMAX reagent (Invitrogen) at 50 μM.

As a result, it was possible to observe that when miR-1-G2U, miR-1-G3U or miR-1-G7U was transfected into the primary cardiomyocyte culture, the size of the cardiomyocytes was increased similar to the myocardial hypertrophy-induced cells (FIG. 14A, +PE). Here, to quantitatively analyze each cell size, 100 or more cells were quantified using the ImageJ program (NIH), and it was analyzed that all of the miR-1 substitutes (miR-1-G2U, miR-1-G3U and miR-1-G7U) synthesized to recognize only a G:Awobble pair site of miR-1 are 1.5 to 1.8-fold bigger than the case in which the control (NT) was transfected. This is a similar level to the size of cardiomyocytes in the phenylephrine (PE)-induced myocardial hypertrophy model, and miR-1 itself shows an effect of reducing cell size in terms of the morphology of the cardiomyocyte, showing that the canonical target inhibitory effect exhibits a function in cell morphology different from G:A wobble-mediated target suppression (FIG. 14B). In addition, to further confirm the myocardial hypertrophy observed in this example at the molecular level, the expression of an atrial naturiuretic peptide (ANP) gene known to be increased in expression as a marker was measured as a relative value, through quantitative polymerase chain reaction (qPCR), as compared with GAPDH (FIG. 14C). As a result, when the miR-1 substitutes (miR-1-G2U, miR-1-G3U and miR-1-G7U) synthesized to recognize only a G:Awobble pair site of the miR-1 was transfected into cardiomyocytes, it was possible to confirm through four repeated experiments that all of them significantly increased an ANP expression level approximately 1.2- to 1.5-fold compared to the control (NT). The increase in ANP expression is less than the increase in size of the phenylephrine (PE)-treated cardiomyocyte experimental group, which is approximately 2-fold higher than the control, which is, however, the opposite of the phenomenon in which ANP expression was significantly reduced when original miR-1 was transfected. This shows that miR-1 exhibits a completely different biological function due to a G:A wobble pair site.

From the above, it was finally seen that the non-canonical target suppressed by the G:A wobble pair in the miRNA seed region can exhibit a biologically different function from the conventional canonical target recognition. This finding indicates that the function caused by a canonical target of miRNA is different from the function caused by a non-canonical target thereof, and according to this, if only the G:A wobble target of the miRNA can be suppressed, only a biological function occurring due to the G:A wobble target will be selectively controlled.

EXAMPLE 15
Observation of Induction of Myocardial Hypertrophy Through miR-133-G4U Expression in Cardiomyocyte Cell Line (H9c2)

In terms of the non-canonical target phenomenon recognized by G:A wobble base pairing acting on the seed region of miRNA, to additionally confirm its biological function in cardiomyocytes, other than the miR-1 described in the previous example, miRNA-GU was applied to miR-133 known to function in cardiomyocytes. The miR-133 is known to have a function of regulating myocyte development and disease pathology and inhibiting myocardial hypertrophy (Nat Med. 2007, 13(5): 613-618; Ikeda S et al, Mol cell boil, 2009, vo129, 2193-2204). Therefore, the inventors transfected a 50 nM duplex prepared with miR-133-G4U (5′-UUUUGUCCCCUUCAACCAGCUG-3′), which is an interference-inducing nucleic acid specifically inhibiting target recognition through the G4 from the 5′ end of a non-canonical GA wobble target site of the miR-133, synthesized by Bionia as described above into a cardiomyocyte cell line (h9c2), and observed a change in cell morphology (FIG. 15A). As a result, miR-133-G4U expression increased the size of the H9c2 cells compared with the control (NT) (FIG. 15A), and as a result of quantitatively analyzing the sizes of 100 or more cells using the ImageJ (NIH) program, it was possible to confirm that the cell size significantly approximately 2-fold increased (FIG. 15B).

Accordingly, it was possible to predict that the decrease in expression of the non-canonical G:A wobble seed target of miR-133, occurring by miR-133-G4U induces myocardial hypertrophy, which is a novel function different from the canonical seed target inhibitory function of miR-133 for inhibiting myocardial hypertrophy.

EXAMPLE 16
Confirmation of Effect of Inhibiting Expression of Non-Canonical GA Wobble Seed Site Gene by miR-122 in Liver Cancer Cells Using Luciferase Reporter

In Example 10 described above, from the finding that miRNA non-canonically binds with a G:A wobble pair seed site, miRNA-GU specifically recognizing only the non-canonical target binding was invented, and it was observed in Examples 11, 12, 13 or 14 that, when this was applied to miR-124, miR-1 or miR-133, an effect different from the conventional function was exhibited. In addition, to confirm whether the expression of a target gene having a non-canonical G:A wobble pair seed site in miRNA is able to be actually inhibited, the function in a myocardial tissue cell line was confirmed for miR-1 in Example 13. In addition, it was attempted to confirm whether the expression of a non-canonical G:A wobble seed target gene can be inhibited in miR-122 specifically expressed in liver tissue and liver cancer cells, using a luciferase reporter system (FIG. 16).

The miR-122 specifically expressed in liver cancer or liver tissue and thus functioning has Gs capable of making a G:A wobble pair in positions 2, 3, 5 and 7 from the 5′ end of the seed sequence (the 1^stto 8^thbases from the 5′end: 5′-UGG AGU GU-3′). Therefore, first, in order to measure the inhibitory efficiency of the corresponding target site capable of being non-canonically recognized through G:A wobble pairing at G2, an inhibitory concentration 50 (IC50) was measured by the same method as described in Example 2 and compared with a canonical seed site (FIG. 16A).

To detect the inhibition of the expression of a non-canonical G:A wobble seed target enabling G:A base pairing with G2 of miR-122, a luciferase reporter vector for an experiment was produced by introducing five sequentially arranged copies of a non-canonical G:A wobble seed site (2G:A) sequence (the 1^stto 9^thbases from the 5′ end: 5′-CAC ACU CAA-3′) for the G2 of miRNA-122 into the 3′ untranslated region (3′ UTR) of a Renilla luciferase gene in a psi-check2 (Promega) vector In addition, a luciferase reporter vector for a canonical seed site (the 1^stto 9^thbases from the 5′ end: 5′-CAC ACU CCA-3′) was simultaneously produced in the same manner as described above.

As a result of measuring an effect of inhibiting the expression of a target gene having a canonical seed site and a non-canonical G:A wobble seed site (2G:A site) at G2 from the 5′ end at various concentrations of miR-122 by a change in activity of a corresponding luciferase reporter using the luciferase reporter vector produced as described above, and examining an IC50 value, the IC50 of the miR-122 was measured to be approximately 0.5 nM for a canonical seed target site, and to be approximately 7 nM for a non-canonical G:A wobble seed site (2G:A site), confirming that the miR-122 inhibits the expression of the gene having the non-canonical G:A wobble seed site (2G:A site), and showing that the efficiency is lower compared to the canonical seed site (FIG. 16A). In addition, when examining a concentration at which the inhibition of gene expression was initiated in a non-canonical G:A wobble seed target using a luciferase reporter, it was confirmed that luciferase enzyme activity is suppressed at 1.5 nM or more (FIG. 16A). This showed an approximately 2-fold difference in inhibitory efficiency from when the miR-122 inhibits the expression of a canonical seed target site.

This experiment was conducted by synthesizing a duplex of miR-122 in the same manner as used in this example, co-transfecting it with a corresponding psi-check2 vector (50 ng) at various concentrations (0, 1, 5, 10 and 25 nM) into approximately 10,000 liver cancer cells (Huh7, KCLB: 60104) according to the manufacturer's protocol using a Lipofectamine 2000 reagent (Invitrogen), and incubating the cells in a 96-well plate, and then luciferase activity was measured in the same manner as used in Example 2.

After confirming that miR-122 inhibits the expression of the non-canonical G:A wobble seed site gene, to confirm whether it is equally regulated by miR-122 present in cells, using a Huh7 cell line, which are liver cancer cells containing miR-122, a luciferase reporter assay was performed on a non-canonical G:A wobble seed site (2G:A) caused by G2 and the G3 from the 5′ end (FIG. 16B). To perform this assay, additionally, a luciferase reporter vector (luc-3G:A site) for a non-canonical G:A wobble seed site (3G:A) caused by G3 from the 5′ end, a luciferase reporter vector for a non-canonical G:A wobble seed site (luc-2G:A, 3G:A site) caused by G2 and G3 from the 5′ end, and a luciferase reporter vector (luc-PM site) measuring a perfectly complementary sequence to the entire sequence of miR-122 were produced in the same manner as described above, and co-transfected with a luciferase reporter vector (luc-2G:A site) for the non-canonical G:A wobble seed site (2G:A) caused by G2 from the 5′ end into a liver cancer cell line to measure luciferase activity (FIG. 16B).

As a result, when a luc-PM site vector was transfected into Huh7 cells known to have miR-122, it was observed that the activity was reduced by approximately 50% compared with the control into which only a luciferase vector was transfected, confirming that there is miR-122 in Huh7 cells, and simultaneously, the miR-122 suppresses the activity of a non-canonical G:A wobble seed target reporter (Luc-3G:A) at G3, and non-canonical G:A wobble seed target reporters (Luc-2G:A, 3G:A) at G2 and G3. In addition, to see whether the suppressive effect is induced by the miR-122, an miR-122 expression inhibitor (hsa-miR-122-5p inhibitor, IH-300591-06-0010) was purchased from Damacon and treated at 50 nM, confirming that all inhibitory phenomena disappeared.

According to the example, it was confirmed that the miR-122 is weaker than a canonical seed site, and can inhibit the expression of the non-canonical G:A wobble seed target caused by G2 from the 5′ end, and miR-122 naturally present in liver cancer cells (Huh7) inhibits the expression of the non-canonical 3G:A wobble seed target gene and the non-canonical 2,3G:A wobble seed target gene based on the 5′ end. Particularly, the inhibitory effect shown in the liver cancer cells (Huh7) disappears due to the treatment of the miR-122 expression inhibitor, which is regulated by miR-122 (FIG. 16B). Therefore, it can be seen that the miR-122 has a function of regulating the inhibition of the expression of a non-canonical G:A wobble seed target gene.

EXAMPLE 17
Confirmation of Phenomenon of Inhibiting Cell Migration in Liver Cancer Cell Line Through Inhibition of Expression of Specific Non-Canonical G:A Wobble Seed Target Gene of miR-122

To examine the function of a non-canonical G:A wobble seed target of miR-122 in liver cancer cells, miRNA-GU complementarily recognizing and suppressing the corresponding non-canonical G:A wobble seed target site was applied to be transfected into liver cancer cells (hepG2), and then a wound healing assay was performed to examine how the cell migration ability among the properties of the liver cancer cells, which is important for cancer metastasis, changes.

The wound healing assay for the liver cell line (hepG2) was performed by first synthesizing miRNA-GU complementarily recognizing the non-canonical 2G:A wobble seed target, the non-canonical 3G:A wobble seed target, or the non-canonical 2,3G:A wobble seed target based on the 5′ end of miR-122 with a corresponding sequence, transfecting the resulting sequence into the hepG2 cells in the same manner as described in the above-described example, making a scratch in a cell layer using a 1,000-μl tip 24 hours after culture, and incubating the cells until 48 hours and observing cell migration in an experimental group, and comparing with cells transfected with a control NT-bpi. The sequences of interference-inducing nucleic acids used in the assay are as follows: (miR-122: 5′-UG GAG UGU GAC AAU GGU GUU UG-3′; miR-122-G2U: 5′-UU GAG UGU GAC AAU GGU GUU UG-3′; miR-122-G3U: 5′-UG UAG UGU GAC AAU GGU GUU UG-3′; miR-122-G2,3U: 5′-UU UAG UGU GAC AAU GGU GUU UG-3′).

As a result, in the case of the control NT-6pi and the miR-122-transfected case, in 48-hour cell culture, it was possible to observe the migration of cells almost completely filling the scratch. However, in experimental groups transfected with miR-122-G2U and miR-122-G2,3U siRNA, it was seen that the cell migration is inhibited, and such an inhibitory phenomenon is most strongly shown in the miR-122-G2,3U siRNA-transfected experimental group (FIG. 17A). As quantitatively measured, in the cases of miR-122-G2U and miR-122-G2,3U siRNA, compared with the control, it was confirmed that the cell migration was inhibited 2- to 3-fold (FIG. 17B). The quantitative analysis was performed to observe the miR-122-mediated cell migration in terms of cell morphology, and the observation result was analyzed using the ImageJ program (NIH). Additionally, as a result of the experiment for the miR-122-G3U siRNA-induced cell migration, a cell migration inhibitory function by miR-122-G3U was not observed (FIG. 17C and 17D).

Based on this, it can be seen that the miR-122-mediated suppression of a non-canonical G:A wobble seed target inhibits the migration of liver cancer cells, indicating that G:A wobble at the 2^ndbase preferentially acts for inhibition, and when the G:A wobble at the 3^rdbase is added, the intensification of the cell migration inhibitory function is induced.

EXAMPLE 18
Confirmation of Cell Cycle Arrest by Regulation of miR-122-G2,3U-Mediated Non-Canonical G:A Wobble Seed Target

Cell migration is closely related to cell division, and widely observed, particularly, in cancer cells (Cancer research, 2004, 64(22):8420-8427). Accordingly, to confirm whether the miR-122-G2,3U-induced inhibition of liver cancer cell migration is related to the regulation of a cell cycle, flow cytometry was performed in the same manner as in Example 12. Here, as a result of measuring the cell cycle of each type of cells after NT-6pi, miR-122, miR-122-G2U, miR-122-3U and miR-122-G2,3U were delivered into the liver cancer cell line (HepG2), in the miR-122-G2,3U-transfected experimental group, it was confirmed that the ratio of the cells in the G0/G1 phase is increased by 52% to 68% on average compared with the control (NP-6pi), and the G2/M-phase cell distribution is significantly low (FIG. 18). However, in the cases of miR-122, miR-122-G2U and miR-122-G3U, compared with the control, significant differences were not observed. Here, the cell cycle analysis was performed by delivering the synthesized corresponding siRNA duplex into HepG at 50 nM and incubating for 24 hours. The analysis was conducted according to the manufacturer's protocol with a Muse Cell Cycle Kit (Catalog No. MCH100106, Millipore) and a Muse Cell Analyzer (Millipore).

Accordingly, it was confirmed that the cancer cell function regulated by miR-122-G2,3U reduces cell division (G2/M), and induces cell cycle arrest (G0/G1), and the result of the example may be interpreted as miR-122 recognizes non-canonical GA wobble seed sites through G:A wobble pairing at both of G2 and G3 based on the 5′ end, and exhibits the function of preventing the progression of a liver cancer cell cycle.

EXAMPLE 19
Observation of Inducing Cell Cycle Arrest by Regulation of miR-122-G2,3U, miR-122-G2,7U-Induced Non-Canonical G:A Wobble Seed Targets

The miR-122 function at a non-canonical G:A wobble seed target identified that miR-122-G2,3U has a function of inhibiting liver cell migration in Example 17, and particularly, it was observed that the inhibition of cell migration induced by the expression of the miR-122-G2,3U is maximized when a regulatory effect of G3 is added to the biological function of the 2^ndbase for regulating a non-canonical G:A wobble seed target (FIG. 17). Accordingly, to examine whether the biological function of suppressing a non-canonical G:A wobble seed target is changed with an additional combination of G2, G3, and G7 in miR-122, an experiment for additionally confirming a cell cycle regulatory function of miR-122-G7U, miR-122-G3,7U or miR-122-G2,7U siRNA.

Here, for the experiment, a liver cancer cell line (HepG2) was used in the same manner as described above, and a corresponding miRNA-GU duplex was prepared in the same manner as in Example 18 above and transfected into the cells at 50 nM. However, in this cell cycle observation, after 24-hour culture, the cells were treated with 100 mg/mL of nocodazole for 16 hours, HepG2 cells at the G2/M phase (division preparation phase/division phase) was synchronized, and the amount of cells in a cell cycle arrest state at G0/G1 was measured using a Muse Cell Analyzer (Millipore) according to how many cells are arrested at G2/M (FIG. 19).

As a result, it was possible to confirm that, in the case of miR-122-G2U, compared with the control NT-6pi, there was no difference in number of cells at G0/G1, which is a cell cycle arrest state, but in the case of miR-122-G2,3U siRNA and miR-122-G2,7U siRNA, when a non-canonical G:A wobble seed target site was regulated in addition to G2, the proportion of the cells in the cell cycle arrest state (G0/G1) increased (FIG. 19A). However, miR-122-G3,7U did not exhibit a significant increase (FIG. 19A). According to quantitative analysis, in the miR-122-G2,3U siRNA-transfected experimental group, compared with the control, the proportion of the cells in a cell cycle arrest state (G0/G1) increased approximately 2-fold or more, and in the case of the miR-122-G2,7U experimental group, it was observed that the proportion of the cells in a cell cycle arrest state (G0/G1) increased approximately 1.5-fold (FIG. 19B).

Based on the above, it was confirmed that cell cycle arrest can be increased through the regulation of non-canonical G:A wobble seed targets mediated by miR-122-G2,3U siRNA and miR-122-G2,7U siRNA.

EXAMPLE 20
Observation of Induction of Cell Cycle Arrest Induced by Regulation of Non-Canonical G:A Wobble Seed Targets Mediated by let-7a-G2U and let-7a-G2,4U

As a representative miRNA having a tumor suppressor function, there is the let-7 family. Since a function of regulating the development process of C. elegans has been reported (Nature, 2000, 403(6772): 901-906), and expression suppressed in various cancer cells (Cancer Res., 2004, 64(11): 3753-3756) and an anticancer function through the regulation of tumorigenesis (Cell, 2005, 120(5): 635-647; Genes Dev. 2007, 21(9):1025-1030) have been reported, based on this, the let-7 family has been studied as significant genes having potential for cancer diagnosis and development of an anticancer agent. Accordingly, the present inventors conducted an experiment to examine the biological function induced by the suppression of the non-canonical G:A wobble seed target of let-7.

The seed sequence from the 1^stto 9^thbases from the 5′ end of let-7a is 5′-UGA GGU AGU-3′, Gs capable of making G:A wobble base pairing are present in positions 2,4, 5, and 8. The inventors focused on G2 and G4, and thus conducted an experiment in the same manner as in Example 19 above, modified them into miRNA-GU to synthesize a sequence, transfected the sequence into a liver cancer cell line, that is, HepG2 cells, and then examined how a cell cycle is affected. The base sequences for the let-7 synthesized and used in the experiment are as follows: (let-7a: 5′-U GAG GUA GUA GGU UGU AUA G UU-3′; let-7a-G2U: 5′-U UAG GUA GUA GGU UGU AUA G UU-3′; let-7a-G4U: 5′-U GAU GUA GUA GGU UGU AUA G UU-3′; let-7a-G2,4U: 5′-U UAU GUA GUA GGU UGU AUA G UU-3′).

As a result, it was observed that, in the case of let-7a, compared with the control NT-6pi, there was no significant difference, but let-7a-G2U and let-7a-G2,4U increased cell cycle arrest at G0/G1, and let-7-G4U even increased the amount of cells at G2/M to make the cell cycle faster (FIG. 20A). Through quantification of the results of four-repeated experiments, it was possible to observe that, in the let-7a-G2U experimental group, compared with the control (Nt-6pi), the distribution of G1/G0-phase cells increased approximately 1.5-fold, and the distribution of G2/M-phase cells is reduced (FIG. 20B). Accordingly, it was possible to confirm that the inhibition of the expression of a non-canonical 2G:A wobble seed target of let-7a induces cell cycle arrest of the HepG2 cells. Subsequently, in the case of G4 present in the seed of let-7a, through a single GA wobble (let-7a-G4U), the induction of cell cycle arrest of HepG2 cells was reduced, but the cell cycle proceeded rapidly to the G2/M phase, resulting in a large number of cells remaining by the treatment with a nocodazole drug. In addition, when a G:A wobble was made at both G2 and G4 (let-7a-G2,4U), it was possible to observe the induction of the cell cycle arrest of HepG2 cells . Such cell cycle arrest was not observed in let-7a-transfected HepG2 cells (FIG. 20B). Therefore, the inhibition of the expression of non-canonical G:A wobble seed targets at the G2, and G2 and G4 of let-7a induced the cell cycle arrest of HepG2 cells, confirming that let-7a has a completely different function from the functions shown by the regulation of HepG2 cells performed by the inventors.

According to the example, let-7 serves to promote cancer cell cycle arrest by suppressing the gene of a non-canonical GA seed site at G2 based on the 5′ end, and it can be seen that, more preferably, when both G2 and G4 are involved in G:A wobble pairing, its effect is the largest. In addition, as the non-canonical GA seed site through G4 based on the 5′ end of let-7 promotes the progression of the cell cycle of cancer cells, it is considered that the proliferation of cancer cells will be induced.

EXAMPLE 21
Observation that Dedifferentiation Induced by Suppression of Non-Canonical G:A Wobble Seed Target Mediated is Promoted by miR-302-4GU and miR-372-4GU, using OCT4 Promoter Reporter

Differentiated cells may acquire differentiation ability again by artificial expression of several transcription factors, and cells finally acquiring pluripotent differentiation ability again, such as embryonic cells, are referred to as induced pluripotent stem cells. It was reported that this technique can produce inducible pluripotent stem cells (iPSs) having differentiation diversity such as stem cells by transfecting four factors (Oct3/4, Sox2, c-Myc, Klf4) into mouse embryonic fibroblasts (MEM) (Cell, 2006, 126 (4): 663-676). Similarly, it has been reported that iPSs can be made by delivering four factors (OCT4, SOX2, NANOG, and LIN28) into human somatic cells (Science, 2007, 318(5858): 1917-1920). This is a field with very high applicability in that, through dedifferentiation, the possibility of regeneration that allows pluripotent differentiation can be created by any cells, including human cells, and after the first finding, there has been a consistent effort to increase iPS production efficiency. As part of this, as one of the methods for efficiently inducing pluripotency with MiRNA-induced pluripotent stem cells (mirPSs) reported, a function of dedifferentiating cells with miRNA such as miR-302a or miR-372 alone or in combination with four factors (Oct3/4, Sox2, c-Myc and Klf4) was reported (RNA, 2008 14(10): 2115-2124; Nat Biotechnol. 2011, 29 (5): 443-448). Therefore, the inventors conducted an experiment to examine whether the inhibition of the expression of non-canonical G:A wobble seed targets of miR-302a and miR-372 can promote a process of dedifferentiating cells.

First, to monitor pluripotency induction, a Oct4 promoter reported to be activated in stem cells and a GFP-containing plasmid expressed depending on the promoter were purchased (Addgene #21319) (FIG. 21A). When such a Oct4 expression reporter vector (pOct4:GFP) is transfected into the differentiated cervical cancer cells (HeLa), GFP is not expressed, whereas when the corresponding cells are dedifferentiated to obtain differentiation ability, GFP is expressed in a Oct4 expression reporter. An experiment was performed on miRNAs such as miR-302a and miR-372, known to cause dedifferentiation, with the Oct4 expression reporter vector. In the seed sequences of miR-302a and miR-372, the sequence of the 2^ndto 8^thbases from the 5′ end is “AA GUG CU”, and thus the non-canonical G:A wobble seed can be made at G4 and G6 from the 5′ end. However, the inventors conducted an experiment by synthesizing miR-302-G4U or miR-372-G4U, focusing on G4.

First, the experiment was conducted by delivering an Oct4 expression reporter vector (pOct4:GFP) into HeLa cells using a Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol, synthesizing a guide strand and a passenger strand according to each of human miR-302 and miR-372 sequences in Bionia as described in the above example to prepare a duplex, and sequentially delivering the duplexes at 50 nM using an RNAiMAX reagent (Invitrogen). In addition, siRNAs specific for the non-canonical G:A wobble seed targets of miR-302 and miR-372 were prepared by substituting a G base with U in the seed in the same manner as in the above example, and then delivered into cells at 50 nM. The base sequences of the nucleic acids used herein are as follows: (miR-302: 5′-UAAGUGCUUCCAUGUUUUGGUGA-3′; miR-302-4GU: 5′-UAAUUGCUUCCAUGUUUUGGUGA-3′; miR-302 passenger strand: 5′-ACUUAAACGUGGAUGUACUUGCU-3′; miR-372: 5′-AAAGUGCUGCGACAUUUGAGCGU-3′; miR-372-G4U: 5′-AAAUUGCUGCGACAUUUGAGCGU-3′, and miR-372 passenger strand: 5′-CCUCAAAUGUGGAGCACUAUUCU-3′). HeLa cells into which a reporter vector and RNA were transfected were incubated in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin for 14 days, and during transfection, incubated in an antibiotic-free complete medium.

As a result, in the experimental group into which miR-302 or miR-372 was delivered independently, compared with the control, colony-forming cell growth was shown, but GFP expression showing the activity of the Oct4 promoter was not observed until the period of 14-day culture. However, in the experimental groups into which siRNAs in which the non-canonical G:A wobble seed targets of miR-302 and miR-372 are suppressed were transfected, GFP expression exhibiting colony-forming cell growth as well as the activity of the Oct4 promoter was observed (FIGS. 21B and 21C, red arrows), and when siRNAs suppressing miR-302 and the non-canonical G:A wobble seed target thereof and miR-372 and the non-canonical G:A wobble seed target thereof were simultaneously delivered into cells, more intensive and larger colonies of GFP-expressing cells were able to be observed (FIGS. 21B and 21C). Based on this, it was able to be seen that the suppression of the non-canonical G:A wobble seed targets of miR-302 and miR-372 may promote dedifferentiation of cells and acquisition of differentiation ability through Oct4 expression.

According to this example, it was possible to observe that miR-372-G4U and miR-302a-G4U may promote the dedifferentiation of cells more efficiently than when the induction of the dedifferentiation of cells is caused by expressing conventional miR-372 and miR-302 alone, and therefore, it was able to be seen that the miR-372-G4U and the miR-302a-G4U may be used as materials for promoting the dedifferentiation of cells.

EXAMPLE 22
Confirmation of Phenomenon in Which 2′ OMe Modification at 6^thBase Based on 5′ End Does Not Suppress Non-Canonical Nucleation Bulge Target of Corresponding RNA Interference Derivative

In the above-described example, a non-canonical nucleation bulge target had a novel biological function completely different from that of a canonical seed target, and thus miRNA-BS, which is an RNA interference-inducing nucleic acid, regulating only the inhibition of the expression of a non-canonical nucleation bulge target was invented, and its function was then confirmed. However, in the case of miRNA-BS, it was possible to confirm that, in order to complementarily arrange a non-canonical nucleation bulge site of the conventional miRNA, its seed sequence should be modified, but a novel nucleation bulge site may be generated again through the modified seed sequence. Accordingly, it is known that there is a need for technology in which an RNA interference-induced nucleic acid such as miRNA-BS recognizes only a canonical seed sequence, but does not recognize a non-canonical nucleation bulge site. Therefore, the inventors conducted an experiment to reduce the pairing strength at position 6, focusing on that the base in position 6 based on the 5′ end is important in the seed sequence for non-canonical nucleation bulge pairing, and the degree of non-canonical nucleation bulge pairing may vary depending on the pairing strength of the base in position 6. That is, a methyl group (2′ OMe) was added at the 2′ position of the ribosyl ring of the 6^thnucleotide of miR-124 to modify the miR-124, and it was observed by a luciferase reporter assay whether the modified interference-inducing nucleic acid (miR-124-6me) has the function of suppressing a non-canonical nucleation bulge target of miR-124. The 2′OMe-modified miR-124 was prepared through synthesis (Bionia), and a luciferase reporter assay was conducted in the same manner as in Example 2 to measure gene inhibitory efficiency by IC50 (FIG. 22A). The luciferase reporter vector used herein was used for a canonical seed site (Luc-seed) and a non-canonical nucleation bulge site (Luc-nucleation bulge) of miR-124.

As a result, it was observed that an inhibitory effect on the non-canonical bulge target gene of miR-124 shown in Example 2 disappeared, whereas the inhibition of gene expression through the canonical seed site still exhibits an excellent inhibitory effect, exhibiting an IC50 of 0.3 nM, even when 2′ OMe was added at the 6^thnucleotide (FIG. 22A). Additionally, when 2′OMe was added to the 6^thnucleotide of miR-1 (miR-1-6me), which is miRNA different from miR-124, in the same manner as described above, and a gene expression inhibitory effect on a target was examined using a luciferase vector including a canonical seed site (Luc-seed) and non-canonical nucleation bulge site (Luc-nucleation bulge) of miR-1 (FIG. 22B), like the result of the miR-124, the inhibitory effect on a miR-1-mediated non-canonical bulge target gene disappeared, whereas it was possible to confirm that the gene expression inhibition through a canonical seed site was excellent, an IC50 of 0.7 nM is shown. According to the example, when 2′OMe modification was applied to the 6^thnucleotide based on the 5′ end of the RNA interference-inducing nucleic acid, it was possible to confirm that the inhibitory effect on the expression of a canonical seed target gene induced by the corresponding RNA interference-inducing nucleic acid is maintained, and the inhibition of the expression of a non-canonical nucleation bulge target gene disappears.

EXAMPLE 23
Confirmation that 2′ OMe Modification at 6^thPosition Based on 5′ End Does Not Suppress Total Non-Canonical Nucleation Bulge Target mRNA in Transcriptome Through RNA-Seq Analysis

In Example 22, it was confirmed that when a 2′OM modification is applied to the 6^thnucleotide of miR-1, the inhibitory effect of the expression of a (miR-1-6me) canonical seed target gene is maintained, and the inhibition of the expression of a non-canonical nucleation bulge target gene is reduced. Accordingly, to confirm whether the miR-1-6me invented by the inventors, actually reduces the function of suppressing the non-canonical bulge target gene of miR-1 in a transcriptome of a cardiomyocyte cell line (h9c2) in which all genes actually related to the miR-1 function are expressed, the miR-1 and the miR-1-6me were transfected into h9c2 cells, and then subjected to RNA-Seq analysis. The RNA-Seq analysis was performed in the same manner as in Example 7 using the 6^thbase based on the 5′ end modified through abasic substitution (NT-6pi) in a cel-miR-67 sequence, which is miRNA of C. elegans as an experimental control. Here, an RNA-Seq library was prepared using a SENSE Total RNA-Seq Library Kit (Lexogen), and sequenced using MiniSeq (Illumina).

Afterward, the sequence data, FASTAQ file, obtained by the analysis was mapped to a mouse genomic sequence (m6) with the TopHat2 program, an expression value (FPKM) was calculated by Cufflink and Cuffdiff programs, normalized to the result in h9c2 cells into which the control NT-6pi was transfected and then expressed as a log2 ratio (fold change) for analysis. Here, to analyze the RNA-Seq result to confirm whether the amount of mRNA of a gene with a miRNA target site is inhibited by the expression of corresponding miRNA, a gene having the canonical seed site (7mer base-paired with a sequence from the 2^ndto 8^thbases from the 5′ end) of miR-1 in the 3′ UTR was selected, and the profile results were comparatively analyzed with a cumulative fraction in order of corresponding miRNA expression-dependent inhibition (FIG. 23A). As a result, it was confirmed that both miR-1 and miR-1-6me can well suppress a gene (seed) having the canonical seed site of miR-1 in the 3′ UTR, compared with the total gene (mRNA) distribution. Afterward, as the gene having the non-canonical bulge site (nuc, 7mer) of miR-1 in the 3′ UTR was also analyzed with a cumulative fraction in the same manner as described above, whether the inhibition of the expression of the non-canonical nucleation bulge target gene of miR-1 was reduced was analyzed by relatively comparing the amounts of corresponding target mRNA in miR-1-transfecetd cells and miR-1-6me-transfected cells (FIG. 23B). As a result, in miR-1, when compared to the expression in miR-1-6me, it was observed that the non-canonical nucleation bulge target gene is still suppressed significantly when relatively compared with total mRNA, confirming that the inhibition of the expression of the corresponding non-canonical nucleation bulge target gene is reduced in miR-1-6me.

According to the result of the above-described example, it was able to be seen that conventional miRNA at the transcriptome level efficiently suppressed a non-canonical bulge target gene with the conventional canonical seed target gene, but miRNA in which a 2′OMe modification was applied to the 6^thnucleotide (miRNA-6me) still suppresses a canonical seed target gene, but not a non-canonical bulge target gene. Accordingly, while maintaining the original intention to specifically suppress only the non-canonical nucleation bulge site of corresponding miRNA by applying the 2′OMe modification applied to the 6^thbase from the 5′ end to miRNA-BS, its side effects can be minimized by completely eliminating non-canonical nucleation bulge pairing that may newly appear.

EXAMPLE 24
Identification of miRNA Binding to Non-Canonical Nucleation Bulge Site Through Ago HITS CLIP Assay

To identify miRNA to which the invention for specifically recognizing and suppressing a non-canonical nucleation bulge site by miRNA can be applied, by analyzing the result by a method of analyzing a miRNA target at the transcriptome level, that is, Ago HITS CLIP assay, miRNA binding to the non-canonical nucleation bulge site was identified. First, Ago HITS-CLIP data obtained from the cerebral cortex of a mouse 13 days after birth (p13) was sequenced in the same manner as in Example 10, confirming that the top 20 expressed miRNAs binding with Argonaute bind with the non-canonical nucleation bulge site of the target mRNA (FIG. 24A). Therefore, when the corresponding miRNA (FIG. 24B) recognizes target mRNA through a base sequence in the seed region, it was able to be seen that the miRNA can bind with target mRNA by canonical base pairing through the seed part as well as a non-canonical nucleation bulge site.

Expanding mouse Ago HITS-CLIP data analysis of the example, other Ago HITS-CLIP results obtained from human brain and cardiac tissues (Boudreau R L et al, 2014, Neuron, 81(2) 294-305, Spengler R M et al, 2016, Nucleic Acids Res, 44(15) 7120-7131) were analyzed in the same manner as in the above-described example, and the frequencies of the canonical seed site and non-canonical nucleation bulge site at which Ago and the corresponding miRNA interacted were calculated (human brain miRNA, Table 1; human heart miRNA, Table 2).

TABLE 1

Seed
Bulge

miRNA family
Seed
Seed site
Bulge site
site #
site #

let-7/98/4458/4500
GAGGUAG
CUACCUC
UAACCUC
2,225
803

miR-125a-5p/125b-5p/351/670/4319
CCCUGAG
CUCAGGG
UCCAGGG
2,117
2,014

miR-124/124ab/506
AAGGCAC
GUGCCUU
UGGCCUU
2,345
2,707

miR-9/9ab
CUUUGGU
ACCAAAG
CCCAAAG
2,593
2,445

miR-29abcd
AGCACCA
UGGUGCU
GGGUGCU
3,680
1,335

miR-103a/107/107ab
GCAGCAU
AUGCUGC
UGGCUGC
2,853
3,094

miR-221/222/222ab/1928
GCUACAU
AUGUAGC
UGGUAGC
1,053
830

miR-26ab/1297/4465
UCAAGUA
UACUUGA
ACCUUGA
1,583
1,391

miR-15abc/16/16abc/195/322/424/497/1907
AGCAGCA
UGCUGCU
GCCUGCU
5,278
2,420

miR-126-3p
CGUACCG
CGGUACG
GGGUACG
97
223

miR-30abcdef/30abe-5p/384-5p
GUAAACA
UGUUUAC
GUUUUAC
2,169
1,509

miR-33ab/33-5p
UGCAUUG
CAAUGCA
AAAUGCA
1,464
3,217

miR-34ac/34bc-5p/449abc/449c-5p
GGCAGUG
CACUGCC
ACCUGCC
2,450
2,360

miR-19ab
GUGCAAA
UUUGCAC
UUUGCAC
1,735
1,735

miR-99ab/100
ACCCGUA
UACGGGU
ACCGGGU
191
370

miR-17/17-5p/20ab/20b-
AAAGUGC
GCACUUU
CAACUUU
1,951
1,946

5p/93/106ab/427/518a-3p/519d

miR-27abc/27a-3p
UCACAGU
ACUGUGA
CUUGUGA
3,054
1,546

miR-218/218a
UGUGCUU
AAGCACA
AGGCACA
2,360
1,515

miR-22/22-3p
AGCUGCC
GGCAGCU
GCCAGCU
2,899
2,213

miR-185/882/3473/4306/4644
GGAGAGA
UCUCUCC
CUUCUCC
2,466
3,044

miR-181abcd/4262
ACAUUCA
UGAAUGU
GAAAUGU
3,504
3,159

miR-338/338-3p
CCAGCAU
AUGCUGG
UGGCUGG
2,605
2,940

miR-127/127-3p
CGGAUCC
GGAUCCG
GAAUCCG
430
344

miR-101/101ab
ACAGUAC
GUACUGU
UAACUGU
1,470
1,537

miR-149
CUGGCUC
GAGCCAG
AGGCCAG
2,663
2,235

miR-23abc/23b-3p
UCACAUU
AAUGUGA
AUUGUGA
3,329
2,134

miR-324-5p
GCAUCCC
GGGAUGC
GGGAUGC
1,175
1,175

miR-24/24ab/24-3p
GGCUCAG
CUGAGCC
UGGAGCC
2,600
2,467

miR-33a-3p/365/365-3p
AAUGCCC
GGGCAUU
GGGCAUU
1,222
1,222

miR-139-5p
CUACAGU
ACUGUAG
CUUGUAG
1,228
971

miR-138/138ab
GCUGGUG
CACCAGC
ACCCAGC
3,067
2,152

miR-143/1721/4770
GAGAUGA
UCAUCUC
CAAUCUC
1,944
802

miR-25/32/92abc/363/363-3p/367
AUUGCAC
GUGCAAU
UGGCAAU
1,274
1,504

miR-574-5p
GAGUGUG
CACACUC
ACCACUC
1,145
936

miR-7/7ab
GGAAGAC
GUCUUCC
UCCUUCC
1,733
2,689

miR-145
UCCAGUU
AACUGGA
ACCUGGA
2,720
3,330

miR-135ab/135a-5p
AUGGCUU
AAGCCAU
AGGCCAU
2,197
1,932

miR-148ab-3p/152
CAGUGCA
UGCACUG
GCCACUG
2,074
2,391

miR-28-5p/708/1407/1653/3139
AGGAGCU
AGCUCCU
GCCUCCU
2,176
2,740

miR-130ac/301ab/301b/301b-

3p/454/721/4295/3666
AGUGCAA
UUGCACU
UGGCACU
1,609
1,526

miR-3132
GGGUAGA
UCUACCC
CUUACCC
947
776

miR-155
UAAUGCU
AGCAUUA
GCCAUUA
1,425
1,095

miR-485-3p
UCAUACA
UGUAUGA
GUUAUGA
1,984
1,133

miR-132/212/212-3p
AACAGUC
GACUGUU
ACCUGUU
1,352
1,670

miR-377
UCACACA
UGUGUGA
GUUGUGA
2,776
1,291

hsa-miR-9-3p
UAAAGCU
AGCUUUA
GCCUUUA
1,731
1,339

miR-374ab
UAUAAUA
UAUUAUA
AUUUAUA
2,134
3,280

miR-129-3p/129ab-3p/129-1-3p/129-2-3p
AGCCCUU
AAGGGCU
AGGGGCU
1,516
1,622

hsa-miR-126-5p
AUUAUUA
UAAUAAU
AAAUAAU
2,223
3,982

miR-425/425-5p/489
AUGACAC
GUGUCAU
UGGUCAU
1,405
1,634

miR-423-3p
GCUCGGU
ACCGAGC
CCCGAGC
607
1,013

miR-144
ACAGUAU
AUACUGU
UAACUGU
1,946
1,537

miR-21/590-5p
AGCUUAU
AUAAGCU
UAAAGCU
951
1,714

miR-31
GGCAAGA
UCUUGCC
CUUUGCC
1,392
2,396

hsa-miR-20b-3p
CUGUAGU
ACUACAG
CUUACAG
1,660
1,484

hsa-let-7d-3p
UAUACGA
UCGUAUA
CGGUAUA
182
108

miR-191
AACGGAA
UUCCGUU
UCCCGUU
439
298

miR-18ab/4735-3p
AAGGUGC
GCACCUU
CAACCUU
1,225
1,140

miR-369-3p
AUAAUAC
GUAUUAU
UAAUUAU
1,804
2,410

hsa-miR-5187-5p
GGGAUGA
UCAUCCC
CAAUCCC
1,543
735

miR-382
AAGUUGU
ACAACUU
CAAACUU
1,709
1,699

miR-485-5p/1698/1703/1962
GAGGCUG
CAGCCUC
AGGCCUC
3,699
1,600

hsa-miR-136-3p
AUCAUCG
CGAUGAU
GAAUGAU
496
1,589

miR-576-3p
AGAUGUG
CACAUCU
ACCAUCU
1,694
1,742

miR-204/204b/211
UCCCUUU
AAAGGGA
AAAGGGA
2,274
2,274

miR-769-5p
GAGACCU
AGGUCUC
GGGUCUC
1,005
973

miR-342-5p/4664-5p
GGGGUGC
GCACCCC
CAACCCC
1,387
1,516

miR-361-5p
UAUCAGA
UCUGAUA
CUUGAUA
1,343
1,105

miR-199ab-3p/3129-5p
CAGUAGU
ACUACUG
CUUACUG
1,295
1,409

miR-142-3p
GUAGUGU
ACACUAC
CAACUAC
752
1,293

miR-299-5p/3563-5p
GGUUUAC
GUAAACC
UAAAACC
716
1,482

miR-193/193b/193a-3p
ACUGGCC
GGCCAGU
GCCCAGU
1,631
1,709

hsa-miR-1277-5p
AAUAUAU
AUAUAUU
UAAUAUU
3,600
2,930

miR-140/140-5p/876-3p/1244
AGUGGUU
AACCACU
ACCCACU
1,312
1,181

hsa-miR-30a/d/e-3p
UUUCAGU
ACUGAAA
CUUGAAA
2,764
2,569

hsa-let-7i-3p
UGCGCAA
UUGCGCA
UGGCGCA
237
450

miR-409-5p/409a
GGUUACC
GGUAACC
GUUAACC
650
498

miR-379/1193-5p/3529
GGUAGAC
GUCUACC
UCCUACC
827
1,186

miR-136
CUCCAUU
AAUGGAG
AUUGGAG
2,349
1,823

miR-154/872
AGGUUAU
AUAACCU
UAAACCU
1,004
1,282

miR-4684-3p
GUUGCAA
UUGCAAC
UGGCAAC
991
1,385

miR-376abd/376b-3p
UCAUAGA
UCUAUGA
CUUAUGA
1,608
1,326

miR-323/323-3p
ACAUUAC
GUAAUGU
UAAAUGU
1,533
3,793

miR-361-3p
CCCCCAG
CUGGGGG
UGGGGGG
2,283
1,483

miR-335/335-5p
CAAGAGC
GCUCUUG
CUUCUUG
1,423
1,916

miR-652
AUGGCGC
GCGCCAU
CGGCCAU
516
680

miR-340-5p
UAUAAAG
CUUUAUA
UUUUAUA
1,832
4,317

miR-423a/423-5p/3184/3573-5p
GAGGGGC
GCCCCUC
CCCCCUC
2,226
2,135

miR-371/373/371b-5p
CUCAAAA
UUUUGAG
UUUUGAG
2,605
2,605

miR-1185/3679-5p
GAGGAUA
UAUCCUC
AUUCCUC
932
1,432

miR-3613-3p
CAAAAAA
UUUUUUG
UUUUUUG
4,481
4,481

miR-548abakhjiwy/548abcd-5p/559
AAAGUAA
UUACUUU
UAACUUU
2,709
2,289

miR-93/93a/105/106a/291a-
AAGUGCU
AGCACUU
GCCACUU
2,008
1,208

3p/294/295/302abcde/372/373/428/519a/520

be/520acd-3p/1378/1420ac

miR-339b/339-5p/3586-5p
CCCUGUC
GACAGGG
ACCAGGG
1,261
1,348

miR-876-5p/3167
GGAUUUC
GAAAUCC
AAAAUCC
1,627
1,856

miR-329/329ab/362-3p
ACACACC
GGUGUGU
GUUGUGU
1,846
1,699

hsa-miR-143-5p
GUGCAGU
ACUGCAC
CUUGCAC
1,540
1,008

miR-582-5p
UACAGUU
AACUGUA
ACCUGUA
1,995
1,457

miR-146ac/146b-5p
GAGAACU
AGUUCUC
GUUUCUC
1,488
1,708

miR-380/380-3p
AUGUAAU
AUUACAU
UUUACAU
1,709
2,532

miR-487a
AUCAUAC
GUAUGAU
UAAUGAU
1,304
1,885

miR-499-3p/499a-3p
ACAUCAC
GUGAUGU
UGGAUGU
1,920
2,367

miR-539/539-5p
GAGAAAU
AUUUCUC
UUUUCUC
2,102
3,454

miR-551a
CGACCCA
UGGGUCG
GGGGUCG
298
394

miR-142-5p
AUAAAGU
ACUUUAU
CUUUUAU
2,158
2,700

hsa-miR-17-3p
CUGCAGU
ACUGCAG
CUUGCAG
2,741
2,227

miR-199ab-5p
CCAGUGU
ACACUGG
CAACUGG
1,927
1,525

miR-542-3p
GUGACAG
CUGUCAC
UGGUCAC
1,780
1,288

miR-1277
ACGUAGA
UCUACGU
CUUACGU
471
326

hsa-miR-29c-5p
GACCGAU
AUCGGUC
UCCGGUC
178
270

miR-3145-3p
GAUAUUU
AAAUAUC
AAAUAUC
1,955
1,955

hsa-miR-106b-3p
CGCACUG
CAGUGCG
AGGUGCG
575
717

hsa-miR-22-5p
GUUCUUC
GAAGAAC
AAAGAAC
3,059
2,606

hsa-miR-144-5p
GAUAUCA
UGAUAUC
GAAUAUC
986
1,136

miR-744/1716
GCGGGGC
GCCCCGC
CCCCCGC
1,475
1,411

hsa-miR-132-5p
CCGUGGC
GCCACGG
CCCACGG
954
829

miR-488
UGAAAGG
CCUUUCA
CUUUUCA
1,880
2,812

hsa-miR-377-5p
GAGGUUG
CAACCUC
AAACCUC
1,793
1,486

miR-501-3 p/502-3p/500/502a
AUGCACC
GGUGCAU
GUUGCAU
922
1,025

miR-486-5p/3107
CCUGUAC
GUACAGG
UAACAGG
1,022
915

miR-450a/451a
UUUGCGA
UCGCAAA
CGGCAAA
253
426

hsa-miR-let7f-2/3p,hsa-miR-1185-3p
UAUACAG
CUGUAUA
UGGUAUA
1,683
1,045

hsa-miR-30c-3p
UGGGAGA
UCUCCCA
CUUCCCA
2,333
2,347

miR-499-5p
UAAGACU
AGUCUUA
GUUCUUA
1,070
1,257

miR-421
UCAACAG
CUGUUGA
UGGUUGA
1,892
1,227

miR-197
UCACCAC
GUGGUGA
UGGGUGA
2,358
1,907

miR-296-5p
GGGCCCC
GGGGCCC
GGGGCCC
1,938
1,938

miR-561
AAAGUUU
AAACUUU
AAACUUU
3,082
3,082

miR-326/330/330-5p
CUCUGGG
CCCAGAG
CCCAGAG
3,106
3,106

miR-214/761/3619-5p
CAGCAGG
CCUGCUG
CUUGCUG
4,995
2,468

miR-612/1285/3187-5p
CUGGGCA
UGCCCAG
GCCCCAG
3,439
3,210

miR-409-3p
AAUGUUG
CAACAUU
AAACAUU
1,798
3,158

miR-378/422a/378bcdefhi
CUGGACU
AGUCCAG
GUUCCAG
1,653
2,031

miR-342-3p
CUCACAC
GUGUGAG
UGGUGAG
1,793
3,079

miR-338-5p
ACAAUAU
AUAUUGU
UAAUUGU
2,412
2,011

miR-625
GGGGGAA
UUCCCCC
UCCCCCC
1,847
1,202

miR-200bc/429/548a
AAUACUG
CAGUAUU
AGGUAUU
2,342
1,840

hsa-miR-376a-5p
UAGAUUC
GAAUCUA
AAAUCUA
942
1,785

hsa-miR-379/411-3p
AUGUAAC
GUUACAU
UUUACAU
1,165
2,532

miR-3126-5p
GAGGGAC
GUCCCUC
UCCCCUC
1,254
2,186

miR-584
UAUGGUU
AACCAUA
ACCCAUA
1,052
622

hsa-miR-let-7a/b/f-3p
UAUACAA
UUGUAUA
UGGUAUA
2,494
1,045

miR-411
AGUAGAC
GUCUACU
UCCUACU
756
1,213

miR-573/3533/3616-5p/3647-5p
UGAAGUG
CACUUCA
ACCUUCA
1,664
1,979

miR-885-5p
CCAUUAC
GUAAUGG
UAAAUGG
1,041
1,692

hsa-miR-99-3p
AAGCUCG
CGAGCUU
GAAGCUU
329
1,793

miR-671-5p
GGAAGCC
GGCUUCC
GCCUUCC
2,019
2,523

miR-876-3p
GGUGGUU
AACCACC
ACCCACC
1,386
1,692

miR-654-3p
AUGUCUG
CAGACAU
AGGACAU
1,927
2,025

hsa-miR-340-3p
CCGUCUC
GAGACGG
AGGACGG
924
772

miR-450b-3p/769-3p
UGGGAUC
GAUCCCA
AUUCCCA
1,320
1,582

miR-3614-5p
CACUUGG
CCAAGUG
CAAAGUG
1,954
2,537

hsa-miR-124-5p
GUGUUCA
UGAACAC
GAAACAC
1,421
1,620

miR-491-5p
GUGGGGA
UCCCCAC
CCCCCAC
2,168
2,505

miR-589
GAGAACC
GGUUCUC
GUUUCUC
1,006
1,708

miR-96/507/1271
UUGGCAC
GUGCCAA
UGGCCAA
1,652
2,566

miR-545
CAGCAAA
UUUGCUG
UUUGCUG
3,335
3,335

miR-548a-3p/548ef/2285a
AAAACUG
CAGUUUU
AGGUUUU
3,014
2,238

miR-30b-3p/3689c/3689a-3p
UGGGAGG
CCUCCCA
CUUCCCA
3,070
2,347

miR-323-5p/1421qns
GGUGGUC
GACCACC
ACCCACC
1,458
1,692

hsa-miR-32-3p
AAUUUAG
CUAAAUU
UAAAAUU
1,603
4,141

miR-3942-5p/4703-5p
AGCAAUA
UAUUGCU
AUUUGCU
1,763
2,331

miR-34b/449c/1360/2682
AGGCAGU
ACUGCCU
CUUGCCU
2,010
1,710

hsa-miR-23a/b-5p
GGGUUCC
GGAACCC
GAAACCC
1,112
1,544

hsa-miR-545-5p
CAGUAAA
UUUACUG
UUUACUG
2,206
2,206

miR-362-5p/500b
AUCCUUG
CAAGGAU
AAAGGAU
1,699
2,110

miR-677/4420
UCACUGA
UCAGUGA
CAAGUGA
2,556
1,859

miR-577
AGAUAAA
UUUAUCU
UUUAUCU
2,323
2,323

miR-3613-5p
GUUGUAC
GUACAAC
UAACAAC
879
912

miR-369-5p
GAUCGAC
GUCGAUC
UCCGAUC
128
172

miR-590-3p
AAUUUUA
UAAAAUU
AAAAAUU
4,141
4,931

miR-127-5p
UGAAGCU
AGCUUCA
GCCUUCA
1,985
2,321

miR-150/5127
CUCCCAA
UUGGGAG
UGGGGAG
1,828
2,905

miR-544/544ab/544-3p
UUCUGCA
UGCAGAA
GCCAGAA
4,175
2,248

hsa-miR-29a-5p
CUGAUUU
AAAUCAG
AAAUCAG
2,516
2,516

miR-873
CAGGAAC
GUUCCUG
UUUCCUG
2,349
3,190

miR-3614-3p
AGCCUUC
GAAGGCU
AAAGGCU
1,923
1,801

miR-186
AAAGAAU
AUUCUUU
UUUCUUU
2,994
6,937

miR-483-3p
CACUCCU
AGGAGUG
GGGAGUG
1,815
1,517

hsa-miR-374a-3p
UUAUCAG
CUGAUAA
UGGAUAA
1,388
1,599

miR-196abc
AGGUAGU
ACUACCU
CUUACCU
1,458
1,248

hsa-miR-145-3p
GAUUCCU
AGGAAUC
GGGAAUC
1,410
891

hsa-miR-29b-2-5p
UGGUUUC
GAAACCA
AAAACCA
2,317
3,094

hsa-miR-221-5p
CCUGGCA
UGCCAGG
GCCCAGG
2,418
3,446

miR-323b-3p
CCAAUAC
GUAUUGG
UAAUUGG
1,014
1,173

hsa-miR-548as-3p
AAAACCC
GGGUUUU
GGGUUUU
1,687
1,687

miR-616
GUCAUUG
CAAUGAC
AAAUGAC
1,505
1,986

miR-330-3p
CAAAGCA
UGCUUUG
GCCUUUG
2,946
2,316

hsa-miR-7-3p
AACAAAU
AUUUGUU
UUUUGUU
3,562
6,317

miR-4525
GGGGGAU
AUCCCCC
UCCCCCC
926
1,202

miR-3064-5p/3085-3p
CUGGCUG
CAGCCAG
AGGCCAG
3,803
2,235

miR-187
CGUGUCU
AGACACG
GAACACG
540
534

hsa-miR-26a-3p
CUAUUCU
AGAAUAG
GAAAUAG
1,190
1,730

miR-452/4676-3p
ACUGUUU
AAACAGU
AAACAGU
2,317
2,317

miR-129-5p/129ab-5p
UUUUUGC
GCAAAAA
CAAAAAA
2,534
3,710

miR-223
GUCAGUU
AACUGAC
ACCUGAC
1,097
1,271

miR-4755-3p
GCCAGGC
GCCUGGC
CCCUGGC
3,108
2,922

miR-1247
CCCGUCC
GGACGGG
GAACGGG
718
554

miR-3129-3p
AACUAAU
AUUAGUU
UUUAGUU
1,133
2,127

hsa-miR-335-3p
UUUUCAU
AUGAAAA
UGGAAAA
4,804
5,029

miR-542-5p
CGGGGAU
AUCCCCG
UCCCCCG
550
763

hsa-miR-181a-3p
CCAUCGA
UCGAUGG
CGGAUGG
432
513

hsa-miR-186-3p
CCCAAAG
CUUUGGG
UUUUGGG
2,255
1,981

hsa-miR-96-3p
AUCAUGU
ACAUGAU
CAAUGAU
1,619
1,601

hsa-miR-27b-5p
GAGCUUA
UAAGCUC
AAAGCUC
658
1,376

miR-491-3p
UUAUGCA
UGCAUAA
GCCAUAA
1,096
770

miR-4687-3p
GGCUGUU
AACAGCC
ACCAGCC
1,686
2,279

hsa-miR-101-5p
AGUUAUC
GAUAACU
AUUAACU
983
1,337

hsa-let-7e-3p
UAUACGG
CCGUAUA
CGGUAUA
158
108

miR-4772-5p
GAUCAGG
CCUGAUC
CUUGAUC
1,197
974

miR-337-3p
UCCUAUA
UAUAGGA
AUUAGGA
950
1,012

hsa-miR-223-5p
GUGUAUU
AAUACAC
AUUACAC
1,015
952

hsa-miR-146a-3p
CUCUGAA
UUCAGAG
UCCAGAG
2,590
2,417

hsa-miR-16/195-3p
CAAUAUU
AAUAUUG
AUUAUUG
2,403
2,178

miR-941
ACCCGGC
GCCGGGU
CCCGGGU
579
655

miR-3677-3p
UCGUGGG
CCCACGA
CCCACGA
622
622

hsa-miR-766-5p
GGAGGAA
UUCCUCC
UCCCUCC
2,713
2,554

miR-299/299-3p/3563-3p
AUGUGGG
CCCACAU
CCCACAU
1,218
1,218

miR-3140-3p
GCUUUUG
CAAAAGC
AAAAAGC
1,704
2,794

miR-532-5p/511
AUGCCUU
AAGGCAU
AGGGCAU
1,676
1,375

hsa-miR-24-5p
GCCUACU
AGUAGGC
GUUAGGC
481
333

hsa-miR-4524a-3p
GAGACAG
CUGUCUC
UGGUCUC
2,248
1,397

miR-4778-5p
AUUCUGU
ACAGAAU
CAAGAAU
2,383
2,138

miR-642b
GACACAU
AUGUGUC
UGGUGUC
1,522
1,749

miR-483-5p
AGACGGG
CCCGUCU
CCCGUCU
571
571

miR-767-5p
GCACCAU
AUGGUGC
UGGGUGC
2,048
1,223

hsa-miR-31-3p
GCUAUGC
GCAUAGC
CAAUAGC
460
686

miR-885-3p
GGCAGCG
CGCUGCC
GCCUGCC
1,707
2,782

miR-4706/4749-5p
GCGGGGA
UCCCCGC
CCCCCGC
1,057
1,411

miR-574-3p
ACGCUCA
UGAGCGU
GAAGCGU
444
423

miR-3173-3p
AAGGAGG
CCUCCUU
CUUCCUU
2,325
2,916

miR-2127/4728-5p
GGGAGGG
CCCUCCC
CCCUCCC
3,357
3,357

hsa-miR-103a-2-5p
GCUUCUU
AAGAAGC
AGGAAGC
3,805
2,616

miR-3591-3p
AACACCA
UGGUGUU
GGGUGUU
2,405
964

miR-766
CUCCAGC
GCUGGAG
CUUGGAG
4,936
2,384

hsa-miR-155-3p
UCCUACA
UGUAGGA
GUUAGGA
1,168
750

hsa-miR-625-3p
ACUAUAG
CUAUAGU
UAAUAGU
663
1,110

hsa-miR-15b-3p
GAAUCAU
AUGAUUC
UGGAUUC
1,454
1,683

miR-522/518e/1422p
AAAUGGU
ACCAUUU
CCCAUUU
1,930
1,550

miR-548d-3p/548acbz
AAAAACC
GGUUUUU
GUUUUUU
2,468
3,983

hsa-miR-452-3p
UCAUCUG
CAGAUGA
AGGAUGA
3,420
3,113

miR-192/215
UGACCUA
UAGGUCA
AGGGUCA
692
989

miR-1551/4524
UAGCAGC
GCUGCUA
CUUGCUA
1,713
911

hsa-miR-425-3p
UCGGGAA
UUCCCGA
UCCCCGA
474
758

miR-3126-3p
AUCUGGC
GCCAGAU
CCCAGAU
1,430
1,956

miR-519a/519bc-3p/291b-3p/1347
AAGUGCA
UGCACUU
GCCACUU
1,869
1,208

miR-450b-5p
UUUGCAA
UUGCAAA
UGGCAAA
2,408
2,581

hsa-miR-125b-2-3p
CACAAGU
ACUUGUG
CUUUGUG
1,485
3,042

miR-2441/4436a
CAGGACA
UGUCCUG
GUUCCUG
2,535
2,349

hsa-miR-5583-3p
AAUAUGG
CCAUAUU
CAAUAUU
1,237
1,789

miR-139-3p
GAGACGC
GCGUCUC
CGGUCUC
530
387

miR-324-3p/1913
CUGCCCC
GGGGCAG
GGGGCAG
2,454
2,454

hsa-miR-141-5p
AUCUUCC
GGAAGAU
GAAAGAU
2,912
2,391

hsa-miR-365a/b-5p
GGGACUU
AAGUCCC
AGGUCCC
1,025
1,007

miR-654-5p/541
GGUGGGC
GCCCACC
CCCCACC
2,064
3,441

hsa-miR-29b-1-5p
CUGGUUU
AAACCAG
AAACCAG
2,401
2,401

miR-563/380-5p
GGUUGAC
GUCAACC
UCCAACC
796
1,134

hsa-miR-16-1-3p
CAGUAUU
AAUACUG
AUUACUG
1,869
1,645

miR-1304
UUGAGGC
GCCUCAA
CCCUCAA
1,448
1,515

miR-216c/1461/4684-5p
UCUCUAC
GUAGAGA
UAAGAGA
1,268
1,571

hsa-miR-2681-5p
UUUUACC
GGUAAAA
GUUAAAA
1,871
2,348

hsa-miR-1307-5p
CGACCGG
CCGGUCG
CGGGUCG
142
184

miR-194
GUAACAG
CUGUUAC
UGGUUAC
1,384
838

miR-296-3p
AGGGUUG
CAACCCU
AAACCCU
1,287
1,644

miR-2681
AUCAUGG
CCAUGAU
CAAUGAU
1,560
1,601

hsa-miR-205-3p
AUUUCAG
CUGAAAU
UGGAAAU
2,657
3,667

miR-888
ACUCAAA
UUUGAGU
UUUGAGU
1,927
1,927

miR-4802-3p
ACAUGGA
UCCAUGU
CCCAUGU
1,689
1,306

hsa-miR-708-3p
AACUAGA
UCUAGUU
CUUAGUU
1,086
956

hsa-let-7a/g-3p
UGUACAG
CUGUACA
UGGUACA
1,922
1,356

miR-762/4492/4498
GGGCUGG
CCAGCCC
CAAGCCC
3,902
1,829

hsa-miR-744-3p
UGUUGCC
GGCAACA
GCCAACA
1,638
1,906

hsa-miR-1914-3p,hsa-miR-5194
GAGGGGU
ACCCCUC
CCCCCUC
1,419
2,135

hsa-miR-148b-5p
AGUUCUG
CAGAACU
AGGAACU
2,295
2,133

miR-615-5p
GGGGUCC
GGACCCC
GAACCCC
1,701
1,133

miR-514/514b-3p
UUGACAC
GUGUCAA
UGGUCAA
1,075
1,388

miR-28-3p
ACUAGAU
AUCUAGU
UCCUAGU
710
818

miR-4423-5p
GUUGCCU
AGGCAAC
GGGCAAC
1,112
1,104

miR-550a
GUGCCUG
CAGGCAC
AGGGCAC
1,628
1,190

hsa-miR-125b-1-3p
CGGGUUA
UAACCCG
AAACCCG
170
314

hsa-miR-506-5p
AUUCAGG
CCUGAAU
CUUGAAU
1,601
1,672

hsa-miR-493-5p
UGUACAU
AUGUACA
UGGUACA
2,262
1,356

hsa-miR-1306-5p
CACCUCC
GGAGGUG
GAAGGUG
3,462
3,178

hsa-miR-561-5p
UCAAGGA
UCCUUGA
CCCUUGA
1,593
1,071

miR-3189-3p
CCUUGGG
CCCAAGG
CCCAAGG
2,190
2,190

miR-675-5p/4466
GGUGCGG
CCGCACC
CGGCACC
808
902

hsa-miR-34a-3p
AAUCAGC
GCUGAUU
CUUGAUU
1,287
1,531

hsa-miR-454-5p
CCCUAUC
GAUAGGG
AUUAGGG
408
522

miR-4796-3p
AAAGUGG
CCACUUU
CAACUUU
1,584
1,946

miR-509-5p/509-3-5p/4418
ACUGCAG
CUGCAGU
UGGCAGU
2,886
2,144

miR-183
AUGGCAC
GUGCCAU
UGGCCAU
1,564
2,389

miR-182
UUGGCAA
UUGCCAA
UGGCCAA
2,373
2,566

hsa-miR-19a/b-5p
GUUUUGC
GCAAAAC
CAAAAAC
1,498
2,170

hsa-miR-212-5p
CCUUGGC
GCCAAGG
CCCAAGG
2,504
2,190

miR-3200-5p
AUCUGAG
CUCAGAU
UCCAGAU
1,917
2,199

miR-3065-3p
CAGCACC
GGUGCUG
GUUGCUG
3,895
2,230

miR-4755-5p
UUCCCUU
AAGGGAA
AGGGGAA
2,544
2,094

hsa-miR-93-3p
CUGCUGA
UCAGCAG
CAAGCAG
2,988
2,872

miR-3130-5p/4482
ACCCAGU
ACUGGGU
CUUGGGU
1,133
1,064

hsa-miR-488-5p
CCAGAUA
UAUCUGG
AUUCUGG
1,210
1,862

hsa-miR-5000-5p
AGUUCAG
CUGAACU
UGGAACU
1,839
1,923

hsa-miR-378a-5p
UCCUGAC
GUCAGGA
UCCAGGA
1,310
3,115

miR-300-5p/4709-3p
UGAAGAG
CUCUUCA
UCCUUCA
2,629
2,301

miR-575/4676-5p
AGCCAGU
ACUGGCU
CUUGGCU
1,768
1,842

hsa-miR-33a-3p
AAUGUUU
AAACAUU
AAACAUU
3,158
3,158

miR-1307
CUCGGCG
CGCCGAG
GCCCGAG
730
1,265

miR-3942-3p
UUCAGAU
AUCUGAA
UCCUGAA
2,201
2,681

miR-4677-5p
UGUUCUU
AAGAACA
AGGAACA
3,452
2,370

miR-339-3p
GAGCGCC
GGCGCUC
GCCGCUC
659
845

miR-548b-3p
AAGAACC
GGUUCUU
GUUUCUU
1,221
2,708

hsa-miR-642b-5p
GUUCCCU
AGGGAAC
GGGGAAC
1,315
1,079

miR-188-5p
AUCCCUU
AAGGGAU
AGGGGAU
1,196
1,000

hsa-miR-652-5p
AACCCUA
UAGGGUU
AGGGGUU
668
863

miR-2114
AGUCCCU
AGGGACU
GGGGACU
1,293
1,145

miR-3688-5p
GUGGCAA
UUGCCAC
UGGCCAC
1,436
2,200

hsa-miR-15a-3p
AGGCCAU
AUGGCCU
UGGGCCU
1,693
2,093

hsa-miR-181c-3p
ACCAUCG
CGAUGGU
GAAUGGU
348
1,407

miR-515-3p/519e
AGUGCCU
AGGCACU
GGGCACU
1,319
1,076

miR-2447/4646-5p
CUGGGAA
UUCCCAG
UCCCCAG
2,541
3,052

miR-122/122a/1352
GGAGUGU
ACACUCC
CAACUCC
904
1,498

miR-532-3p
CUCCCAC
GUGGGAG
UGGGGAG
2,177
2,905

miR-556-3p
UAUUACC
GGUAAUA
GUUAAUA
1,059
1,338

hsa-miR-218-2-3p
AUGGUUC
GAACCAU
AAACCAU
1,126
2,029

miR-643
CUUGUAU
AUACAAG
UAACAAG
1,234
1,168

hsa-miR-92a-2-5p
GGUGGGG
CCCCACC
CCCCACC
3,441
3,441

miR-140-3p
ACCACAG
CUGUGGU
UGGUGGU
2,614
2,715

miR-1245
AGUGAUC
GAUCACU
AUUCACU
952
1,528

hsa-miR-2115-3p
AUCAGAA
UUCUGAU
UCCUGAU
2,231
1,919

miR-93b/512-3p/1186
AGUGCUG
CAGCACU
AGGCACU
2,145
1,319

miR-518bcf/518a-3p/518d-3p
AAAGCGC
GCGCUUU
CGGCUUU
417
493

miR-3200-3p
ACCUUGC
GCAAGGU
CAAAGGU
1,413
1,958

miR-337-5p
AACGGCU
AGCCGUU
GCCCGUU
357
313

hsa-miR-100-3p
AAGCUUG
CAAGCUU
AAAGCUU
1,256
2,216

miR-545/3065/3065-5p
CAACAAA
UUUGUUG
UUUGUUG
3,158
3,158

miR-17-2-3p/4793-3p
CUGCACU
AGUGCAG
GUUGCAG
2,233
1,734

miR-1903/4778-3p
CUUCUUC
GAAGAAG
AAAGAAG
6,522
5,458

hsa-miR-302a-5p
CUUAAAC
GUUUAAG
UUUUAAG
1,279
3,298

hsa-miR-183-3p
UGAAUUA
UAAUUCA
AAAUUCA
1,730
3,049

miR-3144-5p
GGGGACC
GGUCCCC
GUUCCCC
1,177
1,090

hsa-miR-105-3p
CGGAUGU
ACAUCCG
CAAUCCG
633
207

miR-582-3p
AACUGGU
ACCAGUU
CCCAGUU
1,651
1,618

miR-4662a-3p
AAGAUAG
CUAUCUU
UAAUCUU
955
1,512

miR-3140-5p
CCUGAAU
AUUCAGG
UUUCAGG
1,829
2,876

hsa-miR-106a-3p
UGCAAUG
CAUUGCA
AUUUGCA
1,780
2,344

hsa-miR-135a-3p
AUAGGGA
UCCCUAU
CCCCUAU
766
569

miR-345/345-5p
CUGACUC
GAGUCAG
AGGUCAG
1,506
1,626

hsa-miR-196a-3p
GGCAACA
UGUUGCC
GUUUGCC
1,687
1,546

miR-125a-3p/1554
CAGGUGA
UCACCUG
CAACCUG
2,062
2,184

miR-3145-5p
ACUCCAA
UUGGAGU
UGGGAGU
1,578
1,365

miR-676
UGUCCUA
UAGGACA
AGGGACA
731
1,542

miR-3173-5p
GCCCUGC
GCAGGGC
CAAGGGC
1,847
1,530

hsa-miR-148a-5p
AAGUUCU
AGAACUU
GAAACUU
2,623
2,190

hsa-miR-5586-3p
AGAGUGA
UCACUCU
CAACUCU
1,458
1,318

miR-615-3p
CCGAGCC
GGCUCGG
GCCUCGG
819
1,056

miR-3688-3p
AUGGAAA
UUUCCAU
UUUCCAU
2,409
2,409

miR-4662a-5p
UAGCCAA
UUGGCUA
UGGGCUA
1,095
890

miR-4659ab-5p
UGCCAUG
CAUGGCA
AUUGGCA
1,724
1,588

hsa-miR-5586-5p
AUCCAGC
GCUGGAU
CUUGGAU
1,955
1,561

hsa-miR-514a-5p
ACUCUGG
CCAGAGU
CAAGAGU
1,626
1,358

miR-10abc/10a-5p
ACCCUGU
ACAGGGU
CAAGGGU
1,094
850

miR-4709-5p
CAACAGU
ACUGUUG
CUUGUUG
1,765
1,425

hsa-miR-888-3p
ACUGACA
UGUCAGU
GUUCAGU
1,814
1,503

miR-1785/2443/3616-3p
GAGGGCA
UGCCCUC
GCCCCUC
2,268
2,226

miR-3127-5p
UCAGGGC
GCCCUGA
CCCCUGA
2,111
1,778

miR-1188-3p/2467-5p
GAGGCUC
GAGCCUC
AGGCCUC
1,628
1,600

hsa-miR-382-3p
AUCAUUC
GAAUGAU
AAAUGAU
1,589
3,166

miR-660
ACCCAUU
AAUGGGU
AUUGGGU
1,046
859

hsa-miR-301a-5p
CUCUGAC
GUCAGAG
UCCAGAG
1,581
2,417

miR-508-3p
GAUUGUA
UACAAUC
ACCAAUC
753
704

hsa-miR-185-3p
GGGGCUG
CAGCCCC
AGGCCCC
3,623
1,750

hsa-miR-200c-5p,hsa-miR-550a-3p
GUCUUAC
GUAAGAC
UAAAGAC
880
1,448

miR-3605-5p
GAGGAUG
CAUCCUC
AUUCCUC
2,176
1,432

miR-513c/514b-5p
UCUCAAG
CUUGAGA
UUUGAGA
1,417
2,980

miR-490-3p
AACCUGG
CCAGGUU
CAAGGUU
1,423
1,176

miR-520a-5p/525-5p/2464-3p
UCCAGAG
CUCUGGA
UCCUGGA
2,562
3,682

miR-3144-3p
UAUACCU
AGGUAUA
GGGUAUA
1,131
416

hsa-miR-5187-3p
CUGAAUC
GAUUCAG
AUUUCAG
1,788
2,978

miR-3664-3p
CUCAGGA
UCCUGAG
CCCUGAG
2,354
2,643

miR-3189-5p
GCCCCAU
AUGGGGC
UGGGGGC
1,190
1,973

miR-4670-3p
GAAGUUA
UAACUUC
AAACUUC
1,185
1,856

miR-105/105ab
CAAAUGC
GCAUUUG
CAAUUUG
2,029
1,390

miR-1323/5480
CAAAACU
AGUUUUG
GUUUUUG
2,600
2,980

hsa-miR-135b-3p
UGUAGGG
CCCUACA
CCCUACA
1,301
1,301

hsa-miR-5010-3p
UUUGUGU
ACACAAA
CAACAAA
2,055
2,215

miR-493/493b
GAAGGUC
GACCUUC
ACCCUUC
1,486
1,226

miR-3605-3p
CUCCGUG
CACGGAG
ACCGGAG
1,003
523

miR-188-3p
UCCCACA
UGUGGGA
GUUGGGA
2,505
1,204

hsa-miR-449c-3p
UGCUAGU
ACUAGCA
CUUAGCA
666
994

miR-486-3p
GGGGCAG
CUGCCCC
UGGCCCC
3,671
1,979

miR-501-5p
AUCCUUU
AAAGGAU
AAAGGAU
2,110
2,110

miR-4761-5p
CAAGGUG
CACCUUG
ACCCUUG
1,395
1,067

miR-3130-3p
CUGCACC
GGUGCAG
GUUGCAG
2,529
1,734

hsa-miR-202-5p
UCCUAUG
CAUAGGA
AUUAGGA
782
1,012

miR-629
GGGUUUA
UAAACCC
AAAACCC
648
1,532

miR-224
AAGUCAC
GUGACUU
UGGACUU
1,775
2,076

miR-202-3p
GAGGUAU
AUACCUC
UAACCUC
967
803

miR-4772-3p
CUGCAAC
GUUGCAG
UUUGCAG
1,734
3,221

miR-4796-5p
GUCUAUA
UAUAGAC
AUUAGAC
644
649

hsa-miR-551b-5p
AAAUCAA
UUGAUUU
UGGAUUU
3,263
3,018

miR-556-5p
AUGAGCU
AGCUCAU
GCCUCAU
1,783
1,498

hsa-miR-122-3p
ACGCCAU
AUGGCGU
UGGGCGU
472
570

hsa-miR-2116-3p
CUCCCAU
AUGGGAG
UGGGGAG
1,677
2,905

miR-4677-3p
CUGUGAG
CUCACAG
UCCACAG
2,060
2,243

miR-877
UAGAGGA
UCCUCUA
CCCUCUA
1,217
886

hsa-miR-200a/b-5p
AUCUUAC
GUAAGAU
UAAAGAU
1,426
2,480

miR-576-5p
UUCUAAU
AUUAGAA
UUUAGAA
1,902
3,054

miR-490-5p
CAUGGAU
AUCCAUG
UCCCAUG
1,332
1,335

hsa-miR-589-3p
CAGAACA
UGUUCUG
GUUUCUG
2,362
2,464

hsa-miR-548a0/ax-5p
GAAGUAA
UUACUUC
UAACUUC
1,328
1,185

miR-4786-3p
GAAGCCA
UGGCUUC
GGGCUUC
2,438
1,736

hsa-miR-374b-3p
UUAGCAG
CUGCUAA
UGGCUAA
1,295
1,073

hsa-miR-26b-3p
CUGUUCU
AGAACAG
GAAACAG
2,650
2,883

miR-3158-3p
AGGGCUU
AAGCCCU
AGGCCCU
1,924
2,078

miR-4423-3p
UAGGCAC
GUGCCUA
UGGCCUA
947
1,191

miR-518d-5p/519bc-5p520c-5p/523b/526a
UCUAGAG
CUCUAGA
UCCUAGA
873
1,171

miR-548aeajamx
AAAAACU
AGUUUUU
GUUUUUU
3,406
3,983

miR-4707-3p
GCCCGCC
GGCGGGC
GCCGGGC
833
1,405

hsa-miR-138-2-3p
CUAUUUC
GAAAUAG
AAAAUAG
1,730
2,619

hsa-miR-10a-3p
AAAUUCG
CGAAUUU
GAAAUUU
312
2,899

miR-526b
UCUUGAG
CUCAAGA
UCCAAGA
1,981
2,157

miR-3622ab-3p
CACCUGA
UCAGGUG
CAAGGUG
2,068
2,555

hsa-miR-676-5p
CUUCAAC
GUUGAAG
UUUGAAG
1,836
3,633

hsa-miR-873-3p
GAGACUG
CAGUCUC
AGGUCUC
1,486
1,005

hsa-miR-424-3p
AAAACGU
ACGUUUU
CGGUUUU
718
363

hsa-miR-20a-3p
CUGCAUU
AAUGCAG
AUUGCAG
2,577
2,148

hsa-miR-345-3p
CCCUGAA
UUCAGGG
UCCAGGG
1,933
2,014

hsa-miR-660-3p
CCUCCUG
CAGGAGG
AGGGAGG
3,542
2,232

hsa-miR-5004-3p
UUGGAUU
AAUCCAA
AUUCCAA
1,667
1,752

miR-1276
AAAGAGC
GCUCUUU
CUUCUUU
1,806
3,023

hsa-miR-541-5p
AAGGAUU
AAUCCUU
AUUCCUU
1,468
2,068

miR-193a-5p
GGGUCUU
AAGACCC
AGGACCC
1,519
1,556

hsa-miR-5682
UAGCACC
GGUGCUA
GUUGCUA
1,266
926

miR-2115
GCUUCCA
UGGAAGC
GGGAAGC
2,401
1,873

miR-3663-5p
CUGGUCU
AGACCAG
GAACCAG
2,067
1,959

hsa-miR-222-5p
UCAGUAG
CUACUGA
UAACUGA
1,194
1,284

hsa-miR-27a-5p
GGGCUUA
UAAGCCC
AAAGCCC
648
1,483

miR-1618/3940-3p
AGCCCGG
CCGGGCU
CGGGGCU
932
999

miR-4661-3p
AGGAUCC
GGAUCCU
GAAUCCU
1,282
1,248

miR-3177-5p
GUGUACA
UGUACAC
GUUACAC
1,144
685

hsa-miR-18b-3p
GCCCUAA
UUAGGGC
UAAGGGC
510
612

miR-146b-3p
GCCCUGU
ACAGGGC
CAAGGGC
1,163
1,530

hsa-miR-25-5p
GGCGGAG
CUCCGCC
UCCCGCC
1,072
998

miR-4670-5p
AGCGACC
GGUCGCU
GUUCGCU
287
264

hsa-miR-192-3p
UGCCAAU
AUUGGCA
UUUGGCA
1,588
2,139

hsa-miR-548at-5p
AAAGUUA
UAACUUU
AAACUUU
2,289
3,082

miR-659
UUGGUUC
GAACCAA
AAACCAA
1,459
2,761

miR-141/200a
AACACUG
CAGUGUU
AGGUGUU
2,525
1,651

hsa-miR-187-5p
GCUACAA
UUGUAGC
UGGUAGC
1,062
830

miR-1745/3194-3p
GCUCUGC
GCAGAGC
CAAGAGC
2,345
1,790

hsa-miR-193b-5p
GGGGUUU
AAACCCC
AAACCCC
1,133
1,133

hsa-miR-182-3p
GGUUCUA
UAGAACC
AGGAACC
632
1,391

miR-298/2347/2467-3p
GCAGAGG
CCUCUGC
CUUCUGC
3,079
2,700

hsa-miR-130b-5p
CUCUUUC
GAAAGAG
AAAAGAG
3,037
3,023

miR-2319b/3664-5p
ACUCUGU
ACAGAGU
CAAGAGU
1,740
1,358

hsa-miR-18a-3p
CUGCCCU
AGGGCAG
GGGGCAG
2,147
2,454

miR-3064-3p
UGCCACA
UGUGGCA
GUUGGCA
2,551
1,379

miR-4746-3p
GCGGUGC
GCACCGC
CAACCGC
672
553

miR-1893/2277-5p
GCGCGGG
CCCGCGC
CCCGCGC
903
903

miR-3619-3p
GGACCAU
AUGGUCC
UGGGUCC
819
1,045

hsa-miR-138-1-3p
CUACUUC
GAAGUAG
AAAGUAG
1,322
1,623

hsa-miR-92a-1-5p
GGUUGGG
CCCAACC
CCCAACC
1,397
1,397

miR-1954/3158-5p
CUGCAGA
UCUGCAG
CUUGCAG
3,361
2,227

miR-4728-3p
AUGCUGA
UCAGCAU
CAAGCAU
2,248
1,225

miR-3127-3p
CCCCUUC
GAAGGGG
AAAGGGG
1,921
1,436

miR-671-3p
CCGGUUC
GAACCGG
AAACCGG
486
315

miR-2313/3944-5p
GUGCAGC
GCUGCAC
CUUGCAC
2,106
1,008

miR-4661-5p
ACUAGCU
AGCUAGU
GCCUAGU
697
522

miR-509ab/509-3p
GAUUGGU
ACCAAUC
CCCAAUC
704
664

hsa-miR-194-3p
CAGUGGG
CCCACUG
CCCACUG
2,033
2,033

hsa-miR-211-3p
CAGGGAC
GUCCCUG
UCCCCUG
2,263
2,087

miR-1843-5p/4802-5p
AUGGAGG
CCUCCAU
CUUCCAU
1,752
1,757

miR-34b
AAUCACU
AGUGAUU
GUUGAUU
1,976
1,351

hsa-miR-2114-3p
GAGCCUC
GAGGCUC
AGGGCUC
1,445
1,230

hsa-miR-877-3p
CCUCUUC
GAAGAGG
AAAGAGG
4,459
2,689

hsa-miR-218-1-3p
UGGUUCC
GGAACCA
GAAACCA
1,524
2,317

miR-3157-5p
UCAGCCA
UGGCUGA
GGGCUGA
2,470
1,581

miR-502-5p
UCCUUGC
GCAAGGA
CAAAGGA
1,949
2,478

miR-500a
AAUCCUU
AAGGAUU
AGGGAUU
1,913
1,178

miR-3194-5p
GCCAGCC
GGCUGGC
GCCUGGC
2,155
3,108

hsa-miR-1304-3p
CUCACUG
CAGUGAG
AGGUGAG
2,778
4,507

hsa-miR-5004-5p
GAGGACA
UGUCCUC
GUUCCUC
1,991
1,405

miR-453/323b-5p
GGUUGUC
GACAACC
ACCAACC
1,143
1,297

miR-548g
AAACUGU
ACAGUUU
CAAGUUU
2,442
2,106

hsa-miR-92b-5p
GGGACGG
CCGUCCC
CGGUCCC
665
541

miR-523
AACGCGC
GCGCGUU
CGGCGUU
120
164

hsa-miR-584-3p
CAGUUCC
GGAACUG
GAAACUG
2,178
2,803

miR-205/205ab
CCUUCAU
AUGAAGG
UGGAAGG
2,802
2,738

miR-4793-5p
CAUCCUG
CAGGAUG
AGGGAUG
2,588
1,505

hsa-miR-363-5p
GGGUGGA
UCCACCC
CCCACCC
1,794
3,038

hsa-miR-214-5p
GCCUGUC
GACAGGC
ACCAGGC
1,129
1,458

miR-4786-5p
GAGACCA
UGGUCUC
GGGUCUC
1,397
973

miR-3180-5p
UUCCAGA
UCUGGAA
CUUGGAA
2,823
2,446

miR-767-3p
CUGCUCA
UGAGCAG
GAAGCAG
2,665
3,889

hsa-miR-659-5p
GGACCUU
AAGGUCC
AGGGUCC
853
971

miR-1404/2110
UGGGGAA
UUCCCCA
UCCCCCA
2,463
2,334

hsa-miR-548at-3p
AAAACCG
CGGUUUU
GGGUUUU
363
1,687

miR-3944-3p
UCGGGCU
AGCCCGA
GCCCCGA
770
788

miR-3157-3p
UGCCCUA
UAGGGCA
AGGGGCA
585
1,346

hsa-miR-449b-3p
AGCCACA
UGUGGCU
GUUGGCU
2,741
1,225

miR-3677-5p
AGUGGCC
GGCCACU
GCCCACU
1,352
1,219

hsa-miR-191-3p
CUGCGCU
AGCGCAG
GCCGCAG
733
1,254

miR-512-5p
ACUCAGC
GCUGAGU
CUUGAGU
1,449
1,140

miR-1346/3940-5p/4507
UGGGUUG
CAACCCA
AAACCCA
1,275
1,842

miR-4746-5p
CGGUCCC
GGGACCG
GGGACCG
671
671

miR-760-3p/1842
GGCUCUG
CAGAGCC
AGGAGCC
2,769
2,357

miR-3622a-5p
AGGCACG
CGUGCCU
GUUGCCU
831
1,304

miR-3939
ACGCGCA
UGCGCGU
GCCGCGU
277
379

hsa-miR-181a-2-3p
CCACUGA
UCAGUGG
CAAGUGG
2,244
1,847

miR-508-5p/509-5p
ACUCCAG
CUGGAGU
UGGGAGU
2,469
1,365

hsa-miR-500a-3p
UGCACCU
AGGUGCA
GGGUGCA
1,681
948

miR-4761-3p
AGGGCAU
AUGCCCU
UGGCCCU
1,622
2,421

miR-1607/1777b/3180-3p/3196
GGGGCGG
CCGCCCC
CGGCCCC
1,569
1,542

miR-1914
CCUGUGC
GCACAGG
CAACAGG
1,697
1,589

miR-513a-5p
UCACAGG
CCUGUGA
CUUGUGA
2,415
1,546

hsa-miR-196b-3p
CGACAGC
GCUGUCG
CUUGUCG
684
244

hsa-miR-675-3p
UGUAUGC
GCAUACA
CAAUACA
764
957

miR-2116
GUUCUUA
UAAGAAC
AAAGAAC
1,137
2,606

hsa-miR-548aj/g/x-5p
GCAAAAG
CUUUUGC
UUUUUGC
1,945
2,486

miR-4687-5p
AGCCCUC
GAGGGCU
AGGGGCU
1,448
1,622

miR-518a-5p/527
UGCAAAG
CUUUGCA
UUUUGCA
2,349
3,094

miR-4659ab-3p
UUCUUCU
AGAAGAA
GAAAGAA
7,178
4,560

hsa-miR-5001-3p
UCUGCCU
AGGCAGA
GGGCAGA
2,729
1,972

hsa-miR-1247-3p
CCCGGGA
UCCCGGG
CCCCGGG
993
1,380

miR-2890/4707-5p
CCCCGGC
GCCGGGG
CCCGGGG
1,307
1,275

hsa-miR-150-3p
UGGUACA
UGUACCA
GUUACCA
1,539
1,064

miR-513a-3p
AAAUUUC
GAAAUUU
AAAAUUU
2,899
4,273

hsa-miR-629-3p
UUCUCCC
GGGAGAA
GGGAGAA
2,796
2,796

miR-548aaf
AAAACCA
UGGUUUU
GGGUUUU
3,406
1,687

miR-2277-3p
GACAGCG
CGCUGUC
GCCUGUC
753
1,529

hsa-miR-518c-5p
CUCUGGA
UCCAGAG
CCCAGAG
2,417
3,106

miR-3547/3663-3p
GAGCACC
GGUGCUC
GUUGCUC
1,534
884

miR-548k
AAAGUAC
GUACUUU
UAACUUU
1,720
2,289

miR-34bc-3p
AUCACUA
UAGUGAU
AGGUGAU
1,347
1,800

miR-518ef
AAGCGCU
AGCGCUU
GCCGCUU
548
529

miR-3187-3p
UGGCCAU
AUGGCCA
UGGGCCA
1,995
1,886

miR-4749-3p
GCCCCUC
GAGGGGC
AGGGGGC
1,774
1,332

miR-1306/1306-3p
CGUUGGC
GCCAACG
CCCAACG
592
516

miR-3177-3p
GCACGGC
GCCGUGC
CCCGUGC
857
778

hsa-miR-548av-3p
AAACUGC
GCAGUUU
CAAGUUU
2,136
2,106

TABLE 2

Seed
Bulge

miRNA family
Seed
Seed site
Bulge site
site #
site #

let-7/98/4458/4500
GAGGUAG
CUACCUC
UAACCUC
2,240
675

miR-1ab/206/613
GGAAUGU
ACAUUCC
CAAUUCC
1,988
841

miR-27abc/27a-3p
UCACAGU
ACUGUGA
CUUGUGA
2,787
1,052

miR-143/1721/4770
GAGAUGA
UCAUCUC
CAAUCUC
1,889
586

miR-126-3p
CGUACCG
CGGUACG
GGGUACG
87
132

miR-30a bcdef/30a be-5p/384-5p
GUAAACA
UGUUUAC
GUUUUAC
2,334
1,157

miR-125a-5p/125b-5p/351/670/4319
CCCUGAG
CUCAGGG
UCCAGGG
1,531
1,331

miR-23abc/23b-3p
UCACAUU
AAUGUGA
AUUGUGA
2,516
1,385

miR-133abc
UUGGUCC
GGACCAA
GAACCAA
1,781
1,224

miR-15abc/16/16abc/195/322/424/497/1907
AGCAGCA
UGCUGCU
GCCUGCU
4,434
1,730

miR-29abcd
AGCACCA
UGGUGCU
GGGUGCU
3,104
869

miR-499-5p
UAAGACU
AGUCUUA
GUUCUUA
858
866

miR-99ab/100
ACCCGUA
UACGGGU
ACCGGGU
199
215

miR-26ab/1297/4465
UCAAGUA
UACUUGA
ACCUUGA
1,500
1,045

miR-21/590-5p
AGCUUAU
AUAAGCU
UAAAGCU
715
1,218

miR-208ab/208ab-3p
UAAGACG
CGUCUUA
GUUCUUA
257
866

miR-22/22-3p
AGCUGCC
GGCAGCU
GCCAGCU
2,314
1,623

miR-221/222/222ab/1928
GCUACAU
AUGUAGC
UGGUAGC
769
576

miR-181abcd/4262
ACAUUCA
UGAAUGU
GAAAUGU
2,452
1,976

miR-24/24ab/24-3p
GGCUCAG
CUGAGCC
UGGAGCC
2,063
1,763

miR-25/32/92abc/363/363-3p/367
AUUGCAC
GUGCAAU
UGGCAAU
1,138
1,097

miR-199ab-3p/3129-5p
CAGUAGU
ACUACUG
CUUACUG
1,041
1,020

miR-103a/107/107ab
GCAGCAU
AUGCUGC
UGGCUGC
2,381
2,029

miR-148ab-3p/152
CAGUGCA
UGCACUG
GCCACUG
1,531
1,707

miR-199ab-5p
CCAGUGU
ACACUGG
CAACUGG
1,414
1,071

miR-28-5p/708/1407/1653/3139
AGGAGCU
AGCUCCU
GCCUCCU
1,530
1,754

miR-374ab
UAUAAUA
UAUUAUA
AUUUAUA
1,538
2,233

miR-19ab
GUGCAAA
UUUGCAC
UUUGCAC
1,522
1,522

miR-126-5p
AUUAUUA
UAAUAAU
AAAUAAU
1,540
2,731

miR-145
UCCAGUU
AACUGGA
ACCUGGA
2,058
2,353

miR-193/193b/193a-3p
ACUGGCC
GGCCAGU
GCCCAGU
1,146
1,237

miR-423a/423-5p/3184/3573-5p
GAGGGGC
GCCCCUC
CCCCCUC
1,532
1,468

miR-33a-3p/365/365-3p
AAUGCCC
GGGCAUU
GGGCAUU
922
922

miR-17/17-5p/20ab/20b-
AAAGUGC
GCACUUU
CAACUUU
1,507
1,298

5p/93/106ab/427/518a-3p/519d

miR-101/101ab
ACAGUAC
GUACUGU
UAACUGU
1,086
1,106

miR-499-3p/499a-3p
ACAUCAC
GUGAUGU
UGGAUGU
1,337
1,511

miR-214/761/3619-5p
CAGCAGG
CCUGCUG
CUUGCUG
3,842
1,626

miR-34ac/34bc-5p/449abc/449c-5p
GGCAGUG
CACUGCC
ACCUGCC
1,563
1,526

miR-185/882/3473/4306/4644
GGAGAGA
UCUCUCC
CUUCUCC
1,695
2,052

miR-378/422a/378bcdefhi
CUGGACU
AGUCCAG
GUUCCAG
1,028
1,168

miR-218/218a
UGUGCUU
AAGCACA
AGGCACA
1,578
970

miR-139-5p
CUACAGU
ACUGUAG
CUUGUAG
932
655

miR-130ac/301ab/301b/301b-
AGUGCAA
UUGCACU
UGGCACU
1,253
1,100

3p/454/721/4295/3666

miR-224
AAGUCAC
GUGACUU
UGGACUU
1,161
1,481

miR-30e-3p, miR-30d-3p, miR-30a-3p
UUUCAGU
ACUGAAA
CUUGAAA
2,001
2,071

miR-146ac/146b-5p
GAGAACU
AGUUCUC
GUUUCUC
969
1,180

miR-652
AUGGCGC
GCGCCAU
CGGCCAU
386
396

miR-1307
CUCGGCG
CGCCGAG
GCCCGAG
516
778

miR-210
UGUGCGU
ACGCACA
CGGCACA
356
310

miR-22-5p
GUUCUUC
GAAGAAC
AAAGAAC
1,863
1,757

miR-338/338-3p
CCAGCAU
AUGCUGG
UGGCUGG
2,173
1,923

miR-324-5p
GCAUCCC
GGGAUGC
GGGAUGC
767
767

miR-128/128ab
CACAGUG
CACUGUG
ACCUGUG
2,113
1,721

miR-191
AACGGAA
UUCCGUU
UCCCGUU
379
248

miR-423-3p
GCUCGGU
ACCGAGC
CCCGAGC
419
675

miR-425/425-5p/489
AUGACAC
GUGUCAU
UGGUCAU
949
1,110

miR-378a-5p
UCCUGAC
GUCAGGA
UCCAGGA
755
1,946

miR-127/127-3p
CGGAUCC
GGAUCCG
GAAUCCG
233
216

miR-142-3p
GUAGUGU
ACACUAC
CAACUAC
609
1,059

miR-208b-5p, miR-208a-5p
AGCUUUU
AAAAGCU
AAAAGCU
1,680
1,680

miR-486-5p/3107
CCUGUAC
GUACAGG
UAACAGG
759
645

let-7d-3p
UAUACGA
UCGUAUA
CGGUAUA
114
94

miR-33ab/33-5p
UGCAUUG
CAAUGCA
AAAUGCA
1,075
2,362

miR-124/124ab/506
AAGGCAC
GUGCCUU
UGGCCUU
1,350
1,469

miR-342-3p
CUCACAC
GUGUGAG
UGGUGAG
1,189
1,910

miR-574-5p
GAGUGUG
CACACUC
ACCACUC
910
619

miR-18ab/4735-3p
AAGGUGC
GCACCUU
CAACCUU
943
881

miR-190/190ab
GAUAUGU
ACAUAUC
CAAUAUC
601
775

miR-150/5127
CUCCCAA
UUGGGAG
UGGGGAG
1,055
1,471

miR-223
GUCAGUU
AACUGAC
ACCUGAC
840
977

miR-144
ACAGUAU
AUACUGU
UAACUGU
1,549
1,106

miR-10abc/10a-5p
ACCCUGU
ACAGGGU
CAAGGGU
690
541

miR-143-5p
GUGCAGU
ACUGCAC
CUUGCAC
974
746

miR-149
CUGGCUC
GAGCCAG
AGGCCAG
1,861
1,451

miR-1296
UAGGGCC
GGCCCUA
GCCCCUA
429
432

let-7i-3p
UGCGCAA
UUGCGCA
UGGCGCA
199
312

miR-17-3p
CUGCAGU
ACUGCAG
CUUGCAG
1,827
1,391

miR-450a/451a
UUUGCGA
UCGCAAA
CGGCAAA
202
281

miR-193a-5p
GGGUCUU
AAGACCC
AGGACCC
909
970

miR-9/9ab
CUUUGGU
ACCAAAG
CCCAAAG
2,035
1,496

miR-1307-5p
CGACCGG
CCGGUCG
CGGGUCG
97
117

miR-132/212/212-3p
AACAGUC
GACUGUU
ACCUGUU
856
1,211

miR-197
UCACCAC
GUGGUGA
UGGGUGA
1,444
1,110

miR983p, let7f-1-3p, let-7b-3p, let-7a-3p
UAUACAA
UUGUAUA
UGGUAUA
1,849
759

miR-219-5p/508/508-3p/4782-3p
GAUUGUC
GACAAUC
ACCAAUC
574
679

miR-24-2-5p, miR-24-1-5p
GCCUACU
AGUAGGC
GUUAGGC
325
196

miR-144-5p
GAUAUCA
UGAUAUC
GAAUAUC
734
754

miR-28-3p
ACUAGAU
AUCUAGU
UCCUAGU
475
610

miR-140/140-5p/876-3p/1244
AGUGGUU
AACCACU
ACCCACU
945
803

miR-133a-5p
GCUGGUA
UACCAGC
ACCCAGC
1,081
1,375

miR-598/598-3p
ACGUCAU
AUGACGU
UGGACGU
318
566

miR-490-3p
AACCUGG
CCAGGUU
CAAGGUU
924
768

miR-29c-5p
GACCGAU
AUCGGUC
UCCGGUC
94
229

miR-361-5p
UAUCAGA
UCUGAUA
CUUGAUA
862
818

miR-106b-3p
CGCACUG
CAGUGCG
AGGUGCG
439
397

miR-93/93a/105/106a/291a-
AAGUGCU
AGCACUU
GCCACUU
1,488
917

3p/294/295/302a bcde/372/373/428/519a/

520be/520acd-3p/1378/1420ac

miR-574-3p
ACGCUCA
UGAGCGU
GAAGCGU
417
255

miR-421
UCAACAG
CUGUUGA
UGGUUGA
1,422
876

miR-99b-3p, miR-99a-3p
AAGCUCG
CGAGCUU
GAAGCUU
278
1,241

miR-186
AAAGAAU
AUUCUUU
UUUCUUU
2,090
4,426

miR-181a-2-3p
CCACUGA
UCAGUGG
CAAGUGG
1,516
1,105

miR-1404/2110
UGGGGAA
UUCCCCA
UCCCCCA
1,607
1,743

miR-135ab/135a-5p
AUGGCUU
AAGCCAU
AGGCCAU
1,459
1,241

miR-362-5p/500b
AUCCUUG
CAAGGAU
AAAGGAU
1,159
1,206

miR-153
UGCAUAG
CUAUGCA
UAAUGCA
818
999

miR-377
UCACACA
UGUGUGA
GUUGUGA
1,954
910

miR-550a
GUGCCUG
CAGGCAC
AGGGCAC
1,047
736

miR-369-5p
GAUCGAC
GUCGAUC
UCCGAUC
92
97

miR-140-3p
ACCACAG
CUGUGGU
UGGUGGU
1,756
1,913

miR-6827-3p, miR-340-3p
CCGUCUC
GAGACGG
AGGACGG
538
450

miR-214-5p
GCCUGUC
GACAGGC
ACCAGGC
737
1,076

miR-27b-5p
GAGCUUA
UAAGCUC
AAAGCUC
414
921

miR-296-5p
GGGCCCC
GGGGCCC
GGGGCCC
1,212
1,212

miR-154/872
AGGUUAU
AUAACCU
UAAACCU
707
982

miR-204/204b/211
UCCCUUU
AAAGGGA
AAAGGGA
1,254
1,254

miR-145-3p
GAUUCCU
AGGAAUC
GGGAAUC
858
601

miR-20a-3p
CUGCAUU
AAUGCAG
AUUGCAG
1,775
1,369

miR-483-3p
CACUCCU
AGGAGUG
GGGAGUG
1,019
932

miR-155
UAAUGCU
AGCAUUA
GCCAUUA
1,024
773

miR-23b-5p, miR-23a-5p
GGGUUCC
GGAACCC
GAAACCC
812
977

miR-1277
ACGUAGA
UCUACGU
CUUACGU
326
194

miR-664/664b
AUUCAUU
AAUGAAU
AUUGAAU
1,850
1,189

miR-887
UGAACGG
CCGUUCA
CGGUUCA
218
213

miR-495/1192
AACAAAC
GUUUGUU
UUUUGUU
1,700
4,230

miR-369-3p
AUAAUAC
GUAUUAU
UAAUUAU
1,234
1,440

miR-361-3p
CCCCCAG
CUGGGGG
UGGGGGG
1,372
836

miR-92b-5p
GGGACGG
CCGUCCC
CGGUCCC
478
422

miR-339b/339-5p/3586-5p
CCCUGUC
GACAGGG
ACCAGGG
719
957

miR-340-5p
UAUAAAG
CUUUAUA
UUUUAUA
1,351
2,941

miR-136-3p
AUCAUCG
CGAUGAU
GAAUGAU
302
910

miR-503
AGCAGCG
CGCUGCU
GCCUGCU
1,093
1,730

miR-1287
GCUGGAU
AUCCAGC
UCCCAGC
1,074
1,958

miR-654-3p
AUGUCUG
CAGACAU
AGGACAU
1,386
1,351

miR-532-5p/511
AUGCCUU
AAGGCAU
AGGGCAU
1,058
908

miR-1-5p
CAUACUU
AAGUAUG
AGGUAUG
1,424
1,229

miR-493-5p
UGUACAU
AUGUACA
UGGUACA
1,770
1,176

miR-744/1716
GCGGGGC
GCCCCGC
CCCCCGC
1,134
952

miR-3591-3p
AACACCA
UGGUGUU
GGGUGUU
1,492
632

miR-30b-3p/3689c/3689a-3p
UGGGAGG
CCUCCCA
CUUCCCA
2,081
1,684

miR-329/329ab/362-3p
ACACACC
GGUGUGU
GUUGUGU
988
1,103

miR-4524a-3p
GAGACAG
CUGUCUC
UGGUCUC
1,533
1,010

miR-376c/741-5p
ACAUAGA
UCUAUGU
CUUAUGU
932
992

miR-29b-2-5p
UGGUUUC
GAAACCA
AAAACCA
1,592
2,292

miR-193b-5p
GGGGUUU
AAACCCC
AAACCCC
752
752

miR-374c/655
UAAUACA
UGUAUUA
GUUAUUA
1,713
878

miR-335/335-5p
CAAGAGC
GCUCUUG
CUUCUUG
892
1,332

miR-452/4676-3p
ACUGUUU
AAACAGU
AAACAGU
1,589
1,589

miR-96/507/1271
UUGGCAC
GUGCCAA
UGGCCAA
1,198
1,841

miR-494
GAAACAU
AUGUUUC
UGGUUUC
1,415
1,185

miR-136
CUCCAUU
AAUGGAG
AUUGGAG
1,329
1,057

miR-29a-5p
CUGAUUU
AAAUCAG
AAAUCAG
1,660
1,660

miR-142-5p
AUAAAGU
ACUUUAU
CUUUUAU
1,660
1,832

miR-501-3p/502-3p/500/502a
AUGCACC
GGUGCAU
GUUGCAU
546
707

miR-542-5p
CGGGGAU
AUCCCCG
UCCCCCG
346
546

miR-874
UGCCCUG
CAGGGCA
AGGGGCA
1,328
835

miR-660
ACCCAUU
AAUGGGU
AUUGGGU
649
513

miR-138/138ab
GCUGGUG
CACCAGC
ACCCAGC
1,898
1,375

miR-7/7ab
GGAAGAC
GUCUUCC
UCCUUCC
1,236
2,073

miR-101-5p
AGUUAUC
GAUAACU
AUUAACU
600
907

miR-651
UUAGGAU
AUCCUAA
UCCCUAA
585
677

miR-339-3p
GAGCGCC
GGCGCUC
GCCGCUC
448
516

miR-625
GGGGGAA
UUCCCCC
UCCCCCC
1,259
942

miR-584
UAUGGUU
AACCAUA
ACCCAUA
657
437

miR-551a
CGACCCA
UGGGUCG
GGGGUCG
191
232

miR-1301/5047
UGCAGCU
AGCUGCA
GCCUGCA
2,276
1,750

miR-31
GGCAAGA
UCUUGCC
CUUUGCC
979
1,660

miR-381-5p
GCGAGGU
ACCUCGC
CCCUCGC
296
547

miR-345/345-5p
CUGACUC
GAGUCAG
AGGUCAG
846
950

miR-374a-3p
UUAUCAG
CUGAUAA
UGGAUAA
874
928

miR-181a-3p
CCAUCGA
UCGAUGG
CGGAUGG
245
294

miR-8073, miR-221-5p
CCUGGCA
UGCCAGG
GCCCAGG
1,403
1,893

miR-500a-3p
UGCACCU
AGGUGCA
GGGUGCA
959
647

miR-409-5p/409a
GGUUACC
GGUAACC
GUUAACC
351
393

miR-542-3p
GUGACAG
CUGUCAC
UGGUCAC
1,326
878

miR-491-5p
GUGGGGA
UCCCCAC
CCCCCAC
1,514
1,749

miR-6788-5p, miR-30c-2-3p, miR-30c-1-3p
UGGGAGA
UCUCCCA
CUUCCCA
1,484
1,684

miR-216a
AAUCUCA
UGAGAUU
GAAGAUU
1,070
1,489

miR-194
GUAACAG
CUGUUAC
UGGUUAC
1,079
624

miR-941
ACCCGGC
GCCGGGU
CCCGGGU
403
377

miR-300/381/539-3p
AUACAAG
CUUGUAU
UUUGUAU
1,188
2,820

miR-376a-5p
UAGAUUC
GAAUCUA
AAAUCUA
720
1,104

miR-1306-5p
CACCUCC
GGAGGUG
GAAGGUG
2,013
1,894

miR-1249
CGCCCUU
AAGGGCG
AGGGGCG
280
385

miR-485-3p
UCAUACA
UGUAUGA
GUUAUGA
1,234
678

miR-642a
UCCCUCU
AGAGGGA
GAAGGGA
1,083
1,205

miR-532-3p
CUCCCAC
GUGGGAG
UGGGGAG
1,306
1,471

miR-3613-5p
GUUGUAC
GUACAAC
UAACAAC
624
596

miR-424-3p
AAAACGU
ACGUUUU
CGGUUUU
505
224

miR-769-5p
GAGACCU
AGGUCUC
GGGUCUC
603
633

miR-3200-3p
ACCUUGC
GCAAGGU
CAAAGGU
826
1,262

miR-1185-2-3p, miR-1185-1-3p, let-7f-2-3p
UAUACAG
CUGUAUA
UGGUAUA
1,249
759

miR-486-3p
GGGGCAG
CUGCCCC
UGGCCCC
2,110
1,257

let-7g-3p,let-7a-2-3p
UGUACAG
CUGUACA
UGGUACA
1,502
1,176

miR-125b-1-3p
CGGGUUA
UAACCCG
AAACCCG
112
227

let-7e-3p
UAUACGG
CCGUAUA
CGGUAUA
152
94

miR-26b-3p
CUGUUCU
AGAACAG
GAAACAG
1,736
1,874

miR-487a
AUCAUAC
GUAUGAU
UAAUGAU
916
1,120

miR-132-5p
CCGUGGC
GCCACGG
CCCACGG
602
500

miR-382
AAGUUGU
ACAACUU
CAAACUU
1,321
1,177

miR-579
UCAUUUG
CAAAUGA
AAAAUGA
1,423
3,031

miR-766
CUCCAGC
GCUGGAG
CUUGGAG
3,325
1,581

miR-493/493b
GAAGGUC
GACCUUC
ACCCUUC
934
932

miR-326/330/330-5p
CUCUGGG
CCCAGAG
CCCAGAG
1,717
1,717

miR-299-5p/3563-5p
GGUUUAC
GUAAACC
UAAAACC
643
1,034

miR-202-5p
UCCUAUG
CAUAGGA
AUUAGGA
493
630

miR-374b-3p
UUAGCAG
CUGCUAA
UGGCUAA
1,062
731

miR-3605-3p
CUCCGUG
CACGGAG
ACCGGAG
683
270

miR-210-5p
GCCCCUG
CAGGGGC
AGGGGGC
1,060
881

miR-15a-3p
AGGCCAU
AUGGCCU
UGGGCCU
1,079
1,332

miR-382-3p
AUCAUUC
GAAUGAU
AAAUGAU
910
1,945

miR-93-3p
CUGCUGA
UCAGCAG
CAAGCAG
1,960
1,934

miR-195-3p, miR-16-2-3p
CAAUAUU
AAUAUUG
AUUAUUG
1,515
1,474

miR-187
CGUGUCU
AGACACG
GAACACG
334
307

miR-5010-3p
UUUGUGU
ACACAAA
CAACAAA
1,493
1,602

miR-103a-2-5p
GCUUCUU
AAGAAGC
AGGAAGC
2,642
1,824

miR-33a-3p
AAUGUUU
AAACAUU
AAACAUU
2,352
2,352

miR-548a-3p/548ef/2285a
AAAACUG
CAGUUUU
AGGUUUU
2,073
1,444

miR-490-5p
CAUGGAU
AUCCAUG
UCCCAUG
825
986

miR-522/518e/1422p
AAAUGGU
ACCAUUU
CCCAUUU
1,444
1,063

miR-1306/1306-3p
CGUUGGC
GCCAACG
CCCAACG
426
385

miR-376abd/376b-3p
UCAUAGA
UCUAUGA
CUUAUGA
1,089
914

miR-675-5p/4466
GGUGCGG
CCGCACC
CGGCACC
524
570

miR-677/4420
UCACUGA
UCAGUGA
CAAGUGA
1,670
1,276

miR-509-5p/509-3-5p/4418
ACUGCAG
CUGCAGU
UGGCAGU
2,130
1,444

miR-125a-3p/1554
CAGGUGA
UCACCUG
CAACCUG
1,482
1,582

miR-200bc/429/548a
AAUACUG
CAGUAUU
AGGUAUU
1,782
1,010

miR-130b-5p
CUCUUUC
GAAAGAG
AAAAGAG
1,625
1,864

miR-656
AUAUUAU
AUAAUAU
UAAAUAU
1,435
2,879

miR-192/215
UGACCUA
UAGGUCA
AGGGUCA
362
639

miR-337-3p
UCCUAUA
UAUAGGA
AUUAGGA
586
630

miR-548aaf
AAAACCA
UGGUUUU
GGGUUUU
2,131
947

miR-139-3p
GGAGACG
CGUCUCC
GUUCUCC
516
932

miR-337-5p
AACGGCU
AGCCGUU
GCCCGUU
231
223

miR-589
GAGAACC
GGUUCUC
GUUUCUC
631
1,180

miR-3200-5p
AUCUGAG
CUCAGAU
UCCAGAU
1,433
1,530

miR-323/323-3p
ACAUUAC
GUAAUGU
UAAAUGU
920
2,441

miR-488
UGAAAGG
CCUUUCA
CUUUUCA
1,315
1,862

miR-1185/3679-5p
GAGGAUA
UAUCCUC
AUUCCUC
593
1,283

miR-6511b-3p, miR-6511a-3p
CUCACCA
UGGUGAG
GGGUGAG
1,910
1,229

miR-660-3p
CCUCCUG
CAGGAGG
AGGGAGG
1,858
965

miR-203
UGAAAUG
CAUUUCA
AUUUUCA
1,903
2,455

miR-425-3p
UCGGGAA
UUCCCGA
UCCCCGA
391
548

miR-433
UCAUGAU
AUCAUGA
UCCAUGA
1,244
1,137

miR-450b-5p
UUUGCAA
UUGCAAA
UGGCAAA
1,666
1,652

miR-675-3p
UGUAUGC
GCAUACA
CAAUACA
555
729

miR-1180
UUCCGGC
GCCGGAA
CCCGGAA
330
415

miR-342-5p/4664-5p
GGGGUGC
GCACCCC
CAACCCC
970
1,038

miR-597/1970
GUGUCAC
GUGACAC
UGGACAC
800
1,168

miR-26a-2-3p, miR-26a-1-3p
CUAUUCU
AGAAUAG
GAAAUAG
685
989

miR-338-5p
ACAAUAU
AUAUUGU
UAAUUGU
1,765
1,291

miR-324-3p/1913
CUGCCCC
GGGGCAG
GGGGCAG
1,474
1,474

miR-548abakhjiwy/548abcd-5p/559
AAAGUAA
UUACUUU
UAACUUU
2,013
1,608

miR-518a-5p/527
UGCAAAG
CUUUGCA
UUUUGCA
1,746
2,241

miR-9-3p
UAAAGCU
AGCUUUA
GCCUUUA
1,258
911

miR-299/299-3p/3563-3p
AUGUGGG
CCCACAU
CCCACAU
906
906

miR-27a-5p
GGGCUUA
UAAGCCC
AAAGCCC
451
939

miR-491-3p
UUAUGCA
UGCAUAA
GCCAUAA
654
600

miR-671-5p
GGAAGCC
GGCUUCC
GCCUUCC
1,385
1,413

miR-190a-3p
UAUAUAU
AUAUAUA
UAAUAUA
2,181
1,454

miR-1323/5480
CAAAACU
AGUUUUG
GUUUUUG
1,639
1,882

miR-1537
AAACCGU
ACGGUUU
CGGGUUU
261
268

miR-411
AGUAGAC
GUCUACU
UCCUACU
559
1,008

miR-4662a-5p
UAGCCAA
UUGGCUA
UGGGCUA
734
574

miR-411-3p, miR-379-3p
AUGUAAC
GUUACAU
UUUACAU
800
1,934

miR-550a-3p, miR-200c-5p
GUCUUAC
GUAAGAC
UAAAGAC
553
959

miR-141/200a
AACACUG
CAGUGUU
AGGUGUU
1,731
1,002

miR-3613-3p
CAAAAAA
UUUUUUG
UUUUUUG
3,104
3,104

miR-2277-3p
GACAGCG
CGCUGUC
GCCUGUC
521
1,103

miR-545/3065/3065-5p
CAACAAA
UUUGUUG
UUUGUUG
1,984
1,984

miR-500a
AAUCCUU
AAGGAUU
AGGGAUU
1,056
680

miR-545-5p
CAGUAAA
UUUACUG
UUUACUG
1,621
1,621

miR-409-3p
AAUGUUG
CAACAUU
AAACAUU
1,478
2,352

miR-874-5p
GGCCCCA
UGGGGCC
GGGGGCC
1,385
1,095

miR-410/344de/344b-1-3p
AUAUAAC
GUUAUAU
UUUAUAU
963
2,815

miR-576-5p
UUCUAAU
AUUAGAA
UUUAGAA
1,231
1,952

miR-129-5p/129ab-5p
UUUUUGC
GCAAAAA
CAAAAAA
1,602
2,408

miR-370
CCUGCUG
CAGCAGG
AGGCAGG
2,014
1,252

miR-1296-3p
AGUGGGG
CCCCACU
CCCCACU
1,356
1,356

miR-590-3p
AAUUUUA
UAAAAUU
AAAAAUU
2,634
3,067

miR-519a/519bc-3p/291b-3p/1347
AAGUGCA
UGCACUU
GCCACUU
1,434
917

miR-518bcf/518a-3p/518d-3p
AAAGCGC
GCGCUUU
CGGCUUU
286
326

miR-501-5p
AUCCUUU
AAAGGAU
AAAGGAU
1,206
1,206

miR-629
GGGUUUA
UAAACCC
AAAACCC
515
1,101

miR-889
UAAUAUC
GAUAUUA
AUUAUUA
794
1,590

miR-450b-3p/769-3p
UGGGAUC
GAUCCCA
AUUCCCA
820
1,347

miR-1618/3940-3p
AGCCCGG
CCGGGCU
CGGGGCU
599
572

miR-125b-2-3p
CACAAGU
ACUUGUG
CUUUGUG
959
2,232

miR-548d-3p/548acbz
AAAAACC
GGUUUUU
GUUUUUU
1,511
2,520

miR-485-5p/1698/1703/1962
GAGGCUG
CAGCCUC
AGGCCUC
2,388
1,087

miR-127-5p
UGAAGCU
AGCUUCA
GCCUUCA
1,476
1,469

miR-6086, miR-377-5p
GAGGUUG
CAACCUC
AAACCUC
1,385
1,093

miR-183
AUGGCAC
GUGCCAU
UGGCCAU
1,045
1,385

miR-625-3p
ACUAUAG
CUAUAGU
UAAUAGU
485
738

miR-544/544ab/544-3p
UUCUGCA
UGCAGAA
GCCAGAA
2,624
1,422

miR-31-3p
GCUAUGC
GCAUAGC
CAAUAGC
321
480

miR-454-5p
CCCUAUC
GAUAGGG
AUUAGGG
195
309

miR-539/539-5p
GAGAAAU
AUUUCUC
UUUUCUC
1,368
2,373

miR-487b-5p, miR-487a-5p
UGGUUAU
AUAACCA
UAAACCA
751
1,077

miR-545
CAGCAAA
UUUGCUG
UUUGCUG
2,422
2,422

miR-4525
GGGGGAU
AUCCCCC
UCCCCCC
712
942

miR-1304-3p
CUCACUG
CAGUGAG
AGGUGAG
1,694
2,510

miR-4423-5p
GUUGCCU
AGGCAAC
GGGCAAC
638
707

miR-16-1-3p
CAGUAUU
AAUACUG
AUUACUG
1,396
1,461

miR-652-5p
AACCCUA
UAGGGUU
AGGGGUU
337
449

miR-219-2-3p/219-3p
GAAUUGU
ACAAUUC
CAAAUUC
771
1,149

miR-1247
CCCGUCC
GGACGGG
GAACGGG
462
376

miR-181c-3p
ACCAUCG
CGAUGGU
GAAUGGU
333
924

miR-15b-3p
GAAUCAU
AUGAUUC
UGGAUUC
1,007
970

miR-3620
CACCCUG
CAGGGUG
AGGGGUG
1,059
703

miR-885-5p
CCAUUAC
GUAAUGG
UAAAUGG
559
1,086

miR-380/380-3p
AUGUAAU
AUUACAU
UUUACAU
1,360
1,934

miR-379/1193-5p/3529
GGUAGAC
GUCUACC
UCCUACC
609
886

miR-656-5p
GGUUGCC
GGCAACC
GCCAACC
638
937

miR-188-5p
AUCCCUU
AAGGGAU
AGGGGAU
725
591

miR-744-3p
UGUUGCC
GGCAACA
GCCAACA
964
1,457

miR-122/122a/1352
GGAGUGU
ACACUCC
CAACUCC
760
995

miR-671-3p
CCGGUUC
GAACCGG
AAACCGG
320
216

miR 7 2 3p, miR 7 1 3p
AACAAAU
AUUUGUU
UUUUGUU
2,401
4,230

miR-518d-5p/519bc-5p520c-5p/523b/526a
UCUAGAG
CUCUAGA
UCCUAGA
594
668

miR-34a-3p
AAUCAGC
GCUGAUU
CUUGAUU
870
986

miR-504/4725-5p
GACCCUG
CAGGGUC
AGGGGUC
801
417

miR-301b-5p, miR-301a-5p
CUCUGAC
GUCAGAG
UCCAGAG
1,035
1,751

miR-654-5p/541
GGUGGGC
GCCCACC
CCCCACC
1,464
2,305

miR-29b-1-5p
CUGGUUU
AAACCAG
AAACCAG
1,830
1,830

miR-371/373/371b-5p
CUCAAAA
UUUUGAG
UUUUGAG
1,634
1,634

miR-2964/2964a-5p
GAUGUCC
GGACAUC
GAACAUC
1,097
1,143

miR-520a-5p/525-5p/2464-3p
UCCAGAG
CUCUGGA
UCCUGGA
1,639
2,485

miR-450a-2-3p
UUGGGGA
UCCCCAA
CCCCCAA
1,381
1,329

miR-511
UGUCUUU
AAAGACA
AAAGACA
2,007
2,007

miR-676
UGUCCUA
UAGGACA
AGGGACA
478
1,028

miR-3130-5p/4482
ACCCAGU
ACUGGGU
CUUGGGU
727
663

miR-3173-5p
GCCCUGC
GCAGGGC
CAAGGGC
1,195
1,045

miR-296-3p
AGGGUUG
CAACCCU
AAACCCU
832
1,127

miR-708-3p
AACUAGA
UCUAGUU
CUUAGUU
657
703

miR-182
UUGGCAA
UUGCCAA
UGGCCAA
1,649
1,841

miR-511-3p
AUGUGUA
UACACAU
ACCACAU
1,072
1,010

miR-1277-5p
AAUAUAU
AUAUAUU
UAAUAUU
2,437
2,072

miR-3064-3p
UGCCACA
UGUGGCA
GUUGGCA
1,804
877

miR-205/205ab
CCUUCAU
AUGAAGG
UGGAAGG
1,579
1,550

miR-323b-3p
CCAAUAC
GUAUUGG
UAAUUGG
593
647

miR-1893/2277-5p
GCGCGGG
CCCGCGC
CCCGCGC
560
560

miR-3157-5p
UCAGCCA
UGGCUGA
GGGCUGA
1,734
1,060

miR-582-5p
UACAGUU
AACUGUA
ACCUGUA
1,375
1,075

miR-1343
UCCUGGG
CCCAGGA
CCCAGGA
2,022
2,022

miR-365b-5p, miR-365a-5p
GGGACUU
AAGUCCC
AGGUCCC
658
659

miR-146b-3p
GCCCUGU
ACAGGGC
CAAGGGC
790
1,045

miR-124-5p
GUGUUCA
UGAACAC
GAAACAC
1,018
1,146

miR-6505-3p
GACUUCU
AGAAGUC
GAAAGUC
1,061
975

miR-526b
UCUUGAG
CUCAAGA
UCCAAGA
1,429
1,346

miR-548aeajamx
AAAAACU
AGUUUUU
GUUUUUU
2,155
2,520

miR-4794
CUGGCUA
UAGCCAG
AGGCCAG
735
1,451

miR-2114
AGUCCCU
AGGGACU
GGGGACU
785
726

miR-19b-2-5p, miR-19b-1-5p, miR-19a-5p
GUUUUGC
GCAAAAC
CAAAAAC
1,020
1,590

miR-18a-3p
CUGCCCU
AGGGCAG
GGGGCAG
1,404
1,474

miR-185-3p
GGGGCUG
CAGCCCC
AGGCCCC
2,449
1,084

miR-433-5p
ACGGUGA
UCACCGU
CAACCGU
453
226

let-7c-3p
UGUACAA
UUGUACA
UGGUACA
1,747
1,176

miR-134/3118
GUGACUG
CAGUCAC
AGGUCAC
1,108
678

miR-548b-3p
AAGAACC
GGUUCUU
GUUUCUU
825
1,825

miR-876-3p
GGUGGUU
AACCACC
ACCCACC
1,020
1,264

miR-3132
GGGUAGA
UCUACCC
CUUACCC
630
596

miR-10a-3p
AAAUUCG
CGAAUUU
GAAAUUU
177
1,754

miR-6741-3p
CGGCUCU
AGAGCCG
GAAGCCG
489
638

miR-34bc-3p
AUCACUA
UAGUGAU
AGGUGAU
842
1,094

miR-186-3p
CCCAAAG
CUUUGGG
UUUUGGG
1,451
1,349

miR-4661-5p
ACUAGCU
AGCUAGU
GCCUAGU
479
409

miR-370-5p
AGGUCAC
GUGACCU
UGGACCU
1,173
1,444

miR-653-3p
UCACUGG
CCAGUGA
CAAGUGA
1,773
1,276

miR-5001-3p
UCUGCCU
AGGCAGA
GGGCAGA
1,606
1,222

miR-2964a-3p
GAAUUGC
GCAAUUC
CAAAUUC
627
1,149

miR-330-3p
CAAAGCA
UGCUUUG
GCCUUUG
2,124
1,436

miR-579-5p
CGCGGUU
AACCGCG
ACCCGCG
114
192

miR-3064-5p/3085-3p
CUGGCUG
CAGCCAG
AGGCCAG
2,582
1,451

miR-376c-5p, miR-376b-5p
GUGGAUA
UAUCCAC
AUUCCAC
537
1,011

miR-3127-3p
CCCCUUC
GAAGGGG
AAAGGGG
1,091
803

miR-483-5p
AGACGGG
CCCGUCU
CCCGUCU
345
345

miR-129-3p/129ab-3p/129-1-3p/129-2-3p
AGCCCUU
AAGGGCU
AGGGGCU
843
1,016

miR-196abc
AGGUAGU
ACUACCU
CUUACCU
1,243
1,010

miR-576-3p
AGAUGUG
CACAUCU
ACCAUCU
1,248
1,205

miR-552/3097-5p
ACAGGUG
CACCUGU
ACCCUGU
1,448
1,073

miR-4761-5p
CAAGGUG
CACCUUG
ACCCUUG
1,009
678

miR-1745/3194-3p
GCUCUGC
GCAGAGC
CAAGAGC
1,601
1,220

miR-4707-3p
GCCCGCC
GGCGGGC
GCCGGGC
742
1,085

miR-548ay-3p, miR-548at-3p
AAAACCG
CGGUUUU
GGGUUUU
224
947

miR-34b/449c/1360/2682
AGGCAGU
ACUGCCU
CUUGCCU
1,215
1,209

miR-513a-5p
UCACAGG
CCUGUGA
CUUGUGA
1,826
1,052

miR-3145-5p
ACUCCAA
UUGGAGU
UGGGAGU
1,013
832

miR-3158-3p
AGGGCUU
AAGCCCU
AGGCCCU
1,186
1,293

miR-556-5p
AUGAGCU
AGCUCAU
GCCUCAU
1,199
1,040

miR-3194-5p
GCCAGCC
GGCUGGC
GCCUGGC
1,385
1,958

miR-6734-3p
CCUUCCC
GGGAAGG
GGGAAGG
1,402
1,402

miR-523
AACGCGC
GCGCGUU
CGGCGUU
94
152

miR-1910
CAGUCCU
AGGACUG
GGGACUG
1,213
1,071

miR-4670-5p
AGCGACC
GGUCGCU
GUUCGCU
182
203

miR-2115
GCUUCCA
UGGAAGC
GGGAAGC
1,641
1,091

miR-508-5p/509-5p
ACUCCAG
CUGGAGU
UGGGAGU
1,518
832

miR-1245
AGUGAUC
GAUCACU
AUUCACU
701
1,223

miR-556-3p
UAUUACC
GGUAAUA
GUUAAUA
596
908

miR-188-3p
UCCCACA
UGUGGGA
GUUGGGA
1,513
797

miR-651-3p
AAGGAAA
UUUCCUU
UUUCCUU
2,819
2,819

miR-615-3p
CCGAGCC
GGCUCGG
GCCUCGG
496
608

miR-758
UUGUGAC
GUCACAA
UCCACAA
663
1,125

miR-4670-3p
GAAGUUA
UAACUUC
AAACUUC
934
1,371

miR-6874-3p, miR-148b-5p
AGUUCUG
CAGAACU
AGGAACU
1,585
1,447

miR-4778-5p
AUUCUGU
ACAGAAU
CAAGAAU
1,649
1,515

miR-453/323b-5p
GGUUGUC
GACAACC
ACCAACC
768
997

miR-6735-3p
GGCCUGU
ACAGGCC
CAAGGCC
896
1,384

miR-2116-3p
CUCCCAU
AUGGGAG
UGGGGAG
944
1,471

miR-4677-5p
UGUUCUU
AAGAACA
AGGAACA
2,302
1,435

miR-584-3p
CAGUUCC
GGAACUG
GAAACUG
1,412
1,733

miR-885-3p
GGCAGCG
CGCUGCC
GCCUGCC
975
1,819

miR-1287-3p
UCUAGCC
GGCUAGA
GCCUAGA
338
414

miR-2127/4728-5p
GGGAGGG
CCCUCCC
CCCUCCC
2,391
2,391

miR-93b/512-3p/1186
AGUGCUG
CAGCACU
AGGCACU
1,487
923

miR-32-3p
AAUUUAG
CUAAAUU
UAAAAUU
958
2,634

miR-561-5p
UCAAGGA
UCCUUGA
CCCUUGA
1,163
769

miR-766-5p
GGAGGAA
UUCCUCC
UCCCUCC
1,925
1,813

miR-877
UAGAGGA
UCCUCUA
CCCUCUA
843
601

miR-25-5p
GGCGGAG
CUCCGCC
UCCCGCC
734
607

miR-376a-2-5p
GUAGAUU
AAUCUAC
AUUCUAC
610
763

miR-585
GGGCGUA
UACGCCC
ACCGCCC
231
454

miR-3187-3p
UGGCCAU
AUGGCCA
UGGGCCA
1,342
1,478

miR-605
AAAUCCC
GGGAUUU
GGGAUUU
862
862

As a result, it was possible to confirm that miRNAs listed in the tables (Tables 1 and 2) bind with a non-canonical nucleation bulge site in a corresponding tissue. Therefore, when all of the data obtained through the Ago HITS-CLIP assay in the example was summarized, thereby identifying miRNA, and then the present invention for modifying a non-canonical nucleation bulge site to be recognized as a canonical seed site was applied to the miRNA, as shown in Table 3 below, each of a total of 426 sequences (BS sequences) consists of the 2^ndto 7^thnucleotides based on the 5′ end of an RNA interference nucleic acid, and the modified RNA interference nucleic acid will specifically bind to the non-canonical bulge target of the corresponding miRNA and exhibit only the corresponding function.

TABLE 3

BS
SEQ

Bulge

sequence
ID NOs
miRNA family
Seed
Seed site
site

GAGGUU
103
let-7/98/4458/4500
GAGGUA
UACCUC
AACCUC

CCCUGG
104
miR-125a-5p/125b-5p/351/670/4319
CCCUGA
UCAGGG
CCAGGG

AAGGCC
105
miR-124/124ab/506
AAGGCA
UGCCUU
GGCCUU

CUUUGG
106
miR-9/9ab
CUUUGG
CCAAAG
CCAAAG

AGCACC
107
miR-29a bcd
AGCACC
GGUGCU
GGUGCU

GCAGCC
108
miR-103a/107/107ab
GCAGCA
UGCUGC
GGCUGC

GCUACC
109
miR-221/222/222ab/1928
GCUACA
UGUAGC
GGUAGC

UCAAGG
110
miR-26ab/1297/4465
UCAAGU
ACUUGA
CCUUGA

AGCAGG
111
miR-15abc/16/16abc/195/322/424/497/1907
AGCAGC
GCUGCU
CCUGCU

CGUACC
112
miR-126-3p
CGUACC
GGUACG
GGUACG

GUAAAA
113
miR-30abcdef/30abe-5p/384-5p
GUAAAC
GUUUAC
UUUUAC

UGCAUU
114
miR-33ab/33-5p
UGCAUU
AAUGCA
AAUGCA

GGCAGG
115
miR-34ac/34bc-5p/449abc/449c-5p
GGCAGU
ACUGCC
CCUGCC

GUGCAA
116
miR-19ab
GUGCAA
UUGCAC
UUGCAC

ACCCGG
117
miR-99ab/100
ACCCGU
ACGGGU
CCGGGU

AAAGUU
118
miR-17/17-5p/20ab/20b-5p/93/106ab/427/518a-
AAAGUG
CACUUU
AACUUU

3p/519d

UCACAA
119
miR-27abc/27a-3p
UCACAG
CUGUGA
UUGUGA

UGUGCC
120
miR-218/218a
UGUGCU
AGCACA
GGCACA

AGCUGG
121
miR-22/22-3p
AGCUGC
GCAGCU
CCAGCU

GGAGAA
122
miR-185/882/3473/4306/4644
GGAGAG
CUCUCC
UUCUCC

ACAUUU
123
miR-181abcd/4262
ACAUUC
GAAUGU
AAAUGU

CCAGCC
124
miR-338/338-3p
CCAGCA
UGCUGG
GGCUGG

CGGAUU
125
miR-127/127-3p
CGGAUC
GAUCCG
AAUCCG

ACAGUU
126
miR-101/101ab
ACAGUA
UACUGU
AACUGU

CUGGCC
127
miR-149
CUGGCU
AGCCAG
GGCCAG

GCAUCC
128
miR-324-5p
GCAUCC
GGAUGC
GGAUGC

GGCUCC
129
miR-24/24ab/24-3p
GGCUCA
UGAGCC
GGAGCC

AAUGCC
130
miR-33a-3p/365/365-3p
AAUGCC
GGCAUU
GGCAUU

CUACAA
131
miR-139-5p
CUACAG
CUGUAG
UUGUAG

GCUGGG
132
miR-138/138ab
GCUGGU
ACCAGC
CCCAGC

GAGAUU
133
miR-143/1721/4770
GAGAUG
CAUCUC
AAUCUC

AUUGCC
134
miR-25/32/92abc/363/363-3p/367
AUUGCA
UGCAAU
GGCAAU

GAGUGG
135
miR-574-5p
GAGUGU
ACACUC
CCACUC

GGAAGG
136
miR-7/7ab
GGAAGA
UCUUCC
CCUUCC

UCCAGG
137
miR-145
UCCAGU
ACUGGA
CCUGGA

AUGGCC
138
miR-135ab/135a-5p
AUGGCU
AGCCAU
GGCCAU

CAGUGG
139
miR-148ab-3p/152
CAGUGC
GCACUG
CCACUG

AGGAGG
140
miR-28-5p/708/1407/1653/3139
AGGAGC
GCUCCU
CCUCCU

AGUGCC
141
miR-130ac/301ab/301b/301b-3p/454/721/4295/3666
AGUGCA
UGCACU
GGCACU

GGGUAA
142
miR-3132
GGGUAG
CUACCC
UUACCC

UAAUGG
143
miR-155
UAAUGC
GCAUUA
CCAUUA

UCAUAA
144
miR-485-3p
UCAUAC
GUAUGA
UUAUGA

AACAGG
145
miR-132/212/212-3p
AACAGU
ACUGUU
CCUGUU

UAAAGG
146
hsa-miR-9-3p
UAAAGC
GCUUUA
CCUUUA

UAUAAA
147
miR-374ab
UAUAAU
AUUAUA
UUUAUA

AGCCCC
148
miR-129-3p/129ab-3p/129-1-3p/129-2-3p
AGCCCU
AGGGCU
GGGGCU

AUUAUU
149
hsa-miR-126-5p
AUUAUU
AAUAAU
AAUAAU

AUGACC
150
miR-425/425-5p/489
AUGACA
UGUCAU
GGUCAU

GCUCGG
151
miR-423-3p
GCUCGG
CCGAGC
CCGAGC

AGCUUU
152
miR-21/590-5p
AGCUUA
UAAGCU
AAAGCU

GGCAAA
153
miR-31
GGCAAG
CUUGCC
UUUGCC

CUGUAA
154
hsa-miR-20b-3p
CUGUAG
CUACAG
UUACAG

UAUACC
155
hsa-let-7d-3p
UAUACG
CGUAUA
GGUAUA

AACGGG
156
miR-191
AACGGA
UCCGUU
CCCGUU

AAGGUU
157
miR-18ab/4735-3p
AAGGUG
CACCUU
AACCUU

AUAAUU
158
miR-369-3p
AUAAUA
UAUUAU
AAUUAU

GGGAUU
159
hsa-miR-5187-5p
GGGAUG
CAUCCC
AAUCCC

AAGUUU
160
miR-382
AAGUUG
CAACUU
AAACUU

GAGGCC
161
miR-485-5p/1698/1703/1962
GAGGCU
AGCCUC
GGCCUC

AUCAUU
162
hsa-miR-136-3p
AUCAUC
GAUGAU
AAUGAU

AGAUGG
163
miR-576-3p
AGAUGU
ACAUCU
CCAUCU

UCCCUU
164
miR-204/204b/211
UCCCUU
AAGGGA
AAGGGA

GAGACC
165
miR-769-5p
GAGACC
GGUCUC
GGUCUC

GGGGUU
166
miR-342-5p/4664-5p
GGGGUG
CACCCC
AACCCC

UAUCAA
167
miR-361-5p
UAUCAG
CUGAUA
UUGAUA

CAGUAA
168
miR-199ab-3p/3129-5p
CAGUAG
CUACUG
UUACUG

GUAGUU
169
miR-142-3p
GUAGUG
CACUAC
AACUAC

GGUUUU
170
miR-299-5p/3563-5p
GGUUUA
UAAACC
AAAACC

ACUGGG
171
miR-193/193b/193a-3p
ACUGGC
GCCAGU
CCCAGU

AAUAUU
172
hsa-miR-1277-5p
AAUAUA
UAUAUU
AAUAUU

AGUGGG
173
miR-140/140-5p/876-3p/1244
AGUGGU
ACCACU
CCCACU

UUUCAA
174
hsa-miR-30a/d/e-3p
UUUCAG
CUGAAA
UUGAAA

UGCGCC
175
hsa-let-7i-3p
UGCGCA
UGCGCA
GGCGCA

GGUUAA
176
miR-409-5p/409a
GGUUAC
GUAACC
UUAACC

GGUAGG
177
miR-379/1193-5p/3529
GGUAGA
UCUACC
CCUACC

CUCCAA
178
miR-136
CUCCAU
AUGGAG
UUGGAG

AGGUUU
179
miR-154/872
AGGUUA
UAACCU
AAACCU

GUUGCC
180
miR-4684-3p
GUUGCA
UGCAAC
GGCAAC

CCCCCC
181
miR-361-3p
CCCCCA
UGGGGG
GGGGGG

CAAGAA
182
miR-335/335-5p
CAAGAG
CUCUUG
UUCUUG

GAGGGG
183
miR-423a/423-5p/3184/3573-5p
GAGGGG
CCCCUC
CCCCUC

CUCAAA
184
miR-371/373/371b-5p
CUCAAA
UUUGAG
UUUGAG

GAGGAA
185
miR-1185/3679-5p
GAGGAU
AUCCUC
UUCCUC

CAAAAA
186
miR-3613-3p
CAAAAA
UUUUUG
UUUUUG

AAGUGG
187
miR-93/93a/105/106a/291a-
AAGUGC
GCACUU
CCACUU

3p/294/295/302abcde/372/373/428/519a/520be/520

acd-3p/1378/1420ac

GGAUUU
188
miR-876-5p/3167
GGAUUU
AAAUCC
AAAUCC

ACACAA
189
miR-329/329ab/362-3p
ACACAC
GUGUGU
UUGUGU

UACAGG
190
miR-582-5p
UACAGU
ACUGUA
CCUGUA

GAGAAA
191
miR-146ac/146b-5p
GAGAAC
GUUCUC
UUUCUC

AUGUAA
192
miR-380/380-3p
AUGUAA
UUACAU
UUACAU

ACAUCC
193
miR-499-3p/499a-3p
ACAUCA
UGAUGU
GGAUGU

CGACCC
194
miR-551a
CGACCC
GGGUCG
GGGUCG

AUAAAA
195
miR-142-5p
AUAAAG
CUUUAU
UUUUAU

CUGCAA
196
hsa-miR-17-3p
CUGCAG
CUGCAG
UUGCAG

CCAGUU
197
miR-199ab-5p
CCAGUG
CACUGG
AACUGG

GUGACC
198
miR-542-3p
GUGACA
UGUCAC
GGUCAC

ACGUAA
199
miR-1277
ACGUAG
CUACGU
UUACGU

GACCGG
200
hsa-miR-29c-5p
GACCGA
UCGGUC
CCGGUC

GAUAUU
201
miR-3145-3p
GAUAUU
AAUAUC
AAUAUC

CGCACC
202
hsa-miR-106b-3p
CGCACU
AGUGCG
GGUGCG

GUUCUU
203
hsa-miR-22-5p
GUUCUU
AAGAAC
AAGAAC

GCGGGG
204
miR-744/1716
GCGGGG
CCCCGC
CCCCGC

CCGUGG
205
hsa-miR-132-5p
CCGUGG
CCACGG
CCACGG

UGAAAA
206
miR-488
UGAAAG
CUUUCA
UUUUCA

AUGCAA
207
miR-501-3p/502-3p/500/502a
AUGCAC
GUGCAU
UUGCAU

CCUGUU
208
miR-486-5p/3107
CCUGUA
UACAGG
AACAGG

UUUGCC
209
miR-450a/451a
UUUGCG
CGCAAA
GGCAAA

UGGGAA
210
hsa-miR-30c-3p
UGGGAG
CUCCCA
UUCCCA

UAAGAA
211
miR-499-5p
UAAGAC
GUCUUA
UUCUUA

UCAACC
212
miR-421
UCAACA
UGUUGA
GGUUGA

UCACCC
213
miR-197
UCACCA
UGGUGA
GGGUGA

GGGCCC
214
miR-296-5p
GGGCCC
GGGCCC
GGGCCC

CUCUGG
215
miR-326/330/330-5p
CUCUGG
CCAGAG
CCAGAG

CAGCAA
216
miR-214/761/3619-5p
CAGCAG
CUGCUG
UUGCUG

CUGGGG
217
miR-612/1285/3187-5p
CUGGGC
GCCCAG
CCCCAG

AAUGUU
218
miR-409-3p
AAUGUU
AACAUU
AACAUU

CUGGAA
219
miR-378/422a/378bcdefhi
CUGGAC
GUCCAG
UUCCAG

CUCACC
220
miR-342-3p
CUCACA
UGUGAG
GGUGAG

ACAAUU
221
miR-338-5p
ACAAUA
UAUUGU
AAUUGU

GGGGGG
222
miR-625
GGGGGA
UCCCCC
CCCCCC

AAUACC
223
miR-200bc/429/548a
AAUACU
AGUAUU
GGUAUU

UAGAUU
224
hsa-miR-376a-5p
UAGAUU
AAUCUA
AAUCUA

UAUGGG
225
miR-584
UAUGGU
ACCAUA
CCCAUA

AGUAGG
226
miR-411
AGUAGA
UCUACU
CCUACU

UGAAGG
227
miR-573/3533/3616-5p/3647-5p
UGAAGU
ACUUCA
CCUUCA

CCAUUU
228
miR-885-5p
CCAUUA
UAAUGG
AAAUGG

AAGCUU
229
hsa-miR-99-3p
AAGCUC
GAGCUU
AAGCUU

GGUGGG
230
miR-876-3p
GGUGGU
ACCACC
CCCACC

AUGUCC
231
miR-654-3p
AUGUCU
AGACAU
GGACAU

CCGUCC
232
hsa-miR-340-3p
CCGUCU
AGACGG
GGACGG

CACUUU
233
miR-3614-5p
CACUUG
CAAGUG
AAAGUG

GUGUUU
234
hsa-miR-124-5p
GUGUUC
GAACAC
AAACAC

GUGGGG
235
miR-491-5p
GUGGGG
CCCCAC
CCCCAC

UUGGCC
236
miR-96/507/1271
UUGGCA
UGCCAA
GGCCAA

AAAACC
237
miR-548a-3p/548ef/2285a
AAAACU
AGUUUU
GGUUUU

AAUUUU
238
hsa-miR-32-3p
AAUUUA
UAAAUU
AAAAUU

AGCAAA
239
miR-3942-5p/4703-5p
AGCAAU
AUUGCU
UUUGCU

AGGCAA
240
miR-34b/449c/1360/2682
AGGCAG
CUGCCU
UUGCCU

GGGUUU
241
hsa-miR-23a/b-5p
GGGUUC
GAACCC
AAACCC

AUCCUU
242
miR-362-5p/500b
AUCCUU
AAGGAU
AAGGAU

UCACUU
243
miR-677/4420
UCACUG
CAGUGA
AAGUGA

AGAUAA
244
miR-577
AGAUAA
UUAUCU
UUAUCU

GUUGUU
245
miR-3613-5p
GUUGUA
UACAAC
AACAAC

GAUCGG
246
miR-369-5p
GAUCGA
UCGAUC
CCGAUC

CUCCCC
247
miR-150/5127
CUCCCA
UGGGAG
GGGGAG

UUCUGG
248
miR-544/544ab/544-3p
UUCUGC
GCAGAA
CCAGAA

CUGAUU
249
hsa-miR-29a-5p
CUGAUU
AAUCAG
AAUCAG

CAGGAA
250
miR-873
CAGGAA
UUCCUG
UUCCUG

AGCCUU
251
miR-3614-3p
AGCCUU
AAGGCU
AAGGCU

AAAGAA
252
miR-186
AAAGAA
UUCUUU
UUCUUU

CACUCC
253
miR-483-3p
CACUCC
GGAGUG
GGAGUG

UUAUCC
254
hsa-miR-374a-3p
UUAUCA
UGAUAA
GGAUAA

AGGUAA
255
miR-196abc
AGGUAG
CUACCU
UUACCU

GAUUCC
256
hsa-miR-145-3p
GAUUCC
GGAAUC
GGAAUC

UGGUUU
257
hsa-miR-29b-2-5p
UGGUUU
AAACCA
AAACCA

CCUGGG
258
hsa-miR-221-5p
CCUGGC
GCCAGG
CCCAGG

CCAAUU
259
miR-323b-3p
CCAAUA
UAUUGG
AAUUGG

GUCAUU
260
miR-616
GUCAUU
AAUGAC
AAUGAC

CAAAGG
261
miR-330-3p
CAAAGC
GCUUUG
CCUUUG

AACAAA
262
hsa-miR-7-3p
AACAAA
UUUGUU
UUUGUU

CGUGUU
263
miR-187
CGUGUC
GACACG
AACACG

CUAUUU
264
hsa-miR-26a-3p
CUAUUC
GAAUAG
AAAUAG

ACUGUU
265
miR-452/4676-3p
ACUGUU
AACAGU
AACAGU

UUUUUU
266
miR-129-5p/129ab-5p
UUUUUG
CAAAAA
AAAAAA

GUCAGG
267
miR-223
GUCAGU
ACUGAC
CCUGAC

GCCAGG
268
miR-4755-3p
GCCAGG
CCUGGC
CCUGGC

CCCGUU
269
miR-1247
CCCGUC
GACGGG
AACGGG

AACUAA
270
miR-3129-3p
AACUAA
UUAGUU
UUAGUU

UUUUCC
271
hsa-miR-335-3p
UUUUCA
UGAAAA
GGAAAA

CGGGGG
272
miR-542-5p
CGGGGA
UCCCCG
CCCCCG

CCAUCC
273
hsa-miR-181a-3p
CCAUCG
CGAUGG
GGAUGG

CCCAAA
274
hsa-miR-186-3p
CCCAAA
UUUGGG
UUUGGG

GAGCUU
275
hsa-miR-27b-5p
GAGCUU
AAGCUC
AAGCUC

UUAUGG
276
miR-491-3p
UUAUGC
GCAUAA
CCAUAA

GGCUGG
277
miR-4687-3p
GGCUGU
ACAGCC
CCAGCC

AGUUAA
278
hsa-miR-101-5p
AGUUAU
AUAACU
UUAACU

GAUCAA
279
miR-4772-5p
GAUCAG
CUGAUC
UUGAUC

UCCUAA
280
miR-337-3p
UCCUAU
AUAGGA
UUAGGA

GUGUAA
281
hsa-miR-223-5p
GUGUAU
AUACAC
UUACAC

CAAUAA
282
hsa-miR-16/195-3p
CAAUAU
AUAUUG
UUAUUG

UCGUGG
283
miR-3677-3p
UCGUGG
CCACGA
CCACGA

GGAGGG
284
hsa-miR-766-5p
GGAGGA
UCCUCC
CCCUCC

AUGUGG
285
miR-299/299-3p/3563-3p
AUGUGG
CCACAU
CCACAU

GCUUUU
286
miR-3140-3p
GCUUUU
AAAAGC
AAAAGC

AUGCCC
287
miR-532-5p/511
AUGCCU
AGGCAU
GGGCAU

GCCUAA
288
hsa-miR-24-5p
GCCUAC
GUAGGC
UUAGGC

AUUCUU
289
miR-4778-5p
AUUCUG
CAGAAU
AAGAAU

GACACC
290
miR-642b
GACACA
UGUGUC
GGUGUC

AGACGG
291
miR-483-5p
AGACGG
CCGUCU
CCGUCU

GCACCC
292
miR-767-5p
GCACCA
UGGUGC
GGGUGC

GCUAUU
293
hsa-miR-31-3p
GCUAUG
CAUAGC
AAUAGC

ACGCUU
294
miR-574-3p
ACGCUC
GAGCGU
AAGCGU

AAGGAA
295
miR-3173-3p
AAGGAG
CUCCUU
UUCCUU

GGGAGG
296
miR-2127/4728-5p
GGGAGG
CCUCCC
CCUCCC

GCUUCC
297
hsa-miR-103a-2-5p
GCUUCU
AGAAGC
GGAAGC

AACACC
298
miR-3591-3p
AACACC
GGUGUU
GGUGUU

ACUAUU
299
hsa-miR-625-3p
ACUAUA
UAUAGU
AAUAGU

GAAUCC
300
hsa-miR-15b-3p
GAAUCA
UGAUUC
GGAUUC

AAAUGG
301
miR-522/518e/1422p
AAAUGG
CCAUUU
CCAUUU

AAAAAA
302
miR-548d-3p/548acbz
AAAAAC
GUUUUU
UUUUUU

UCAUCC
303
hsa-miR-452-3p
UCAUCU
AGAUGA
GGAUGA

UGACCC
304
miR-192/215
UGACCU
AGGUCA
GGGUCA

UAGCAA
305
miR-1551/4524
UAGCAG
CUGCUA
UUGCUA

UCGGGG
306
hsa-miR-425-3p
UCGGGA
UCCCGA
CCCCGA

AUCUGG
307
miR-3126-3p
AUCUGG
CCAGAU
CCAGAU

CACAAA
308
hsa-miR-125b-2-3p
CACAAG
CUUGUG
UUUGUG

CUGCCC
309
miR-324-3p/1913
CUGCCC
GGGCAG
GGGCAG

AUCUUU
310
hsa-miR-141-5p
AUCUUC
GAAGAU
AAAGAU

GGGACC
311
hsa-miR-365a/b-5p
GGGACU
AGUCCC
GGUCCC

CUGGUU
312
hsa-miR-29b-1-5p
CUGGUU
AACCAG
AACCAG

GGUUGG
313
miR-563/380-5p
GGUUGA
UCAACC
CCAACC

UUGAGG
314
miR-1304
UUGAGG
CCUCAA
CCUCAA

UCUCUU
315
miR-216c/1461/4684-5p
UCUCUA
UAGAGA
AAGAGA

UUUUAA
316
hsa-miR-2681-5p
UUUUAC
GUAAAA
UUAAAA

GUAACC
317
miR-194
GUAACA
UGUUAC
GGUUAC

AGGGUU
318
miR-296-3p
AGGGUU
AACCCU
AACCCU

AUUUCC
319
hsa-miR-205-3p
AUUUCA
UGAAAU
GGAAAU

ACUCAA
320
miR-888
ACUCAA
UUGAGU
UUGAGU

ACAUGG
321
miR-4802-3p
ACAUGG
CCAUGU
CCAUGU

UGUACC
322
hsa-let-7a/g-3p
UGUACA
UGUACA
GGUACA

GGGCUU
323
miR-762/4492/4498
GGGCUG
CAGCCC
AAGCCC

UGUUGG
324
hsa-miR-744-3p
UGUUGC
GCAACA
CCAACA

AGUUCC
325
hsa-miR-148b-5p
AGUUCU
AGAACU
GGAACU

UUGACC
326
miR-514/514b-3p
UUGACA
UGUCAA
GGUCAA

ACUAGG
327
miR-28-3p
ACUAGA
UCUAGU
CCUAGU

GUGCCC
328
miR-550a
GUGCCU
AGGCAC
GGGCAC

CGGGUU
329
hsa-miR-125b-1-3p
CGGGUU
AACCCG
AACCCG

AUUCAA
330
hsa-miR-506-5p
AUUCAG
CUGAAU
UUGAAU

CACCUU
331
hsa-miR-1306-5p
CACCUC
GAGGUG
AAGGUG

CCUUGG
332
miR-3189-3p
CCUUGG
CCAAGG
CCAAGG

GGUGCC
333
miR-675-5p/4466
GGUGCG
CGCACC
GGCACC

AAUCAA
334
hsa-miR-34a-3p
AAUCAG
CUGAUU
UUGAUU

CCCUAA
335
hsa-miR-454-5p
CCCUAU
AUAGGG
UUAGGG

ACUGCC
336
miR-509-5p/509-3-5p/4418
ACUGCA
UGCAGU
GGCAGU

GUUUUU
337
hsa-miR-19a/b-5p
GUUUUG
CAAAAC
AAAAAC

UUCCCC
338
miR-4755-5p
UUCCCU
AGGGAA
GGGGAA

CUGCUU
339
hsa-miR-93-3p
CUGCUG
CAGCAG
AAGCAG

ACCCAA
340
miR-3130-5p/4482
ACCCAG
CUGGGU
UUGGGU

CCAGAA
341
hsa-miR-488-5p
CCAGAU
AUCUGG
UUCUGG

UCCUGG
342
hsa-miR-378a-5p
UCCUGA
UCAGGA
CCAGGA

AGCCAA
343
miR-575/4676-5p
AGCCAG
CUGGCU
UUGGCU

CUCGGG
344
miR-1307
CUCGGC
GCCGAG
CCCGAG

UUCAGG
345
miR-3942-3p
UUCAGA
UCUGAA
CCUGAA

UGUUCC
346
miR-4677-5p
UGUUCU
AGAACA
GGAACA

GAGCGG
347
miR-339-3p
GAGCGC
GCGCUC
CCGCUC

AAGAAA
348
miR-548b-3p
AAGAAC
GUUCUU
UUUCUU

GUUCCC
349
hsa-miR-642b-5p
GUUCCC
GGGAAC
GGGAAC

AUCCCC
350
miR-188-5p
AUCCCU
AGGGAU
GGGGAU

AACCCC
351
hsa-miR-652-5p
AACCCU
AGGGUU
GGGGUU

AGUCCC
352
miR-2114
AGUCCC
GGGACU
GGGACU

GUGGCC
353
miR-3688-5p
GUGGCA
UGCCAC
GGCCAC

AGGCCC
354
hsa-miR-15a-3p
AGGCCA
UGGCCU
GGGCCU

ACCAUU
355
hsa-miR-181c-3p
ACCAUC
GAUGGU
AAUGGU

GGAGUU
356
miR-122/122a/1352
GGAGUG
CACUCC
AACUCC

UAUUAA
357
miR-556-3p
UAUUAC
GUAAUA
UUAAUA

AUGGUU
358
hsa-miR-218-2-3p
AUGGUU
AACCAU
AACCAU

CUUGUU
359
miR-643
CUUGUA
UACAAG
AACAAG

ACCACC
360
miR-140-3p
ACCACA
UGUGGU
GGUGGU

AGUGAA
361
miR-1245
AGUGAU
AUCACU
UUCACU

AUCAGG
362
hsa-miR-2115-3p
AUCAGA
UCUGAU
CCUGAU

AAAGCC
363
miR-518bcf/518a-3p/518d-3p
AAAGCG
CGCUUU
GGCUUU

ACCUUU
364
miR-3200-3p
ACCUUG
CAAGGU
AAAGGU

CAACAA
365
miR-545/3065/3065-5p
CAACAA
UUGUUG
UUGUUG

CUUCUU
366
miR-1903/4778-3p
CUUCUU
AAGAAG
AAGAAG

CUUAAA
367
hsa-miR-302a-5p
CUUAAA
UUUAAG
UUUAAG

UGAAUU
368
hsa-miR-183-3p
UGAAUU
AAUUCA
AAUUCA

GGGGAA
369
miR-3144-5p
GGGGAC
GUCCCC
UUCCCC

AACUGG
370
miR-582-3p
AACUGG
CCAGUU
CCAGUU

AAGAUU
371
miR-4662a-3p
AAGAUA
UAUCUU
AAUCUU

CCUGAA
372
miR-3140-5p
CCUGAA
UUCAGG
UUCAGG

UGCAAA
373
hsa-miR-106a-3p
UGCAAU
AUUGCA
UUUGCA

AUAGGG
374
hsa-miR-135a-3p
AUAGGG
CCCUAU
CCCUAU

CUGACC
375
miR-345/345-5p
CUGACU
AGUCAG
GGUCAG

CAGGUU
376
miR-125a-3p/1554
CAGGUG
CACCUG
AACCUG

ACUCCC
377
miR-3145-5p
ACUCCA
UGGAGU
GGGAGU

UGUCCC
378
miR-676
UGUCCU
AGGACA
GGGACA

GCCCUU
379
miR-3173-5p
GCCCUG
CAGGGC
AAGGGC

AGAGUU
380
hsa-miR-5586-3p
AGAGUG
CACUCU
AACUCU

CCGAGG
381
miR-615-3p
CCGAGC
GCUCGG
CCUCGG

AUGGAA
382
miR-3688-3p
AUGGAA
UUCCAU
UUCCAU

UAGCCC
383
miR-4662a-5p
UAGCCA
UGGCUA
GGGCUA

UGCCAA
384
miR-4659ab-5p
UGCCAU
AUGGCA
UUGGCA

AUCCAA
385
hsa-miR-5586-5p
AUCCAG
CUGGAU
UUGGAU

ACUCUU
386
hsa-miR-514a-5p
ACUCUG
CAGAGU
AAGAGU

ACCCUU
387
miR-10abc/10a-5p
ACCCUG
CAGGGU
AAGGGU

ACUGAA
388
hsa-miR-888-3p
ACUGAC
GUCAGU
UUCAGU

UCAGGG
389
miR-3127-5p
UCAGGG
CCCUGA
CCCUGA

GAUUGG
390
miR-508-3p
GAUUGU
ACAAUC
CCAAUC

GGGGCC
391
hsa-miR-185-3p
GGGGCU
AGCCCC
GGCCCC

GUCUUU
392
hsa-miR-200c-5p,hsa-miR-550a-3p
GUCUUA
UAAGAC
AAAGAC

UCUCAA
393
miR-513c/514b-5p
UCUCAA
UUGAGA
UUGAGA

AACCUU
394
miR-490-3p
AACCUG
CAGGUU
AAGGUU

CUGAAA
395
hsa-miR-5187-3p
CUGAAU
AUUCAG
UUUCAG

CUCAGG
396
miR-3664-3p
CUCAGG
CCUGAG
CCUGAG

GCCCCC
397
miR-3189-5p
GCCCCA
UGGGGC
GGGGGC

GAAGUU
398
miR-4670-3p
GAAGUU
AACUUC
AACUUC

CAAAUU
399
miR-105/105ab
CAAAUG
CAUUUG
AAUUUG

UGUAGG
400
hsa-miR-135b-3p
UGUAGG
CCUACA
CCUACA

UUUGUU
401
hsa-miR-5010-3p
UUUGUG
CACAAA
AACAAA

GAAGGG
402
miR-493/493b
GAAGGU
ACCUUC
CCCUUC

CUCCGG
403
miR-3605-3p
CUCCGU
ACGGAG
CCGGAG

UCCCAA
404
miR-188-3p
UCCCAC
GUGGGA
UUGGGA

UGCUAA
405
hsa-miR-449c-3p
UGCUAG
CUAGCA
UUAGCA

CAAGGG
406
miR-4761-5p
CAAGGU
ACCUUG
CCCUUG

AAGUCC
407
miR-224
AAGUCA
UGACUU
GGACUU

GUCUAA
408
miR-4796-5p
GUCUAU
AUAGAC
UUAGAC

AAAUCC
409
hsa-miR-551b-5p
AAAUCA
UGAUUU
GGAUUU

AUGAGG
410
miR-556-5p
AUGAGC
GCUCAU
CCUCAU

ACGCCC
411
hsa-miR-122-3p
ACGCCA
UGGCGU
GGGCGU

CUGUGG
412
miR-4677-3p
CUGUGA
UCACAG
CCACAG

UAGAGG
413
miR-877
UAGAGG
CCUCUA
CCUCUA

UUCUAA
414
miR-576-5p
UUCUAA
UUAGAA
UUAGAA

CAUGGG
415
miR-490-5p
CAUGGA
UCCAUG
CCCAUG

CAGAAA
416
hsa-miR-589-3p
CAGAAC
GUUCUG
UUUCUG

GAAGCC
417
miR-4786-3p
GAAGCC
GGCUUC
GGCUUC

UUAGCC
418
hsa-miR-374b-3p
UUAGCA
UGCUAA
GGCUAA

CUGUUU
419
hsa-miR-26b-3p
CUGUUC
GAACAG
AAACAG

AGGGCC
420
miR-3158-3p
AGGGCU
AGCCCU
GGCCCU

UAGGCC
421
miR-4423-3p
UAGGCA
UGCCUA
GGCCUA

UCUAGG
422
miR-518d-5p/519bc-5p520c-5p/523b/526a
UCUAGA
UCUAGA
CCUAGA

GCCCGG
423
miR-4707-3p
GCCCGC
GCGGGC
CCGGGC

AAAUUU
424
hsa-miR-10a-3p
AAAUUC
GAAUUU
AAAUUU

UCUUGG
425
miR-526b
UCUUGA
UCAAGA
CCAAGA

CUUCAA
426
hsa-miR-676-5p
CUUCAA
UUGAAG
UUGAAG

CCUCCC
427
hsa-miR-660-3p
CCUCCU
AGGAGG
GGGAGG

UUGGAA
428
hsa-miR-5004-3p
UUGGAU
AUCCAA
UUCCAA

GGGUCC
429
miR-193a-5p
GGGUCU
AGACCC
GGACCC

UCAGUU
430
hsa-miR-222-5p
UCAGUA
UACUGA
AACUGA

AGGAUU
431
miR-4661-3p
AGGAUC
GAUCCU
AAUCCU

GGCGGG
432
hsa-miR-25-5p
GGCGGA
UCCGCC
CCCGCC

AGCGAA
433
miR-4670-5p
AGCGAC
GUCGCU
UUCGCU

UUGGUU
434
miR-659
UUGGUU
AACCAA
AACCAA

GCUCUU
435
miR-1745/3194-3p
GCUCUG
CAGAGC
AAGAGC

GGUUCC
436
hsa-miR-182-3p
GGUUCU
AGAACC
GGAACC

GCAGAA
437
miR-298/2347/2467-3p
GCAGAG
CUCUGC
UUCUGC

CUCUUU
438
hsa-miR-130b-5p
CUCUUU
AAAGAG
AAAGAG

GCGGUU
439
miR-4746-3p
GCGGUG
CACCGC
AACCGC

GCGCGG
440
miR-1893/2277-5p
GCGCGG
CCGCGC
CCGCGC

GGACCC
441
miR-3619-3p
GGACCA
UGGUCC
GGGUCC

CUACUU
442
hsa-miR-138-1-3p
CUACUU
AAGUAG
AAGUAG

AUGCUU
443
miR-4728-3p
AUGCUG
CAGCAU
AAGCAU

CCCCUU
444
miR-3127-3p
CCCCUU
AAGGGG
AAGGGG

CCGGUU
445
miR-671-3p
CCGGUU
AACCGG
AACCGG

CAGGGG
446
hsa-miR-211-3p
CAGGGA
UCCCUG
CCCCUG

GAGCCC
447
hsa-miR-2114-3p
GAGCCU
AGGCUC
GGGCUC

CCUCUU
448
hsa-miR-877-3p
CCUCUU
AAGAGG
AAGAGG

UCAGCC
449
miR-3157-5p
UCAGCC
GGCUGA
GGCUGA

UCCUUU
450
miR-502-5p
UCCUUG
CAAGGA
AAAGGA

AAUCCC
451
miR-500a
AAUCCU
AGGAUU
GGGAUU

AAACUU
452
m iR-548g
AAACUG
CAGUUU
AAGUUU

AACGCC
453
miR-523
AACGCG
CGCGUU
GGCGUU

CAGUUU
454
hsa-miR-584-3p
CAGUUC
GAACUG
AAACUG

CCUUCC
455
miR-205/205ab
CCUUCA
UGAAGG
GGAAGG

CAUCCC
456
miR-4793-5p
CAUCCU
AGGAUG
GGGAUG

GGGUGG
457
hsa-miR-363-5p
GGGUGG
CCACCC
CCACCC

GCCUGG
458
hsa-miR-214-5p
GCCUGU
ACAGGC
CCAGGC

UUCCAA
459
miR-3180-5p
UUCCAG
CUGGAA
UUGGAA

UGGGGG
460
miR-1404/2110
UGGGGA
UCCCCA
CCCCCA

UGCCCC
461
miR-3157-3p
UGCCCU
AGGGCA
GGGGCA

CUGCGG
462
hsa-miR-191-3p
CUGCGC
GCGCAG
CCGCAG

UGGGUU
463
miR-1346/3940-5p/4507
UGGGUU
AACCCA
AACCCA

CGGUCC
464
miR-4746-5p
CGGUCC
GGACCG
GGACCG

ACGCGG
465
miR-3939
ACGCGC
GCGCGU
CCGCGU

CCACUU
466
hsa-miR-181a-2-3p
CCACUG
CAGUGG
AAGUGG

UGCACC
467
hsa-miR-500a-3p
UGCACC
GGUGCA
GGUGCA

CGACAA
468
hsa-miR-196b-3p
CGACAG
CUGUCG
UUGUCG

UGUAUU
469
hsa-miR-675-3p
UGUAUG
CAUACA
AAUACA

GCAAAA
470
hsa-miR-548aj/g/x-5p
GCAAAA
UUUUGC
UUUUGC

UUCUUU
471
miR-4659ab-3p
UUCUUC
GAAGAA
AAAGAA

UCUGCC
472
hsa-miR-5001-3p
UCUGCC
GGCAGA
GGCAGA

CCCGGG
473
hsa-miR-1247-3p
CCCGGG
CCCGGG
CCCGGG

CCCCGG
474
miR-2890/4707-5p
CCCCGG
CCGGGG
CCGGGG

UGGUAA
475
hsa-miR-150-3p
UGGUAC
GUACCA
UUACCA

UUCUCC
476
hsa-miR-629-3p
UUCUCC
GGAGAA
GGAGAA

GACAGG
477
miR-2277-3p
GACAGC
GCUGUC
CCUGUC

GAGCAA
478
miR-3547/3663-3p
GAGCAC
GUGCUC
UUGCUC

AUCACC
479
miR-34bc-3p
AUCACU
AGUGAU
GGUGAU

AAGCGG
480
miR-518ef
AAGCGC
GCGCUU
CCGCUU

UGGCCC
481
miR-3187-3p
UGGCCA
UGGCCA
GGGCCA

CGUUGG
482
miR-1306/1306-3p
CGUUGG
CCAACG
CCAACG

GCACGG
483
miR-3177-3p
GCACGG
CCGUGC
CCGUGC

GGAAUU
484
miR-1ab/206/613
GGAAUG
CAUUCC
AAUUCC

CACAGG
485
miR-128/128ab
CACAGU
ACUGUG
CCUGUG

UAGGGG
486
miR-1296
UAGGGC
GCCCUA
CCCCUA

ACGUCC
487
miR-598/598-3p
ACGUCA
UGACGU
GGACGU

UGAACC
488
miR-887
UGAACG
CGUUCA
GGUUCA

CAUACC
489
miR-1-5p
CAUACU
AGUAUG
GGUAUG

ACAUAA
490
miR-376c/741-5p
ACAUAG
CUAUGU
UUAUGU

UAAUAA
491
miR-374c/655
UAAUAC
GUAUUA
UUAUUA

GAAACC
492
miR-494
GAAACA
UGUUUC
GGUUUC

UUAGGG
493
miR-651
UUAGGA
UCCUAA
CCCUAA

UGCAGG
494
miR-1301/5047
UGCAGC
GCUGCA
CCUGCA

GCGAGG
495
miR-381-5p
GCGAGG
CCUCGC
CCUCGC

AAUCUU
496
miR-216a
AAUCUC
GAGAUU
AAGAUU

AUACAA
497
miR-300/381/539-3p
AUACAA
UUGUAU
UUGUAU

CGCCCC
498
miR-1249
CGCCCU
AGGGCG
GGGGCG

UCAUUU
499
miR-579
UCAUUU
AAAUGA
AAAUGA

AUAUUU
500
miR-656
AUAUUA
UAAUAU
AAAUAU

UCAUGG
501
miR-433
UCAUGA
UCAUGA
CCAUGA

UUCCGG
502
miR-1180
UUCCGG
CCGGAA
CCGGAA

GUGUCC
503
miR-597/1970
GUGUCA
UGACAC
GGACAC

UAUAUU
504
miR-190a-3p
UAUAUA
UAUAUA
AAUAUA

AAACCC
505
miR-1537
AAACCG
CGGUUU
GGGUUU

GGCCCC
506
miR-874-5p
GGCCCC
GGGGCC
GGGGCC

AUAUAA
507
miR-410/344de/344b-1-3p
AUAUAA
UUAUAU
UUAUAU

CCUGCC
508
miR-370
CCUGCU
AGCAGG
GGCAGG

GAAUUU
509
miR-219-2-3p/219-3p
GAAUUG
CAAUUC
AAAUUC

CACCCC
510
miR-3620
CACCCU
AGGGUG
GGGGUG

GACCCC
511
miR-504/4725-5p
GACCCU
AGGGUC
GGGGUC

GAUGUU
512
miR-2964/2964a-5p
GAUGUC
GACAUC
AACAUC

UUGGGG
513
miR-450a-2-3p
UUGGGG
CCCCAA
CCCCAA

UGUCUU
514
miR-511
UGUCUU
AAGACA
AAGACA

GACUUU
515
miR-6505-3p
GACUUC
GAAGUC
AAAGUC

ACGGUU
516
miR-433-5p
ACGGUG
CACCGU
AACCGU

CGGCUU
517
miR-6741-3p
CGGCUC
GAGCCG
AAGCCG

AGGUCC
518
miR-370-5p
AGGUCA
UGACCU
GGACCU

CGCGGG
519
miR-579-5p
CGCGGU
ACCGCG
CCCGCG

GUGGAA
520
miR-376c-5p,miR-376b-5p
GUGGAU
AUCCAC
UUCCAC

ACAGGG
521
miR-552/3097-5p
ACAGGU
ACCUGU
CCCUGU

CAGUCC
522
miR-1910
CAGUCC
GGACUG
GGACUG

UUGUGG
523
miR-758
UUGUGA
UCACAA
CCACAA

GGCCUU
524
miR-6735-3p
GGCCUG
CAGGCC
AAGGCC

GUAGAA
525
miR-376a-2-5p
GUAGAU
AUCUAC
UUCUAC

GGGCGG
526
miR-585
GGGCGU
ACGCCC
CCGCCC

AACCGG
527
miR-451
AACCGU
ACGGUU
CCGGUU

UAUUGG
528
miR-137/137ab
UAUUGC
GCAAUA
CCAAUA

Through the Ago HITS-CLIP assay in the above example, miRNA binding to a non-canonical bulge target in several tissue cells was identified, and when the present invention for modifying miRNA to recognize a non-canonical nucleation bulge site as a canonical seed site is applied, a total of 426 sequences (BS sequences) were prepared, and therefore, technology for exhibiting only the function of suppressing the non-canonical target of miRNA was completed.

EXAMPLE 25
Analysis of Sequence Variation in miRNA Sequence Database (TCGA) of Cancer Patients, and Thereby Identifying miRNA Binding to Non-Canonical G:A Wobble Seed Site

According to the above examples, it was confirmed that miRNA binds to a non-canonically G:A wobble site, thereby inhibiting the expression of a target gene, and therefore, in the present invention, a miRNA sequence modification method (miRNA-BS) of modifying a sequence to recognize a non-canonical G:A wobble site as a canonical seed site was developed. If a miRNA function is changed through wobble pairing between G in the conventional miRNA seed sequence and A of target mRNA, based on the idea that such a mechanism can be shown in a disease such as a tumor through sequence variation of miRNA, a result of sequencing miRNA from tissue of a cancer patient was analyzed (miRNA-Seq). Here, as 8,105 sequences were obtained from the miRNA sequencing-related TCGA database of cancer patients, and additionally, 468 sequences were obtained through cancer-related miRNA sequencing from the Gene Expression Omnibus (GEO) database, a total of 8,573 tumor miRNA sequencing results obtained thereby were mapped using the bowtie2 program in the same manner as in Example 10, and then sites with variations were analyzed. At this time, the miRNA sequencing data used in the analysis for each cancer type is as shown in FIG. 25A.

As a result of examining the distribution of the most frequent variations (top 10,000) and the least variations (bottom 10,000) by classifying variation fractions by position based on the 5′ end of miRNA (FIG. 25B), it was possible to observe that the most frequent variations (top 10,000) mainly occur in the seed region (between the 2′ to the 8^thbases) of miRNA. In addition, examining the most frequent variations (top 100) and the least variations (bottom 100) on a heatmap (FIG. 25C), it was able to be seen that the variations are concentrated in the seed region as in the examples above. Examining the sequencing result with DNA through reverse transcription of the miRNA by analyzing it with A, T, C and G, the sequence variation in the tumor miRNA which tends to be shown in the seed region was detected at only G in the seed sequence (FIG. 25D). Such tendency was also confirmed from that, in addition to top 100 variations, all variations mainly occur at G (FIG. 25E).

This may be a phenomenon in which G of miRNA recognizing the non-canonical G:A wobble noted by the inventors is changed to U to more tightly bind to A of the target mRNA on the opposite side, and as a result of analyzing which bases Gs, which show the higher variation rate, were changed to (FIG. 25F), in most of the corresponding results in which the miRNA is sequenced with DNA through reverse transcription, it was observed that G changes to T. This result indicates that miRNA pairs with A in a target by changing its G to U for G present in the seed region to well recognize a non-canonical G:A wobble seed target as a canonical seed in actual cancer tissue, and therefore, this type of variation indicates how to apply the sequencing method that allows recognition of a non-canonical G:A wobble seed sequence as a canonical seed sequence, developed in the present invention, to which G at which position of which miRNA. Accordingly, putting all of the results together, miRNA with a normalized variation frequency per cancer patient of 2 or more was selected (see Table 4). As shown in Table 4, a total of 335 sequences (sequence (G>U)) were designed to have variations from the 2^ndnucleotide based on the 5′ end of an RNA interference nucleic acid, and the modified RNA interference nucleic acids will only interact with a non-canonical G:A bulge target for the corresponding miRNA and exhibit only a corresponding function.

TABLE 4

Sequence
SEQ

G > T

(G > U)
ID NOs
miRNA name
Seed
Position
read/patient

UGAAUG
529
hsa-miR-1-3p
GGAAUG
2
17.1

UUAACA
530
hsa-miR-194-5p
GUAACA
2
96.3

UGGUCU
531
hsa-miR-193a-5p
GGGUCU
2
12

UAAUCA
532
hsa-miR-15b-3p
GAAUCA
2
2.2

UUCUUA
533
hsa-miR-200c-5p
GUCUUA
2
4.2

UCCUGU
534
hsa-miR-214-5p
GCCUGU
2
2.8

UUGACU
535
hsa-miR-134-5p
GUGACU
2
10.3

UAUUCC
536
hsa-miR-145-3p
GAUUCC
2
2.2

UUUCUU
537
hsa-miR-22-5p
GUUCUU
2
2.5

UCUCGG
538
hsa-miR-423-3p
GCUCGG
2
6.3

UAGACU
539
hsa-miR-873-3p
GAGACU
2
4.5

UGAGUG
540
hsa-miR-122-5p
GGAGUG
2
837.8

UAGAUG
541
hsa-miR-143-3p
GAGAUG
2
322.7

UAGGCU
542
hsa-miR-485-5p
GAGGCU
2
2.2

UGUUAC
543
hsa-miR-409-5p
GGUUAC
2
8.1

UGCUCA
544
hsa-miR-24-3p
GGCUCA
2
65.6

UUCAGU
545
hsa-miR-223-3p
GUCAGU
2
12.7

UAUAUC
546
hsa-miR-144-5p
GAUAUC
2
16.4

UGUAGA
547
hsa-miR-379-5p
GGUAGA
2
47.2

UAGAAC
548
hsa-miR-146b-5p/hsa-miR-146a-5p
GAGAAC
2
21.2

UAGAAA
549
hsa-miR-539-5p
GAGAAA
2
3.3

UGGCCC
550
hsa-miR-296-5p
GGGCCC
2
2.7

UCACCA
551
hsa-miR-767-5p
GCACCA
2
10.1

UGCAGU
552
hsa-miR-34a-5p/hsa-miR-34c-5p
GGCAGU
2
8.9

UAGGUA
553
hsa-let-7f-5p/hsa-let-7d-5p/hsa-let-7b-5p/hsa-
GAGGUA
2
320.1

let-7a-5p/hsa-let-7e-5p/hsa-miR-202-3p/hsa-

let-71-5p/hsa-miR-98-5p/hsa-let-7c-5p/hsa-let-

7g-5p

UUGCCU
554
hsa-miR-1271-3p
GUGCCU
2
2

UCUGGU
555
hsa-miR-138-5p
GCUGGU
2
5.9

UUGCAA
556
hsa-miR-19b-3p/hsa-miR-19a-3p
GUGCAA
2
4.1

UGGCUU
557
hsa-miR-27a-5p
GGGCUU
2
2.2

UCCCUG
558
hsa-miR-146b-3p
GCCCUG
2
7.6

UGAAGA
559
hsa-miR-7-5p
GGAAGA
2
2.3

UAGGGG
560
hsa-miR-423-5p
GAGGGG
2
3.7

UCAUCC
561
hsa-miR-324-5p
GCAUCC
2
2.6

UGGUUU
562
hsa-miR-629-5p
GGGUUU
2
3.3

UGAGAC
563
hsa-miR-139-3p
GGAGAC
2
2.3

UUAAAC
564
hsa-miR-30d-5p/hsa-miR-30e-5p/hsa-miR-
GUAAAC
2
1004.2

30a-5p/hsa-miR-30c-5p/hsa-miR-30b-5p

UCUACA
565
hsa-miR-221-3p/hsa-miR-222-3p
GCUACA
2
6.3

UAUUGG
566
hsa-miR-509-3p
GAUUGG
2
54

UAGACC
567
hsa-miR-769-5p
GAGACC
2
3.9

UUAGUG
568
hsa-miR-142-3p
GUAGUG
2
5.2

UGAGAG
569
hsa-miR-185-5p
GGAGAG
2
8.9

UAUUGU
570
hsa-miR-508-3p/hsa-miR-219a-5p
GAUUGU
2
211.9

UGCAAG
571
hsa-miR-31-5p
GGCAAG
2
4.2

UCAGCA
572
hsa-miR-103a-3p/hsa-miR-107
GCAGCA
2
757.3

UUGACA
573
hsa-miR-542-3p
GUGACA
2
7.3

UAAUUG
574
hsa-miR-219a-2-3p
GAAUUG
2
29.7

AUCACC
575
hsa-miR-29c-3p/hsa-miR-29a-3p/hsa-miR-29b-3p
AGCACC
3
11

CUGGUU
576
hsa-miR-125b-1-3p
CGGGUU
3
8.3

GUAAGA
577
hsa-miR-7-5p
GGAAGA
3
2.3

AUUAGA
578
hsa-miR-411-5p
AGUAGA
3
2.7

AUGUAG
579
hsa-miR-196a-5p/hsa-miR-196b-5p
AGGUAG
3
3.3

AUGCAC
580
hsa-miR-3622a-5p
AGGCAC
3
2

UUAAGC
581
hsa-miR-127-5p
UGAAGC
3
4.6

AUCUGC
582
hsa-miR-22-3p
AGCUGC
3
335.7

UUCAUA
583
hsa-miR-153-3p
UGCAUA
3
3.1

GUGUCU
584
hsa-miR-193a-5p
GGGUCU
3
5.8

GUAGAG
585
hsa-miR-185-5p
GGAGAG
3
3.1

GUAAUG
586
hsa-miR-1-3p
GGAAUG
3
10.2

AUCAGC
587
hsa-miR-15b-5p/hsa-miR-16-5p/hsa-miR-424-5p
AGCAGC
3
5.6

UUUACA
588
hsa-let-7g-3p/hsa-miR-493-5p/hsa-let-7c-3p
UGUACA
3
2.7

UUCGCA
589
hsa-let-7i-3p
UGCGCA
3
2.4

UUUGCU
590
hsa-miR-218-5p
UGUGCU
3
3.6

CUACCG
591
hsa-miR-1307-5p
CGACCG
3
3.3

CUGAUC
592
hsa-miR-127-3p
CGGAUC
3
10.5

UUUGCG
593
hsa-miR-210-3p
UGUGCG
3
13.6

CUUGUC
594
hsa-miR-187-3p
CGUGUC
3
2.1

UUCCAA
595
hsa-miR-192-3p
UGCCAA
3
2.2

UUACCU
596
hsa-miR-192-5p
UGACCU
3
73.2

GUCAGU
597
hsa-miR-34a-5p/hsa-miR-34c-5p
GGCAGU
3
3.1

AUCUUA
598
hsa-miR-21-5p
AGCUUA
3
738.6

UUCACC
599
hsa-miR-500a-3p
UGCACC
3
2.7

GUGUUU
600
hsa-miR-629-5p
GGGUUU
3
2.9

GUUAGA
601
hsa-miR-379-5p
GGUAGA
3
22.1

UUAAAU
602
hsa-miR-203a-3p
UGAAAU
3
200.1

GUCUCA
603
hsa-miR-24-3p
GGCUCA
3
10.5

UUGGAG
604
hsa-miR-30c-2-3p
UGGGAG
3
6.2

UUAAAG
605
hsa-miR-488-3p
UGAAAG
3
2

AUUGCA
606
hsa-miR-301a-3p/hsa-miR-301b-3p
AGUGCA
3
5

CUUACC
607
hsa-miR-126-3p
CGUACC
3
15.8

GUAGUG
608
hsa-miR-122-5p
GGAGUG
3
60.4

GUGCUU
609
hsa-miR-27a-5p
GGGCUU
3
2.8

CUUUUC
610
hsa-miR-26b-3p
CUGUUC
4
3

CUUCCC
611
hsa-miR-324-3p
CUGCCC
4
2.3

CAUCAC
612
hsa-miR-3065-3p
CAGCAC
4
4

GAUGGG
613
hsa-miR-423-5p
GAGGGG
4
11.4

GUUUUC
614
hsa-miR-124-5p
GUGUUC
4
3.3

CUUACU
615
hsa-miR-345-5p
CUGACU
4
6

CCUAGC
616
hsa-miR-615-3p
CCGAGC
4
2

AUUGCU
617
hsa-miR-889-5p/hsa-miR-135a-5p/hsa-miR-
AUGGCU
4
15.8

135b-5p

GGUUCU
618
hsa-miR-193a-5p
GGGUCU
4
35.4

AAUGUG
619
hsa-miR-18a-5p
AAGGUG
4
3.3

CGUGUU
620
hsa-miR-125b-1-3p
CGGGUU
4
57

AGUAGC
621
hsa-miR-708-5p/hsa-miR-28-5p
AGGAGC
4
10.5

AAUUCA
622
hsa-miR-224-5p
AAGUCA
4
2.9

AAUCUU
623
hsa-miR-100-3p
AAGCUU
4
9.5

CAUGAA
624
hsa-miR-873-5p
CAGGAA
4
6.7

UAUCCA
625
hsa-miR-4662a-5p
UAGCCA
4
7.5

AAUCUC
626
hsa-miR-99b-3p/hsa-miR-99a-3p
AAGCUC
4
9.3

ACUGUG
627
hsa-miR-433-5p
ACGGUG
4
2.3

GUUACA
628
hsa-miR-542-3p
GUGACA
4
20.9

GAUGAU
629
hsa-miR-3605-5p
GAGGAU
4
11.8

GCUGGG
630
hsa-miR-744-5p
GCGGGG
4
4.2

UAUGGC
631
hsa-miR-1296-5p
UAGGGC
4
26.2

UUUGUC
632
hsa-miR-133a-3p
UUGGUC
4
11.7

AAUUUG
633
hsa-miR-382-5p
AAGUUG
4
6.8

AUUACA
634
hsa-miR-425-5p
AUGACA
4
20.9

GAUGUU
635
hsa-miR-377-5p
GAGGUU
4
2.2

CGUAUC
636
hsa-miR-127-3p
CGGAUC
4
219.1

GGUGCG
637
hsa-miR-3180-3p
GGGGCG
4
2.4

GAUAUG
638
hsa-miR-143-3p
GAGAUG
4
936.3

UUUUGA
639
hsa-miR-758-3p
UUGUGA
4
4.9

CUUCUG
640
hsa-miR-93-3p
CUGCUG
4
2.9

GGUGCC
641
hsa-miR-128-2-5p
GGGGCC
4
4.2

GUUACU
642
hsa-miR-134-5p
GUGACU
4
29.2

AGUUUA
643
hsa-miR-154-5p
AGGUUA
4
3.5

AGUCAC
644
hsa-miR-3622a-5p
AGGCAC
4
5.1

AAUGCA
645
hsa-miR-124-3p
AAGGCA
4
38.7

GGUCUU
646
hsa-miR-27a-5p
GGGCUU
4
5

CAUUGG
647
hsa-miR-194-3p
CAGUGG
4
8.7

UUUUUC
648
hsa-miR-375
UUGUUC
4
1084.1

AAUUUC
649
hsa-miR-148a-5p
AAGUUC
4
4.4

GCUCGG
650
hsa-miR-2277-5p
GCGCGG
4
2.9

GAUACC
651
hsa-miR-769-5p
GAGACC
4
26.3

CUUCAG
652
hsa-miR-17-3p
CUGCAG
4
52.2

GAUACU
653
hsa-miR-873-3p
GAGACU
4
9.9

CUUCAA
654
hsa-miR-4772-3p
CUGCAA
4
2.3

AGUUUU
655
hsa-miR-329-5p
AGGUUU
4
2.1

UUUGCA
656
hsa-miR-182-5p/hsa-miR-96-5p
UUGGCA
4
598.4

GAUGCU
657
hsa-miR-2467-5p/hsa-miR-485-5p
GAGGCU
4
12.7

CUUGCU
658
hsa-miR-149-5p
CUGGCU
4
11.8

UGUUUU
659
hsa-miR-29b-2-5p
UGGUUU
4
4.8

ACUCCA
660
hsa-miR-122-3p
ACGCCA
4
5

AAUUGC
661
hsa-miR-302a-3p/hsa-miR-520a-3p/hsa-miR-
AAGUGC
4
29.3

519b-3p/hsa-miR-520b/hsa-miR-519c-3p/hsa-

miR-520c-3p/hsa-miR-519a-3p

AUUCCU
662
hsa-miR-532-5p
AUGCCU
4
194.9

CCUUGG
663
hsa-miR-132-5p
CCGUGG
4
2.3

AAUGAU
664
hsa-miR-541-5p
AAGGAU
4
2.8

CCUGUU
665
hsa-miR-671-3p
CCGGUU
4
3.6

GGUCCC
666
hsa-miR-296-5p
GGGCCC
4
3

AAUCGC
667
hsa-miR-518e-3p
AAGCGC
4
10.8

UGUUUA
668
hsa-miR-487a-5p
UGGUUA
4
2.5

GAUAAC
669
hsa-miR-589-5p/hsa-miR-146b-5p/hsa-miR-
GAGAAC
4
60.3

146a-5p

AGUUAG
670
hsa-miR-196b-5p/hsa-miR-196a-5p
AGGUAG
4
34.4

GGUGCA
671
hsa-miR-486-3p
GGGGCA
4
2

GGUUUU
672
hsa-miR-629-5p
GGGUUU
4
8.7

CUUGAC
673
hsa-miR-378a-3p
CUGGAC
4
105.2

GAUCUU
674
hsa-miR-27b-5p
GAGCUU
4
3.2

GCUCCU
675
hsa-miR-6720-3p
GCGCCU
4
2.3

ACUCUC
676
hsa-miR-574-3p
ACGCUC
4
16.6

CUUAUU
677
hsa-miR-29a-5p
CUGAUU
4
2.6

UGUGAG
678
hsa-miR-30c-2-3p/hsa-miR-30c-1-3p
UGGGAG
4
22.2

CAUUAG
679
hsa-miR-199b-3p
CAGUAG
4
32.1

GAUUGU
680
hsa-miR-574-5p
GAGUGU
4
3.4

GAUAAA
681
hsa-miR-539-5p
GAGAAA
4
4.7

CUUUGA
682
hsa-miR-4677-3p
CUGUGA
4
3.1

AUUUCU
683
hsa-miR-654-3p
AUGUCU
4
2.9

AUUGCG
684
hsa-miR-652-3p
AUGGCG
4
7.8

GUUCAA
685
hsa-miR-19a-3p/hsa-miR-19b-3p
GUGCAA
4
16.9

GAUGUA
686
hsa-let-7c-5p/hsa-miR-98-5p/hsa-let-7g-
GAGGUA
4
1300.2

5p/hsa-let-7f-5p/hsa-miR-202-3p/hsa-let-7b-

5p/hsa-let-7e-5p/hsa-let-7a-5p/hsa-let-7d-

5p/hsa-let-7i-5p

GAUCAC
687
hsa-miR-3663-3p
GAGCAC
4
11.7

CAUUGC
688
hsa-miR-152-3p/hsa-miR-148b-3p/hsa-miR-
CAGUGC
4
1529.4

148a-3p

GGUGUU
689
hsa-miR-193b-5p
GGGGUU
4
2.3

AUUCAC
690
hsa-miR-502-3p/hsa-miR-501-3p
AUGCAC
4
5.5

AUUUGG
691
hsa-miR-299-3p
AUGUGG
4
4.5

AGUUGU
692
hsa-miR-140-5p
AGUGGU
5
4.4

UUGUCA
693
hsa-miR-96-5p/hsa-miR-182-5p
UUGGCA
5
25.1

ACUUGC
694
hsa-miR-193b-3p
ACUGGC
5
6.9

AAUUCC
695
hsa-miR-365a-3p
AAUGCC
5
4.9

CCUUUA
696
hsa-miR-486-5p
CCUGUA
5
7.4

CGGUUU
697
hsa-miR-125b-1-3p
CGGGUU
5
3.9

UGUUCG
698
hsa-miR-210-3p
UGUGCG
5
11.7

GAAUGU
699
hsa-miR-493-3p
GAAGGU
5
2.8

AAAUUA
700
hsa-miR-548am-5p
AAAGUA
5
3

UGUUCU
701
hsa-miR-218-5p
UGUGCU
5
4.4

AAAUUG
702
hsa-miR-20b-5p/hsa-miR-20a-5p/hsa-miR-93-
AAAGUG
5
24.7

5p/hsa-miR-17-5p/hsa-miR-106b-5p

GGUUGG
703
hsa-miR-541-3p
GGUGGG
5
3.8

ACUUUU
704
hsa-miR-452-5p
ACUGUU
5
2.4

CCUUGC
705
hsa-miR-221-5p
CCUGGC
5
6.3

AAAUCG
706
hsa-miR-518f-3p
AAAGCG
5
2

CCUUCU
707
hsa-miR-370-3p
CCUGCU
5
6.5

GCAUCA
708
hsa-miR-107/hsa-miR-103a-3p
GCAGCA
5
57.4

GGAUUG
709
hsa-miR-122-5p
GGAGUG
5
96.6

CCAUCA
710
hsa-miR-338-3p
CCAGCA
5
7.3

AAUUUU
711
hsa-miR-409-3p
AAUGUU
5
7.8

AAGUCA
712
hsa-miR-124-3p
AAGGCA
5
2.7

GAGUUA
713
hsa-let-7d-5p/hsa-let-7g-5p/hsa-let-7i-5p/hsa-
GAGGUA
5
23.9

let-7f-5p/hsa-let-7e-5p/hsa-let-7a-5p/hsa-let-

7b-5p/hsa-let-7c-5p

AGUUCA
714
hsa-miR-130b-3p/hsa-miR-301a-3p/hsa-miR-
AGUGCA
5
4

130a-3p/hsa-miR-301b-3p

AGUUCU
715
hsa-miR-512-3p
AGUGCU
5
2.7

AACUGA
716
hsa-miR-191-5p
AACGGA
5
6.8

ACUUCA
717
hsa-miR-509-3-5p
ACUGCA
5
38.2

AUUUCA
718
hsa-miR-92a-3p/hsa-miR-92b-3p/hsa-miR-
AUUGCA
5
41

363-3p/hsa-miR-25-3p/hsa-miR-32-5p

AAGUUG
719
hsa-miR-18a-5p
AAGGUG
5
7

AUGUCA
720
hsa-miR-183-5p
AUGGCA
5
8.1

GCUUGU
721
hsa-miR-138-5p
GCUGGU
5
2.8

CUCUGC
722
hsa-miR-1307-3p
CUCGGC
5
2.1

GAGUGG
723
hsa-miR-423-5p
GAGGGG
5
3.5

UAAUAC
724
hsa-miR-499a-5p/hsa-miR-208a-3p
UAAGAC
5
4.5

CUGUAC
725
hsa-miR-378a-3p
CUGGAC
5
8.8

AAAUAA
726
hsa-miR-186-5p
AAAGAA
5
3.9

UUUUCA
727
hsa-miR-450b-5p
UUUGCA
5
2.3

UUUUCG
728
hsa-miR-450a-5p
UUUGCG
5
5.1

GUAUUG
729
hsa-miR-142-3p
GUAGUG
5
2.5

ACAUUA
730
hsa-miR-101-3p/hsa-miR-144-3p
ACAGUA
5
8.9

AAAUCU
731
hsa-miR-320a
AAAGCU
5
6.6

CCAUUG
732
hsa-miR-199b-5p/hsa-miR-199a-5p
CCAGUG
5
9.2

UAGUGC
733
hsa-miR-1296-5p
UAGGGC
5
2.9

GGAUAG
734
hsa-miR-185-5p
GGAGAG
5
2.6

AUGUCU
735
hsa-miR-135a-5p
AUGGCU
5
3.1

AGUAUA
736
hsa-miR-411-5p
AGUAGA
6
2.5

UCCAUU
737
hsa-miR-145-5p
UCCAGU
6
4.2

UCAAUU
738
hsa-miR-26a-5p
UCAAGU
6
6.1

ACUGUC
739
hsa-miR-193b-3p
ACUGGC
6
2.1

GGCAUU
740
hsa-miR-34c-5p
GGCAGU
6
7.1

GGUAUA
741
hsa-miR-379-5p
GGUAGA
6
2.1

CCCUUA
742
hsa-miR-125b-5p
CCCUGA
6
3.8

CCUGUC
743
hsa-miR-221-5p
CCUGGC
6
3.1

UCUUUA
744
hsa-miR-526b-5p
UCUUGA
6
4.4

GGAAUA
745
hsa-miR-7-5p
GGAAGA
6
4.4

AGCAUC
746
hsa-miR-16-5p/hsa-miR-15b-5p/hsa-miR-424-
AGCAGC
6
4.7

5p/hsa-miR-15a-5p

UAAAUC
747
hsa-miR-9-3p
UAAAGC
6
4.6

GGGUUG
748
hsa-miR-363-5p
GGGUGG
6
3

AUCUUG
749
hsa-miR-1298-3p
AUCUGG
6
11

GAUUUG
750
hsa-miR-509-3p
GAUUGG
6
4.4

GCGGUG
751
hsa-miR-744-5p
GCGGGG
6
2.1

CAGUUC
752
hsa-miR-148a-3p
CAGUGC
6
36.8

AAGUUC
753
hsa-miR-302a-3p
AAGUGC
6
9.8

UAGGUC
754
hsa-miR-1296-5p
UAGGGC
6
3.3

GAGGUG
755
hsa-miR-423-5p
GAGGGG
6
3.7

CUUUUG
756
hsa-miR-9-5p
CUUUGG
6
40

GCUGUU
757
hsa-miR-138-5p
GCUGGU
6
3.7

AGCUUC
758
hsa-miR-22-3p
AGCUGC
6
47

ACUAUA
759
hsa-miR-28-3p
ACUAGA
6
3.4

GAUUUU
760
hsa-miR-508-3p
GAUUGU
6
8.4

UAUUUC
761
hsa-miR-137
UAUUGC
6
7.2

GGGGUA
762
hsa-miR-5010-5p
GGGGGA
6
2

UCUAUA
763
hsa-miR-523-5p
UCUAGA
6
2.5

AACGUA
764
hsa-miR-191-5p
AACGGA
6
5

CACAUU
765
hsa-miR-128-3p
CACAGU
6
2.8

CCAGUU
766
hsa-miR-199a-5p/hsa-miR-199b-5p
CCAGUG
7
6.4

CCACUU
767
hsa-miR-181a-2-3p
CCACUG
7
5.3

UCACAU
768
hsa-miR-27a-3p/hsa-miR-27b-3p
UCACAG
7
5.4

UAUACU
769
hsa-let-7d-3p
UAUACG
7
3.3

UUUUUU
770
hsa-miR-129-5p
UUUUUG
7
2.2

UGUGCU
771
hsa-miR-210-3p
UGUGCG
7
3.6

GAAUUU
772
hsa-miR-219a-2-3p
GAAUUG
7
9.2

AAAACU
773
hsa-miR-424-3p
AAAACG
7
2.7

AAGGUU
774
hsa-miR-18a-5p
AAGGUG
7
5.1

UGAAAU
775
hsa-miR-488-3p
UGAAAG
7
2.6

GGAAUU
776
hsa-miR-1-3p
GGAAUG
7
3.8

CCAUCU
777
hsa-miR-181a-3p
CCAUCG
7
2.4

CAGUAU
778
hsa-miR-199b-3p
CAGUAG
7
7.9

ACCCUU
779
hsa-miR-10a-5p
ACCCUG
7
3

AGGUAU
780
hsa-miR-196b-5p
AGGUAG
7
2.1

GGUUGU
781
hsa-miR-92a-1-5p
GGUUGG
7
2.2

AGACGU
782
hsa-miR-483-5p
AGACGG
7
2.2

CUGCAU
783
hsa-miR-17-3p
CUGCAG
7
2.1

GGGUGU
784
hsa-miR-363-5p
GGGUGG
7
2

CUUUGU
785
hsa-miR-9-5p
CUUUGG
7
63.3

AAACCU
786
hsa-miR-1537-3p
AAACCG
7
2

AAAGUU
787
hsa-miR-106b-5p/hsa-miR-20a-5p/hsa-miR-
AAAGUG
7
8.4

17-5p/hsa-miR-93-5p

GAGAUU
788
hsa-miR-143-3p
GAGAUG
7
33.4

UUUCAU
789
hsa-miR-30a-3p/hsa-miR-30e-3p
UUUCAG
7
8.7

GCUCGU
790
hsa-miR-423-3p
GCUCGG
7
2.1

AUCUGU
791
hsa-miR-1298-3p
AUCUGG
7
3.7

CGACCU
792
hsa-miR-1307-5p
CGACCG
7
3.5

GAGGGU
793
hsa-miR-423-5p
GAGGGG
7
2.8

GGAGUU
794
hsa-miR-122-5p
GGAGUG
7
53.7

UUAUCAU
795
hsa-miR-374a-3p
UUAUCAG
8
4.9

GGUGCGU
796
hsa-miR-675-5p
GGUGCGG
8
2

GGUUGGU
797
hsa-miR-92a-1-5p
GGUUGGG
8
2.1

CGACCGU
798
hsa-miR-1307-5p
CGACCGG
8
6.7

AGCAGCU
799
hsa-miR-503-5p
AGCAGCG
8
15.3

GGCUCAU
800
hsa-miR-24-3p
GGCUCAG
8
4.9

UAUAAAU
801
hsa-miR-340-5p
UAUAAAG
8
3.6

ACUGCAU
802
hsa-miR-509-3-5p
ACUGCAG
8
6.3

GGCAGUU
803
hsa-miR-34a-5p/hsa-miR-34c-5p
GGCAGUG
8
4.2

UCUUGAU
804
hsa-miR-526b-5p
UCUUGAG
8
6.3

UGAAAUU
805
hsa-miR-203a-3p
UGAAAUG
8
18.8

UGCAUAU
806
hsa-miR-153-3p
UGCAUAG
8
3.4

UAAGACU
807
hsa-miR-208a-3p
UAAGACG
8
9.8

AAUACUU
808
hsa-miR-200b-3p/hsa-miR-200c-3p
AAUACUG
8
15.6

UCUAGAU
809
hsa-miR-518f-5p/hsa-miR-523-5p
UCUAGAG
8
6.4

ACUAUAU
810
hsa-miR-625-3p
ACUAUAG
8
2.9

GUAACAU
811
hsa-miR-194-5p
GUAACAG
8
12.9

UGUACAU
812
hsa-let-7g-3p
UGUACAG
8
2.2

AUCUGGU
813
hsa-miR-1298-3p
AUCUGGG
8
2.1

ACUCUGU
814
hsa-miR-514a-5p
ACUCUGG
8
2.6

AGACGGU
815
hsa-miR-483-5p
AGACGGG
8
12

CGUACCU
816
hsa-miR-126-3p
CGUACCG
8
13.7

CACAGUU
817
hsa-miR-128-3p
CACAGUG
8
12.1

AUACAAU
818
hsa-miR-381-3p
AUACAAG
8
7.1

AAAGCUU
819
hsa-miR-320a
AAAGCUG
8
4.1

UCUCAAU
820
hsa-miR-513c-5p/hsa-miR-514b-5p
UCUCAAG
8
4.3

GCUGGUU
821
hsa-miR-138-5p
GCUGGUG
8
3.4

UCCAGAU
822
hsa-miR-520a-5p
UCCAGAG
8
4.4

CCCUGAU
823
hsa-miR-125b-5p/hsa-miR-125a-5p
CCCUGAG
8
21.3

AACACUU
824
hsa-miR-141-3p
AACACUG
8
2.4

UGCCCUU
825
hsa-miR-874-3p
UGCCCUG
8
3.5

UCCUAUU
826
hsa-miR-202-5p
UCCUAUG
8
22.5

ACCACAU
827
hsa-miR-140-3p
ACCACAG
8
3.2

CCCCCAU
828
hsa-miR-361-3p
CCCCCAG
8
2

UCACAAU
829
hsa-miR-513b-5p
UCACAAG
8
2.5

GUGACUU
830
hsa-miR-134-5p
GUGACUG
8
2.9

UGCAUUU
831
hsa-miR-33a-5p
UGCAUUG
8
2.1

AGUGCUU
832
hsa-miR-512-3p
AGUGCUG
8
2.7

GAGGUAU
833
hsa-let-7a-5p/hsa-let-7c-5p/hsa-let-7b-5p/hsa-
GAGGUAG
8
36.5

let-7d-5p/hsa-let-7f-5p/hsa-let-7e-5p/hsa-let-

7i-5p/hsa-let-7g-5p

UUGUUCU
834
hsa-miR-375
UUGUUCG
8
25.5

AUCAUCU
835
hsa-miR-136-3p
AUCAUCG
8
6.3

ACUCCAU
836
hsa-miR-508-5p
ACUCCAG
8
10.6

AAGGCACU
837
hsa-miR-124-3p
AAGGCACG
9
2.7

UCCCUUUU
838
hsa-miR-204-5p/hsa-miR-211-5p
UCCCUUUG
9
2.6

UGUGCGUU
839
hsa-miR-210-3p
UGUGCGUG
9
3.8

GAGAACUU
840
hsa-miR-146a-5p/hsa-miR-146b-5p
GAGAACUG
9
5.4

GAUUGUAU
841
hsa-miR-508-3p
GAUUGUAG
9
4.9

UCACAUUU
842
hsa-miR-23a-3p
UCACAUUG
9
6.1

GAAUUGUU
843
hsa-miR-219a-2-3p
GAAUUGUG
9
11.4

AACACCAU
844
hsa-miR-21-3p
AACACCAG
9
2.7

GGAGUGUU
845
hsa-miR-122-5p
GGAGUGUG
9
42.5

UAGAGGAU
846
hsa-miR-877-5p
UAGAGGAG
9
2.3

UUUUUGCU
847
hsa-miR-129-5p
UUUUUGCG
9
5.8

UCUUGAGU
848
hsa-miR-526b-5p
UCUUGAGG
9
4.1

CUUAAACU
849
hsa-miR-302a-5p
CUUAAACG
9
13

AUGCCUUU
850
hsa-miR-532-5p
AUGCCUUG
9
3.8

UCCAGAGU
851
hsa-miR-520a-5p
UCCAGAGG
9
2

CUACAGUU
852
hsa-miR-139-5p
CUACAGUG
9
2

ACCCGUAU
853
hsa-miR-99a-5p/hsa-miR-100-5p/hsa-miR-99b-5p
ACCCGUAG
9
34.8

AAAGCUGU
854
hsa-miR-320a
AAAGCUGG
9
2.6

AAUCUCAU
855
hsa-miR-216a-5p
AAUCUCAG
9
5.5

CGGAUCCU
856
hsa-miR-127-3p
CGGAUCCG
9
4.3

CGGGUUAU
857
hsa-miR-125b-1-3p
CGGGUUAG
9
4.7

AUACAAGU
858
hsa-miR-381-3p
AUACAAGG
9
2

UCACAGUU
859
hsa-miR-27a-3p/hsa-miR-27b-3p
UCACAGUG
9
5.6

GGGGGAUU
860
hsa-miR-5010-5p
GGGGGAUG
9
2.5

UGCCCUAU
861
hsa-miR-3157-3p
UGCCCUAG
9
2

CUGCAGUU
862
hsa-miR-17-3p
CUGCAGUG
9
2.4

UCUAGAGU
863
hsa-miR-523-5p
UCUAGAGG
9
2.8

Through the miRNA sequencing variant analyses in the example, variant miRNAs recognizing a non-canonical G:A wobble seed target as a canonical target in various cancer patients were identified, and therefore, when the present invention (miRNA-GU) for modifying a miRNA sequence to recognize a non-canonical G:A wobble seed site as a canonical seed site is applied, a total of 335 sequences (sequence (G>U)) were made (Table 4), thereby completing the technology of exhibiting only the function of suppressing a non-canonical target of miRNA.

INDUSTRIAL APPLICABILITY

When an interference-inducing nucleic acid according to the present invention is used, a biological function exhibited by suppressing a non-canonical target gene of conventional miRNA may be effectively improved, or a part of the functions of the convention miRNA, that is, only a biological function exhibited by suppressing a non-canonical target gene may be selectively exhibited. In addition, cell cycling, differentiation, dedifferentiation, morphology, migration, proliferation or apoptosis may be regulated by the interference-inducing nucleic acid, and thus it is expected to be used in various fields such as pharmaceuticals, cosmetics, etc.

Number	Date	Country	Kind
10-2018-0063054	May 2018	KR	national
10-2018-0063055	May 2018	KR	national
10-2019-0064333	May 2019	KR	national
10-2019-0064334	May 2019	KR	national
10-2019-0064335	May 2019	KR	national
10-2019-0064386	May 2019	KR	national

RNA INTERFERENCE-INDUCING NUCLEIC ACID INHIBITING NONCANONICAL TARGETS OF MICRO RNA, AND USE FOR SAME

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

International Classifications

Abstract

Description

Claims

Priority Claims (6)

PCT Information