Patent Application
20210024915
The present invention relates to the field of molecular biology, and in particular to up-regulating gene expression with a double-stranded small RNA targeting a gene promoter.
Double-stranded small nucleic acid molecules including chemically-synthesized oligoribonucleotides (such as small activating RNA (saRNA)) and naturally occurring oligoribonucleotides (such as micro ribonucleic acid (miRNA)) have been proven to be capable of targeting regulatory sequences (such as promoter sequences) of protein-coding genes in a sequence-specific manner to up-regulate gene expression at the transcriptional and epigenetic level, a phenomenon known as RNA activation (RNAa) (Li, Okino et al. (2006) Proc Natl Acad Sci USA 103:17337-17342; Janowski, Younger et al. (2007) Nat Chem Biol 3:166-173; Place, Li et al. (2008) Proc Natl Acad Sci USA 105:1608-1613; Huang, Place et al. (2012) Nucleic Acids Res 40:1695-1707; Li (2017) Adv Exp Med Biol 983:1-20). Studies have shown that RNA-a is an endogenous molecular mechanism evolutionarily conserved from Caenorhabditis elegans to the human being (Huang, Qin et al. (2010) PLoS One 5:e8848; Seth, Shirayama et al. (2013) Dev Cell 27:656-663; Turner, Jiao et al. (2014) Cell Cycle 13:772-781).
Safe strategies to selectively enhance gene and/or protein production remain a challenge in gene therapy. Viral-based systems have inherent drawbacks including adverse effects on host genome integrity and immunological consequences. RNAa, with the advantages of being able to activate endogenous genes without the risk of altering the genome, represents a new strategy to stimulate target gene expression.
Cyclin-dependent kinase (CDK) inhibitor p21WAF1/CIP1 (p21) is a mediator of several anti-growth pathways and considered a potent tumor suppressor gene in cancer cells (Harper, Adami et al. (1993) Cell 75:805-816). In fact, overexpression of p21 by ectopic vectors or stimulation of endogenous transcription inhibits tumor growth both in vitro and in vivo (Harper, Adami et al. (1993) Cell 75:805-816; Eastham, Hall et al. (1995) Cancer Res 55:5151-5155; Wu, Bellas et al. (1998) J Exp Med 187:1671-1679; Harrington, Spitzweg et al. (2001) J Urol 166:1220-1233). As such, selective activation of p21 can possess therapeutic application for regulating cell growth and treatment of disease (e.g. cancer).
The present invention provides a saRNA, wherein one strand of the saRNA has at least 75% homology or complementarity with any continuous fragment of 16 to 35 nucleotides in length in a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, and 12, and wherein the saRNA activates or upregulates the expression of p21 by targeting the sequence of a human p21 promoter.
In certain embodiments, a target gene sequence of the human p21 is selected from a group consisting of SEQ ID NO: 5-12, wherein the sequence of the human p21 promoter is from −893 bp to −801 bp (SEQ ID NO: 5), −717 bp to −632 bp (SEQ ID NO: 6), −585 bp to −551 bp (SEQ ID NO: 7), −554 bp to −504 bp (SEQ ID NO: 8), −514 bp to −485 bp (SEQ ID NO: 9), −442 bp to −405 bp (SEQ ID NO: 10), −352 bp to −313 bp (SEQ ID NO: 11), or −325 bp to −260 bp (SEQ ID NO: 12) upstream of the transcription start site (TSS) respectively.
In certain embodiments, the saRNA comprises a sense nucleic acid strand and an antisense nucleic acid strand. The sense nucleic acid strand and the antisense nucleic acid strand can contain complementary regions capable of forming a double-stranded nucleic acid structure, and the sense nucleic acid strand or the antisense nucleic acid strand has more than 75%, more than 80%, more than 90%, more than 95%, more than 99%, or 100% homology with any continuous fragment of 16 to 35 nucleotides in length in a sequence of a human p21 promoter.
In certain embodiments, the sense nucleic acid strand and the antisense nucleic acid strand are on two different nucleic acid strands; or the sense nucleic acid strand and the antisense nucleic acid strand are on the same nucleic acid strand, forming a hairpin single-stranded nucleic acid molecule, wherein the complementary regions of the sense nucleic acid strand and the antisense nucleic acid strand form a double-stranded nucleic acid structure.
In certain embodiments, at least one strand of the saRNA has a 3′ overhang of 0 to 6 nucleotides in length; or both strands of the saRNA have a 3′ overhang of 2 to 3 nucleotides in length.
In certain embodiments, the sense nucleic acid strand or the antisense nucleic acid strand has 16 to 35 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides, in length.
In certain embodiments, the sense nucleic acid strand or the antisense nucleic acid strand has at least 75%, such as 80%, 85%, 90%, 95%, 99%, or 100%, homology with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 13-30, 35-46, 59-62, 67-74, 77-80, 85-96, 103-108, 111-118, 121-132, 139-140, 147-180, 185-186, 189-190, 195-198, 201-202, 209-212, 215-218, 225-240, 243-246, 249-258, 261-262, 265-270, 275-280, 283-300, 303-308, 317-320, 323-324, 329-348, 351-352, 357-358, 361-366, 371-392, 399-400, 405-412, 415-416, 419-424, 429-432, 439-442, 447-450, 453-458, and 463-468.
In certain embodiments, the nucleotide sequence of the sense nucleic acid strand or the antisense nucleic acid strand is selected from the group consisting of SEQ ID NOs: 13-30, 35-46, 59-62, 67-74, 77-80, 85-96, 103-108, 111-118, 121-132, 139-140, 147-180, 185-186, 189-190, 195-198, 201-202, 209-212, 215-218, 225-240, 243-246, 249-258, 261-262, 265-270, 275-280, 283-300, 303-308, 317-320, 323-324, 329-348, 351-352, 357-358, 361-366, 371-392, 399-400, 405-412, 415-416, 419-424, 429-432, 439-442, 447-450, 453-458, and 463-468.
The present invention further provides a method for preparing the saRNA as described by any one of the aforementioned examples, wherein the method comprises the following steps: 1) selecting a sequence containing 19 bases as a target site by using the sequence of a promoter of a target gene as a template; 2) synthesizing a RNA sequence having more than 75% homology with the sequence of the target site obtained in step 1) to obtain a sense oligonucleotide strand; 3) synthesizing a sequence complementary with the sense oligonucleotide strand obtained in step 2); and 4) mixing the sense oligonucleotide strand obtained in step 2) and an antisense oligonucleotide strand obtained in step 3) in RNA annealing buffer at a molar ratio of 1:1, heating the mixture, and then naturally cooling the mixture to room temperature, so that a double-stranded saRNA is obtained, wherein the nucleic acid sequence of the human p21 promoter is selected from a group consisting of SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, and 12.
In certain embodiments, at least one nucleotide of the saRNA is a chemically modified nucleotide, and the chemical modification is at least one of the following modifications:
(1) modification of a phosphodiester bond connecting nucleotides in the nucleotide sequence of the saRNA;
(2) modification of 2′-OH of ribose in the nucleotide sequence of the saRNA;
(3) modification of a base in the nucleotide sequence of the saRNA; or
(4) at least one nucleotide in the nucleotide sequence of the small activating nucleic acid molecule being a locked nucleic acid.
In certain embodiments, the expression of p21 is activated or upregulated by at least 10%, such as more than 15%, 20%, 30%, 40%, 50%, 80%, 100%, or 200%.
The present invention further provides a use of the saRNA in the preparation of a formulation for activating or upregulating the expression of p21 in a cell. The saRNA is introduced into the cell directly, or is produced in the cell after a nucleotide sequence encoding the saRNA is introduced into the cell. The cell is a mammal cell, preferably a human cell, and more preferably a human tumor cell. The human cell can be an isolated human cell line or can be present in a human body.
In certain embodiments, the human body is a patient with a tumor caused by insufficient p21 protein expression, wherein administration of an effective amount of the small activating nucleic acid molecule can treat the tumor, and the tumor is preferably a bladder cancer, a prostate cancer, a hepatocellular carcinoma, or a colorectal cancer.
In another aspect, the present invention further provides an isolated p21 saRNA target site, wherein the target site is any continuous 16-35 nucleotide sequence selected from the group consisting of SEQ ID NOs. 5-12.
In yet another aspect, the present invention discloses a method for activating or upregulating the expression of human p21 in a cell, wherein the method comprises administrating the saRNA as described in any one of the aforementioned embodiments to a subject or a cell. The saRNA can be introduced into the cell directly, or can be produced in the cell after a nucleotide sequence encoding the saRNA is introduced into the cell. The cell is a mammalian cell, preferably a human cell, more preferably a human tumor cell, and most preferably a bladder cancer cell, a prostate cancer cell, a hepatocellular carcinoma cell, or a colorectal cancer cell.
The present invention further discloses a composition containing the aforementioned saRNA and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is a liposome, a macromolecular polymer, or a polypeptide.
The present invention further discloses a use of the saRNA or the composition as described above in the preparation of a formulation for activating or upregulating p21 expression. Preferably a use of the saRNA or the composition in the preparation of a formulation is for treating a tumor or a benign proliferative lesion. Preferably, the tumor is a bladder cancer, a prostate cancer, a hepatocellular carcinoma, or a colorectal cancer.
The present invention is further described hereinafter by specific description.
Unless otherwise defined, all the technological and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art covering the present invention.
In the present application, singular forms, such as “a” and “this”, include plural objects, unless otherwise specified clearly in the context.
The term “complementary” as used herein refers to the capability of forming base pairs between two oligonucleotide strands. The base pairs are generally formed by hydrogen bonds between nucleotide units in the antiparallel oligonucleotide strands. The bases of the complementary oligonucleotide strands can be paired in the Watson-Crick manner (such as A to T, A to U, and C to G) or in any other manner allowing the formation of a duplex (such as Hoogsteen or reverse Hoogsteen base pairing). “100% pairing” or “complete complementarity” refers to 100% complementarity, that is, all the nucleotide units of the two strands are bound by hydrogen bonds.
“Complete complementarity” or “100% pairing” means that each nucleotide unit from the first oligonucleotide strand can form a hydrogen bond with the second oligonucleotide strand in the double-stranded region of the double-stranded oligonucleotide molecule, with no base pair being “mispaired”. “Incomplete complementarity” means that not all the nucleotide units of the two strands are bound with each other by hydrogen bonds. For example, for two oligonucleotide strands each of 20 nucleotides in length in the double-stranded region, if only two base pairs in this double-stranded region can be formed through hydrogen bonds, the oligonucleotide strands have a complementarity of 10%. In the same example, if 18 base pairs in this double-stranded region can be formed through hydrogen bonds, the oligonucleotide strands have a complementarity of 90%. “Substantial complementarity” refers to more than about 79%, about 80%, about 85%, about 90%, or about 95% complementarity.
The term “oligonucleotide” as used herein refers to polymers of nucleotides, and includes, but is not limited to, single-stranded or double-stranded molecules of DNA, RNA, DNA/RNA hybrid, oligonucleotide strands containing regularly and irregularly alternating deoxyribosyl portions and ribosyl portions, as well as modified and naturally or unnaturally existing frameworks for such oligonucleotides.
The term “oligoribonucleotide” as used herein refers to an oligonucleotide containing two or more modified or unmodified ribonucleotides and/or analogues thereof.
The terms “oligonucleotide strand” and “oligonucleotide sequence” as used herein can be used interchangeably, referring to a generic term for short nucleotide sequences having less than 50 bases (the nucleic acid can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)). In the present invention, the length of an oligonucleotide strand can be any length from 17 to 30 nucleotides.
The term “gene” as used herein refers to all nucleotide sequences required to encode a polypeptide chain or to transcribe a functional RNA. “Gene” can be an endogenous or fully or partially recombinant gene for a host cell (for example, because an exogenous oligonucleotide and a coding sequence for coding a promoter are introduced into a host cell, or a heterogeneous promoter adjacent to an endogenous coding sequence is introduced into a host cell). For example, the term “gene” includes a nucleic acid sequence composed of exons and introns. Protein-coding sequences are, for example, sequences contained within exons in an open reading frame between an initiation codon and a termination codon, and as used herein, “gene” can comprise a gene regulatory sequence, such as a promoter, an enhancer, and all other sequences known in the art for controlling the transcription, expression or activity of another gene, no matter whether the gene contains a coding sequence or a non-coding sequence. In one case, for example, “gene” can be used to describe a functional nucleic acid containing a regulatory sequence such as a promoter or an enhancer. The expression of a recombinant gene can be controlled by one or more types of heterogenous regulatory sequences.
The term “target gene” as used herein can refer to nucleic acid sequences, transgenes, viral or bacterial sequences, chromosomes or extrachromosomal genes that are naturally present in organisms, and/or can be transiently or stably transfected or incorporated into cells and/or chromatins thereof. The target gene can be a protein-coding gene or a non-protein-coding gene (such as microRNA gene and long non-coding RNA gene). The target gene generally contains a promoter sequence, and the positive regulation for the target gene can be achieved by designing a saRNA having sequence homology with the promoter sequence, characterized as the upregulation of expression of the target gene. “Sequence of a target gene promoter” refers to a non-coding sequence of the target gene, and the reference of the sequence of a target gene promoter in the phrase “complementary with the sequence of a target gene promoter” of the present invention means a coding strand of the sequence, also known as a non-template strand, i.e. a nucleic acid sequence having the same sequence as the coding sequence of the gene. “Target sequence” refers to a sequence fragment in the target gene promoter sequence, which is homologous or complementary with a sense oligonucleotide strand or an antisense oligonucleotide strand of a saRNA.
As used herein, the terms “sense strand” and “sense oligonucleotide strand” can be used interchangeably, and the sense oligonucleotide strand of a saRNA refers to a first ribonucleic acid strand having homology with the coding strand of the promoter sequence of the target gene in the saRNA duplex.
As used herein, the terms “antisense strand” and “antisense oligonucleotide strand” can be used interchangeably, and the antisense oligonucleotide strand of a saRNA refers to a second ribonucleic acid strand complementary with the sense oligonucleotide strand in the saRNA duplex.
The term “coding strand” as used herein refers to a DNA strand in the target gene which cannot be used for transcription, and the nucleotide sequence of this strand is the same as that of RNA produced from transcription (in the RNA, T in DNA is replaced by U). The coding strand of the double-stranded DNA sequence of the target gene promoter described in the present invention refers to a promoter sequence on the same DNA strand as the DNA coding strand of the target gene.
The term “template strand” as used herein refers to the other strand complementary with the coding strand in the double-stranded DNA of the target gene, i.e. the strand that, as a template, can be transcribed into RNA, and this strand is complementary with the transcribed RNA (A to U, G to C). In the process of transcription, RNA polymerase is bound with the template strand, moves along the 3′→5′ direction of the template strand, and catalyzes the synthesis of the RNA along the 5′→3′ direction. The template strand of the double-stranded DNA sequence of the target gene promoter described in the present invention refers to a promoter sequence on the same DNA strand as the DNA template strand of the target gene.
The term “promoter” as used herein refers to a nucleic acid sequence, which does not encode a protein, which plays a regulatory role for the transcription of a protein-coding or RNA-coding nucleic acid sequence by associating with them spatially. Generally, a eukaryotic promoter contains 100 to 5,000 base pairs, although this length range is not intended to limit the term “promoter” as used herein. Although the promoter sequence is generally located at the 5′ terminus of a protein-coding or RNA-coding sequence, in some cases, the promoter sequence also exists in exon and intron sequences.
The term “transcription start site” as used herein refers to a nucleotide marking the transcription start on the template strand of a gene. The transcription start site can appear on the template strand of the promoter region. A gene can have more than one transcription start site.
The term “sequence identity” or “sequence homology” as used herein means that one oligonucleotide strand (sense or antisense) of a saRNA has at least 75% similarity with a region on the coding strand or template strand of the promoter sequence of a target gene.
The term “overhang” as used herein refers to non-base-paired nucleotides at the terminus (5′ or 3′) of an oligonucleotide strand, which is formed by one strand extending out of the other strand in a duplex oligonucleotide. A single-stranded region extending out of the 3′ terminus and/or 5′ terminus of a duplex is referred to as an overhang.
As used herein, the terms “gene activation” or “activating gene expression” can be used interchangeably, and means an increase or upregulation in transcription, translation, expression or activity of a certain nucleic acid as determined by measuring the transcription level, mRNA level, protein level, enzymatic activity, methylation state, chromatin state or configuration, translation level or the activity or state in a cell or biological system of a gene. These activities or states can be determined directly or indirectly. In addition, “gene activation” or “activating gene expression” refers to an increase in activity associated with a nucleic acid sequence, regardless the mechanism of such activation. For example, gene activation occurs at the transcriptional level to increase transcription into RNA and the RNA is translated into a protein, thereby increasing the expression of the protein.
As used herein, the terms “small activating RNA,” “saRNA,” and “small activating nucleic acid molecule” can be used interchangeably, and refer to a ribonucleic acid molecule that can upregulate target gene expression. The saRNA can be composed of a first ribonucleic acid strand (antisense strand, also referred to as antisense oligonucleotide strand) containing a ribonucleotide sequence having sequence homology with the non-coding nucleic acid sequence (e.g., a promotor and an enhancer) of a target gene and a second ribonucleic acid strand (sense strand, also referred to as sense oligonucleotide strand) containing a nucleotide sequence complementary with the first ribonucleic acid strand, wherein the first ribonucleic acid strand and the second ribonucleic acid strand form a duplex. The saRNA can also be comprised of a synthesized or vector-expressed single-stranded RNA molecule that can form a hairpin structure by two complementary regions within the molecule, wherein the first region contains a nucleic acid sequence having sequence homology with the target sequence of a promoter of a gene, and a nucleic acid sequence contained in the second region is complementary with the first region. The length of the duplex region of the saRNA molecule is typically about 10 to about 50, about 12 to about 48, about 14 to about 46, about 16 to about 44, about 18 to about 42, about 20 to about 40, about 22 to about 38, about 24 to about 36, about 26 to about 34, and about 28 to about 32 base pairs, and typically about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 base pairs. In addition, the terms “saRNA”, “small activating RNA”, and “small activating nucleic acid molecule” also contain nucleic acids other than the ribonucleotide, including, but not limited to, modified nucleotides or analogues.
As used herein, the term “hotspot” refers to a promoter region of a gene where targets for functional saRNAs are enriched. In these hotspot regions, at least 60% of the small activating nucleic acid molecules targeting these hotspot regions can induce a 1.5-fold or more change in the mRNA expression of a target gene.
As used herein, the term “p21” refers to the p21WAF1/CIP1 gene, also known as the CDKN1A gene. As a cyclin-dependent kinase (CDK) inhibitor, p21 is an important tumor suppressor gene, also known as “target gene” sometimes. Overexpression of p21 or activation of endogenous p21 transcription has been shown to inhibit the growth of cultured tumor cells and tumors in vivo.
As used herein, the term “synthesis” refers to a method for synthesis of an oligonucleotide, including any method allowing RNA synthesis, such as chemical synthesis, in-vitro transcription, and/or vector-based expression. The present invention provides a method for preparing the small activating nucleic acid molecule, which comprises sequence design and sequence synthesis. The synthesis of the sequence of the small activating nucleic acid molecule can adopt a chemical synthesis or can be entrusted to a biotechnology company specialized in nucleic acid synthesis. Generally speaking, the chemical synthesis comprises the following four steps: (1) synthesis of oligomeric ribonucleotides; (2) deprotection; (3) purification and isolation; (4) desalination and annealing. For example, the specific steps for chemically synthesizing the double-stranded RNA molecule of the present invention, such as saRNA, are as follows:
(1) Synthesis of Oligomeric Ribonucleotides
Synthesis of 1 micromole of RNA was set in an automatic DNA/RNA synthesizer (e.g., Applied Biosystems EXPEDITE8909), and the coupling time of each cycle was also set as 10 to 15 minutes. With a solid phase-bonded 5′-O-p-dimethoxytriphenylmethyl-thymidine substrate as an initiator, one base was bonded to the solid phase substrate in the first cycle, and then, in the nth (19≥n≥2) cycle, one base was bonded to the base bonded in the n−1 cycle. This process was repeated until the synthesis of the whole nucleic acid sequence was completed.
(2) Deprotection
The solid phase substrate bonded with the saRNA was put into a test tube, and 1 ml of a solution of the mixture of ethanol and ammonium hydroxide (volume ratio: 1:3) was added into the test tube. The test tube was then sealed and placed in an incubator, and the mixture was incubated at 25-70° C. for 2 to 30 hours. The solution containing the solid phase substrate bonded with the saRNA was filtered, and the filtrate was collected. The solid phase substrate was rinsed with double distilled water twice (1 ml each time), and the filtrate was collected. The eluents were combined and collected, and dried under vacuum for 1 to 12 hours. Then, 1 ml of a solution of tetrabutylammonium fluoride in tetrahydrofuran (1 M) was added. After 4 to 12 hours of standing at room temperature, 2 ml of n-butanol was then added. Precipitate was collected to obtain a single-stranded crude product of saRNA by high-speed centrifugation.
(3) Purification and Isolation
The obtained crude product of saRNA was dissolved in 2 ml of aqueous ammonium acetate solution with a concentration of 1 mol/ml, and the solution was separated by a reversed-phase C18 column of high pressure liquid chromatography to obtain a purified single-stranded product of saRNA.
(4) Desalination and Annealing
Salts were removed by gel filtration (size exclusion chromatography). A single sense oligomeric ribonucleic acid strand and a single antisense oligomeric ribonucleic acid strand were mixed into 1 to 2 ml of buffer (10 mM Tris, pH=7.5-8.0, 50 mM NaCl) at a molar ratio of 1:1. The solution was heated to 95° C., and was then slowly cooled to room temperature to obtain a solution containing saRNA.
Materials and Methods
Cell Culture and Transfection
Cell lines RT4, KU-7, T24, J82, TCCSUP, and HT-1197 were cultured in the modified McCoy's 5A medium (Gibco); cell lines 5637, PC3, and Bel-7402 were cultured in RPMI1640 medium (Gibco); and the cell line UM-UC-3 was cultured in basal medium (Gibco). All the media contained 10% bovine calf serum (Sigma-Aldrich) and 1% penicillin/streptomycin (Gibco). The cells were cultured at 5% CO2 and 37° C. Double-stranded RNA molecules designed in the experiment were transfected into cells using RNAiMax (Invitrogen, Carlsbad, Calif.) according to the instructions provided by the manufacturer at the concentration of 10 nM (unless otherwise specified).
Cells were seeded into a 6-well plate with 2-3×105 cells/well and were reverse transfected with oligonucleotide duplexes. At the end of the transfection, total cellular RNA was isolated using an RNeasy Plus Mini kit (Qiagen; Hilden, Germany) according to its manual. The isolated RNA (1 μg) was reverse transcribed into cDNA by using a PrimeScript RT kit containing gDNA Eraser (Takara, Shlga, Japan). The resulted cDNA was amplified in an ABI 7500 Fast Real-time PCR System (Applied Biosystems; Foster City, Calif.) using SYBR Premix Ex Taq II (Takara, Shlga, Japan) reagents and target gene specific primers. The reaction conditions were: 95° C. for 3 seconds, 60° C. for 30 seconds, and 40 cycles. Amplification of GAPDH served as an internal control. All primer sequences are listed in Table 1.
Assay of Cell Proliferation Cells were plated into a 96-well plate with 2-4×103 cells/well, cultured overnight, and transfected with oligonucleotide duplexes. Three days after transfection, the CCK8 reagent (Dojindo; Rockville, Md.) was used to assay the cell proliferation according to its manual. Briefly, 10 μL of CCK8 reagent was added into each well on the plate which was then incubated at 37° C. for 1 hour. Absorbance for each well on the plate was measured at 450 nm by a microplate reader.
QuantiGene 2.0 Assay
Cells were plated into a 96-well plate and transfected with oligonucleotide duplexes. 72 hours after transfection, the mRNA levels of target genes were quantitatively assayed with a QuantiGene 2.0 kit (AffyMetrix; Santa Clara, Calif.). QuantiGene 2.0 assay is based on hybridization technology, wherein mRNA levels were directly quantified with gene-specific probes. The experimental procedure is briefly described as follows: a lysis solution was added to lyse the transfected cells, and the cell lysates were added into a capture well plate coated with probe for CDKN1A (p21) and HPRT1 which serves as a housekeeping gene for hybridizing overnight at 55° C. In order to enhance the hybridization signal, hybridizations with 2.0 PreAMP Probe, 2.0 AMP Probe, and 2.0 Label Probe were conducted sequentially in 100 μL of a corresponding buffer solution (provided by Quantigene 2.0 kit). All the hybridizations were conducted with shaking at 50-55° C. for 1 hour. After the last wash step, 2.0 Substrate was added to the solution and incubated at room temperature for 5 minutes. Optical signals were detected with an Infinite 200 PRO plate reader (Tecan, Switzerland).
Statistical Analysis
Results were represented as mean±standard deviation. One-way analysis of variance was carried out with GraphPad Prism software (GraphPad Software), and then a Tukey's t test was conducted for pairwise comparisons. A statistical significance was set as *p<0.05, **p<0.01, and ***p<0.001.
The present invention is further illustrated by the following examples. These examples are provided merely for illustration purposes and shall not be interpreted to limit the scope or content of the present invention in any way.
The 1 kb promoter sequence (
The aforementioned double-stranded RNA molecules were transfected into PC3 prostate cancer cells at a final concentration of 10 nM, and 72 hours after transfection, the p21 mRNA level was detected with the QuantiGene 2.0 kit. The fold change of p21 mRNA level induced by each double-stranded RNA molecule relative to the blank control was calculated and plotted in
Among the 439 screened double-stranded RNA molecules, 132 double-stranded RNA molecules (30.1%) could induce at least a 2-fold change in p21 mRNA level, and 229 (52.4%) double-stranded RNA molecules could induce at least a 1.5-fold change in p21 mRNA level. These double-stranded RNA molecules that upregulated p21 mRNA expression by more than 10% were functional saRNAs. The targets for these functional saRNAs were scattered in the entire p21 promoter region. However, 8 discrete regions showed enrichment for saRNA targets and these regions are considered as saRNA “hotspots.” The hotspot is defined as a region containing at least 10 corresponding saRNAs, wherein at least 60% of them could induce a 1.5-fold or more change in p21 mRNA expression (
These hotspots include: hotspot 1 having a corresponding target sequence from −893 bp to −801 bp in the p21 promoter sequence, shown as SEQ ID NO: 93, wherein 44 functional saRNAs (Table 3,
hotspot 2 (Table 3,
hotspot 3 (Table 3,
hotspot 4 (Table 3,
hotspot 5 (Table 3,
hotspot 6 (Table 3,
hotspot 7 (Table 3,
and hotspot 8 (Table 3,
In order to verify the QuantiGene 2.0 assay results, the 439 double-stranded RNA molecules were divided into four bins according to their activities in inducing p21 mRNA expression, and 5 double-stranded RNA molecules were randomly selected from each bin and transfected into PC3 cells at a concentration of 10 nM. 72 hours after transfection, total cellular RNA was extracted from the transfected cells and reverse transcribed into cDNA which was amplified by RT-qPCR to determine p21 mRNA level. p21 mRNA expression levels for cells transfected with each of the saRNAs determined by the two methods showed a significant correlation (R2=0.82) (
Taken together, the above data indicates that saRNAs can be designed to target selected regions in the p21 promoter to induce p21 expression with certain regions being more sensitive and containing higher percentages of functional saRNA targets.
In order to further evaluate the effect of p21 saRNAs in inducing p21 mRNA expression and suppressing cancer cell proliferation, the saRNAs (RAG1-431, RAG1-553, and RAG1-688) screened by QuantiGene 2.0 were transfected into cancer cell lines including KU-7 (bladder cancer), HCT116 (colon cancer), and HepG2 (hepatocellular carcinoma). The result showed that in all the aforementioned cell lines, all saRNAs can induce at least a two-fold change in the p21 mRNA expression levels and suppress cell proliferation, indicating functional activation of p21 protein. Specifically, RAG1-431, RAG1-553, and RAG1-688 were individually transfected into KU-7 cells, caused a 14.0-, 36.9- and 31.9-fold change in the mRNA expression of p21, and exhibited a 71.7%, 60.7% and 67.4% cell survival rate respectively relative to blank control (Mock) (
All disclosures of each patent literature and scientific literature cited herein are incorporated herein by reference for all purposes.
The present invention can be implemented in other specific forms without departing from its fundamental characteristics. Therefore, the aforementioned examples shall be considered as illustrative rather than restrictive to the present invention described herein. The scope of the present invention is represented by the appended claims rather than the above specification, and is intended to cover all changes falling into the meanings and scopes of equivalents of the claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201810316672.1 | Apr 2018 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2019/082126 | 4/10/2019 | WO | 00 |
