Patent Application
20100305188
The present invention relates to an agent for suppressing or promoting cell proliferation, a diagnostic reagent or therapeutic drug for a disease resulting from a cell proliferation abnormality, a method of suppressing or promoting cell proliferation, a method of suppressing or promoting the expression of a target gene for a nucleic acid, and a method of screening an gent for suppressing or promoting cell proliferation, based on a nucleic acid.
microRNA (miRNA), which is one kind of nucleic acids, is a small non-coding single-stranded RNA of about 22 nucleotides that is not translated into a protein, and has known as being present in many types in organisms, including humans (non-patent documents 1 and 2). A miRNA is produced from a gene transcribed to a single or clustered miRNA precursor. Specifically, first, a primary-miRNA (pri-miRNA), which is a primary transcript, is transcribed from the gene, then, in stepwise processing from the pri-miRNA to a mature type micro-RNA, a precursor-miRNA (pre-miRNA) of about 70 nucleotides having a characteristic hairpin structure is produced from the pri-miRNA. Furthermore, the mature type miRNA is produced from the pre-miRNA by Dicer-mediated processing (non-patent document 3).
A mature type miRNA is thought to be involved in the post-transcriptional control of gene expression by complementarily binding to a mRNA for a target to suppress the translation of the mRNA, or to degrade the mRNA.
Although the mechanism in which miRNAs suppress the expression of target mRNAs has not been clarified in full, its outline is being elucidated through recent years' research. A miRNA suppresses the expression of the mRNA for a target by binding to a partially complementary sequence in the 3′-untranslational region (3′-UTR) of the target mRNA to suppress the translation thereof, or to degrade the target mRNA. Although the complementarity may not be complete, it has been shown that the complementarity of the 2nd to 8th nucleotides from the 5′-end of the miRNA is particularly important; this region is sometimes called the “seed sequence” of the miRNA (non-patent document 4). It has been shown that miRNAs having a same seed sequence suppress the expression of a same target mRNA even if their other sequences differ. Therefore, by making a sequence complementary to a sequence present at the 3′-end of an optionally chosen mRNA as the seed sequence, an RNA having miRNA-like activity can be designed. Usually, the term miRNA, unlike siRNA, often refers exclusively to an RNA “that occurs naturally in cells”, so a miRNA-like sequence designed as described above is sometimes particularly referred to as “an artificial miRNA”.
As of August 2007, in the miRNA database miRBase (http://microrna.sanger.ac.uk/), 533 species of miRNAs were registered for humans, and 5,071 species for all organisms. Of the miRNAs expressed in mammals, including humans, only some have their physiological functions elucidated to date, including miR-181, which is involved in hematopoietic lineage differentiation (non-patent document 5), miR-375, which is involved in insulin secretion (non-patent document 6), and the like; many have their bioactivities unclarified. However, studies using nematodes or Drosophila have shown that miRNAs play various important roles in the development and differentiation in organisms, and a report of the relation to human diseases has been reported suggesting a profound relation to cancers (non-patent document 7).
Because miRNAs are involved in the control of the expression of a wide variety of genes, abnormalities of miRNAs are supposed to be involved in various human diseases. Particularly in cancers, research has been advanced; it has been reported that in many cancers, the expression of miRNAs differs from that in normal tissues, that classification of cancers is enabled by expression profile analyses of miRNAs, and the like (non-patent document 8). It is also known that about half of the human miRNAs that have been found so far are present in chromosome aberrations or fragile portions of chromosomes known in human cancers (non-patent document 9).
Examples of relationships between cancers and miRNAs that have been reported so far include the finding that a miR-15a/miR-16 cluster is contained in the chromosome 13q14, which is deleted in B cell chronic lymphatic leukemia (B-CLL), the deletion being supposed to be a cause of B-CLL (non-patent document 10), and the finding that in B-CLL, the expression of miR-29 and miR-181 has also been decreased, one of the targets being Tcl1, which is known as a carcinogenic gene (non-patent document 11). In lung cancer, it is known that the expression of Let-7, which is a miRNA, has been decreased, one of the targets thereof being Ras, which is known as a carcinogenic gene (non-patent documents 12 and 13). It is also known that the expression of miRNAs such as miR-127 and miR-124a has been decreased due to gene modification by high methylation in cancers, and Bc16 and Cdk6, which are known as carcinogenic genes, serve as targets thereof (non-patent documents 14 and 15). Many miRNAs have their expression decreased in cancer cells; conversely, however, there are some miRNAs with gene amplification or overexpression in cancer cells. For example, in regions where gene amplification is seen in malignant lymphoma, a cluster consisting of six species of miRNAs (miR-17-92) is present; it has been known that when this miRNA cluster gene is forcedly expressed in a mouse model of human B cell lymphoma, the onset of lymphoma is promoted (non-patent document 16). It has also been shown that a gene called BIC, which has been regarded as a cancer gene candidate that does not encode a protein and is overexpressed in Hodgkin lymphoma, encodes miR-155 (non-patent document 17).
As stated above, relationships between cancers and miRNAs have recently been reported in many cases, but many of them are manifested as expression abnormalities in cancer cells, with a few reports showing functions of miRNAs, which include reports that the proliferation of cancer cell lines was inhibited by forcing cancer cell lines to express Let-7, miR-143, or miR-145 (non-patent documents 18, 19, and 20), reports that the cell cycles of cancer cell lines were halted by forcing the cancer cell lines to express miR-16 or miR-34 (non-patent documents 21 and 22), and a report that cell transformation was induced by forcing the cells to express miR-372 or miR-373 (non-patent document 23). There are only a few reports of suppressing cancer growth in animal models by administering a miRNA or a precursor thereof from outside the body to increase the expression of the miRNA, or by administering an antisense oligonucleotide thereof to decrease the expression of the miRNA; these reports include a report that tumorigenesis of a transplanted colorectal cancer cell line was suppressed by administering miR-34a (non-patent document 24) and a report that tumorigenesis of a transplanted breast cancer cell line was suppressed by administering an antisense of miR-21 (non-patent document 25).
It is expected that by identifying miRNAs expressed in various human organs, and analyzing the functions thereof to elucidate their relations to diseases, new therapeutic drugs and diagnostic reagents will be developed.
In particular, finding a miRNA that causes cancer cell proliferation or suppression is expected not only to help to understand the mechanisms of carcinogenesis, but also to lead to the development of diagnostic reagent and therapeutic drugs for human cancers, and new diagnostic methods and therapies for cancers based thereon. Furthermore, regarding diseases other than cancers, the same is expected to contribute to the development of diagnostic reagents and therapeutic drugs for diseases caused by abnormal proliferation of cells, tissue hyperplasia and the like, such as arteriosclerosis, rheumatoid arthritis, prostatic hyperplasia, blood vessel restenosis after percutaneous transvascular coronary angioplasty, fibroid lung, glomerulonephritis, and autoimmune diseases, and diagnostic methods and therapies based thereon.
It is an object of the present invention to provide nucleic acids that are useful in controlling cell proliferation and methods of utilizing the same.
The present invention relates to the following (1) to (32).
According to the present invention, an agent for suppressing or promoting cell proliferation, a diagnostic reagent or therapeutic drug for a disease resulting from a cell proliferation abnormality, an agent for inducing apoptosis, an agent for suppressing or promoting the expression of a target gene for a nucleic acid such as a miRNA, and a method of suppressing or promoting cell proliferation can be provided.
The nucleic acid to be used in the present invention may be any molecule, as far as it is a molecule resulting from polymerization of a nucleotide or a molecule functionally equivalent to the nucleotide; for example, an RNA, which is a ribonucleotide polymer, a DNA, which is a deoxyribonucleotide polymer, a mixed polymer of RNA and DNA, and a nucleotide polymer, including a nucleotide analogue, can be mentioned; furthermore, the nucleic acid may be a nucleotide polymer, including a nucleic acid derivative. In addition, the nucleic acid in the present invention may be a single-stranded nucleic acid or a double-stranded nucleic acid. Double-stranded nucleic acids include double-stranded nucleic acids wherein one strand hybridizes with the other strand under stringent conditions.
In the present invention, the nucleotide analogue may be any molecule, as far as it is a molecule prepared by modifying a ribonucleotide, a deoxyribonucleotide, an RNA or a DNA in order to improve the nuclease resistance thereof, to stabilize the same, to increase the affinity thereof for a complementary chain nucleic acid, to increase the cell permeability thereof, or to visualize the same, compared with the RNA or DNA; for example, a nucleotide analogue modified at the sugar moiety thereof, a nucleotide analogue modified at phosphoric acid diester bond and the like can be mentioned.
The nucleotide analogue modified at the sugar moiety thereof may be any one, as far as an optionally chosen chemical structural substance has been added to, or substituted for, a portion or all of the chemical structure of the sugar of the nucleotide; for example, a nucleotide analogue substituted by 2′-O-methylribose, a nucleotide analogue substituted by 2′-O-propylribose, a nucleotide analogue substituted by 2′-methoxyethoxyribose, a nucleotide analogue substituted by 2′-O-methoxyethylribose, a nucleotide analogue substituted by 2′-O-[2-(guanidium)ethyl]ribose, a nucleotide analogue substituted by 2′-O-fluororibose, a bridged nucleic acid (BNA) having two cyclic structures as a result of introduction of a bridging structure into the sugar moiety, more specifically a locked nucleic acid (LNA) wherein the oxygen atom at the 2′ position and the carbon atom at the 4′ position have been bridged via methylene, and an ethylene bridged nucleic acid) (ENA) [Nucleic Acid Research, 32, e175 (2004)] can be mentioned, and a peptide nucleic acid (PNA) [Acc. Chem. Res., 32, 624 (1999)], an oxypeptide nucleic acid (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001)], and a peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)] and the like can also be mentioned.
The nucleotide analogue modified at phosphoric acid diester bond may be any one, as far as an optionally chosen chemical substance has been added to, or substituted for, a portion or all of the chemical structure of the phosphoric acid diester bond of the nucleotide; for example, a nucleotide analogue substituted by a phosphorothioate bond, a nucleotide analogue substituted by an N3′-P5′ phosphoamidate bond, and the like can be mentioned [SAIBO KOGAKU, 16, 1463-1473 (1997)] [RNAi Method and Antisense Method, Kodansha (2005)].
The nucleic acid derivative may be any molecule, as far as it is a molecule prepared by adding another chemical substance to the nucleic acid in order to improve the nuclease resistance thereof, to stabilize the same, to increase the affinity thereof for a complementary chain nucleic acid, to increase the cell permeability thereof, or to visualize the same, compared with the nucleic acid; for example, a 5′-polyamine conjugated derivative, a cholesterol conjugated derivative, a steroid conjugated derivative, a bile acid conjugated derivative, a vitamin conjugated derivative, a Cy5 conjugated derivative, a Cy3 conjugated derivative, a 6-FAM conjugated derivative, a biotin conjugated derivative and the like can be mentioned.
As examples of nucleic acids, the following nucleic acids (a) to (k) can be mentioned.
As the above-described nucleic acid, a miRNA is preferably used. A miRNA refers to a single-stranded RNA 17 to 28 nucleotides long. The peripheral genomic sequence, including a miRNA sequence has a sequence capable of forming a hairpin structure, and the miRNA can be cut out from either one strand of the hairpin. A miRNA complementarily binds to a mRNA serving as a target therefor to suppress the translation of the mRNA or promote the degradation of the mRNA, thereby mediating the post-translational control of gene expression.
As examples of miRNAs, human miRNAs consisting of a nucleotide sequence shown by any one of SEQ ID NO:1-129, 803, 819, 916, 917, 925 and 2057-2114 can be mentioned. Furthermore, as miRNAs having the same function as the function of human miRNAs consisting of a nucleotide sequence shown by any one of SEQ ID NO:1-129, 803, 819, 916, 917, 925 and 2057-2114, nucleic acids consisting of a nucleotide sequence shown by any one of SEQ ID NO:130-802, 822, 823, 826, 827, 829-840, 842, 843, 846-848, 850, 851, 2115-2122, 2212-2261 and 2315-2321, which are orthologues of the human miRNAs, can be mentioned. As specific examples of orthologues of the human miRNA of SEQ ID NO:1, those consisting of a nucleotide sequence shown by any one of SEQ ID NO:130-139 and 2212 can be mentioned. A table of correspondence of miRNAs consisting of a nucleotide sequence shown by any one of SEQ ID NO:1-129, 803, 819, 916, 917, 925 and 2057-2114 and orthologues thereof is shown in Table 1 (from Table 1-1 to Table 1-4). The biological species shown in the uppermost fields of Table 1 are as follows: has, Homo sapiens, human; mmu, Mus musculus, mouse; rno, Rattus norvegicus, rat; xla, Xenopus laevis, African clawed toad; xtr, Xenopus tropicalis, tropical clawed toad; gga, Gallus gallus, chicken; cfa, Canis familiaris, dog; mdo, Monodelphis domestica, gray short-tailed opossum; age, Ateles geoffroyi, black-handed spider monkey; ila, Lagothrix lagotricha, Humboldt's wooly monkey; sla, Saguinus labiatus, red-bellied tamarin; mml, Macaca mulatta, rhesus monkey; mne, Macaca nemestrina, pig-tailed macaque; ggo, Gorilla gorilla, gorilla; ppa, Pan paniscus, bonobo; ptr, Pan troglodytes, chimpanzee; ppy, Pongo pygmaeus, orangutan; lca, Lemur catta, ring-tailed lemur; cgr, Cricetulus griseus, Chinese hamster; bta, Bos taurus, cattle; oar, Ovis aries, sheep; ssc, Sus scrofa, swine; dre, Danio rerio, zebrafish; fru, Fugu rubripes, tiger puffer; tni, Tetraodon nigroviridis, green spotted puffer. Because human-derived miRNAs and orthologues thereof have high sequence identity, they are considered to possess similar functions. Also because miRNAs in the same subgroup as classified by alphabetical figures added to the ends of their names also share high sequence identity, they are considered to possess similar functions.
Regarding the mechanism in which a miRNA suppresses the translation of the mRNA of a target gene therefor, it is known that a mRNA having a nucleotide sequence complementary to the 2nd to 8th nucleotides from the 5′-end of the miRNA is recognized as a miRNA target gene [Current Biology, 15, R458-R460 (2005)]. By this mechanism, the expression of the mRNA is suppressed by the miRNA. Therefore, miRNAs having the same nucleotide sequence at the 2nd to 8th nucleotides from the 5′-end suppress the expression of the same mRNA to exhibit the same function. As miRNAs having the same nucleotide sequence at the 2nd to 8th nucleotides from the 5′-end as the nucleotide sequence of a miRNA consisting of a nucleotide sequence shown by any one of SEQ ID NO:1-129, 803, 819, 916, 917, 925 and 2057-2114, nucleic acids consisting of a nucleotide sequence shown by any one of SEQ ID NO:804-818, 820-915, 918-924, 926-939 and 2123-2132 can be mentioned. As a specific example of the miRNA consisting of the nucleotide sequence shown by SEQ ID NO:4, the miRNA consisting of the nucleotide sequence shown by SEQ ID NO:804 can be mentioned. Artificial miRNAs are also included in the miRNAs. A table of correspondence of miRNAs having the same nucleotide sequence at the 2nd to 8th nucleotides from the 5′-end as the nucleotide sequence of a miRNA consisting of a nucleotide sequence shown by any one of SEQ ID NO:1-129, 803, 819, 916, 917, 925 and 2057-2114 is shown in Table 2 (from Table 2-1 to Table 2-4). Because miRNAs having a same seed sequence are considered to share the same target nucleotide sequence, they are considered to possess similar functions.
As the above-described nucleic acids, miRNA precursors can also be used preferably. A miRNA precursor is a nucleic 5 acid about 50 to about 200 nucleotides long, more preferably about 70 to about 100 nucleotides long, including the above-described nucleic acid, and capable of forming a hairpin structure. A miRNA is produced from a miRNA precursor via processing by a protein called Dicer.
As examples of miRNA precursors for the human miRNAs of SEQ ID NO:1, nucleic acids consisting of a nucleotide sequence shown by SEQ ID NO:940 or 941 can be mentioned. For the miRNAs consisting of a nucleotide sequence shown by any one of SEQ ID NO:2-939, 2057-2132, 2212-2261 and 2315-2321, nucleic acids consisting of a nucleotide sequence shown by any one of SEQ ID NO:942-2056, 2133-2211, 2262-2314 and 2322-2328 can be mentioned. A table of correspondence of miRNAs and miRNA precursors in the present invention is shown in Table 3 (from Table 3-1 to Table 3-17). In the present invention, miRNA precursors include artificial miRNA precursors.
In the present invention, a nucleic acid having an identity of 90% or more to a nucleotide sequence shown by any one of SEQ ID NO:1-2328 means a nucleic acid having an identity of at least 90% or more, preferably 93% or more, more preferably 95% or more, still more preferably 96% or more, particularly preferably 97% or more, most preferably 98% or more, to a nucleic acid consisting of a nucleotide sequence shown by any one of SEQ ID NO:1-2328, as calculated using an analytical software program such as BLAST [J. Mol. Biol., 215, 403 (1990)] or FASTA [Methods in Enzymology, 183, 63 (1990)].
The stringent conditions in the above refer to those under which signals can be detected by adding a membrane having one chain blotted and the other chain labeled with 32P-ATP to a Hybridization buffer consisting of 7.5 mL, 0.6 mL of 1 M Na2HPO4 (pH 7.2), 21 mL of 10% SDS, 0.6 mL of 50×Denhardt's solution, and 0.3 mL of 10 mg/mL sonicated salmon sperm DNA, carrying out a reaction at 50° C. overnight, thereafter washing the membrane with 5×SSC/5% SDS liquid at 50° C. for 10 minutes, and further washing the same with 1×SSC/1% SDS solution at 50° C. for 10 minutes, thereafter taking out the membrane, and exposing it to an X-ray film.
The method of detecting the expression of nucleic acid such as a miRNA using a nucleic acid may be any method, as far as the presence of nucleic acid such as a miRNA or miRNA precursor in a sample can be detected; for example, (1) Northern hybridization, (2) dot blot hybridization, (3) in situ hybridization, (4) quantitative PCR, (5) differential hybridization, (6) microarray, (7) ribonuclease protection assay and the like can be mentioned.
The method of detecting a mutation of nucleic acid such as a miRNA using a nucleic acid may be any method, as far as it enables detection of a mutation of the nucleotide sequence of nucleic acid such as a miRNA or miRNA precursor in a sample; for example, a method wherein a hetero-double-strand formed by hybridization of a nucleic acid having a non-mutated nucleotide sequence and a nucleic acid having a mutated nucleotide sequence are detected, or a method wherein a sample-derived nucleotide sequence is directly sequenced to detect the presence or absence of a mutation and the like can be mentioned.
A vector that expresses a nucleic acid may be any vector, as far as it is designed to biosynthesize a nucleic acid such as miRNA when introduced to, and transcribed in, a cell. Specifically, as examples of vectors capable of expressing a nucleic acid such as miRNA in a cell, pcDNA6.2-GW/miR (manufactured by Invitrogen), pSilencer 4.1-CMV (manufactured by Ambion), pSINsi-hH1 DNA (manufactured by Takara Bio Inc.), pSINsi-hU6 DNA (manufactured by Takara Bio Inc.), pENTR/U6 (manufactured by Invitrogen) and the like can be mentioned.
The method of suppressing the expression of a gene having a target nucleotide sequence of nucleic acid such as miRNA (hereinafter to be referred to as target gene) may be any method, as far as it suppresses the expression of a gene having a target nucleotide sequence by means of the activity to suppress the expression of a mRNA having the target nucleotide sequence of nucleic acid such as miRNA. Here, to suppress the expression encompasses a case where the translation of a mRNA is suppressed, and a case where cleavage or degradation of a mRNA results in a decreased amount of protein translated from the mRNA. Specifically, as substances that suppress the expression of a mRNA having the target nucleotide sequence, nucleic acids such as an siRNA and an antisense oligonucleotides can be mentioned. The siRNA can be prepared on the basis of information on the continuous sequence of the mRNA [Genes Dev., 13, 3191 (1999)]. The number of nucleotide residues constituting one strand of the siRNA is preferably 17 to 30 residues, more preferably 18 to 25 residues, still more preferably 19 to 23 residues.
MiRNAs include artificial miRNAs that are single-stranded RNAs 17 to 28 nucleotides long comprising as the 2nd to 8th nucleotides a sequence that is complementary to a consecutive, 7-nucleotide sequence present in the 3′-untranslational region of the mRNA of the target gene. In the case of an RNA comprising a sequence extended anteriorly and posteriorly that forms a hairpin structure, wherein the miRNA sequence can be cut out in cells from either one strand of the hairpin structure by the biosynthetic pathway of the miRNA, the extended sequence is called an artificial miRNA precursor. With a gene whose expression is to be suppressed as the target gene, an artificial miRNA and artificial miRNA precursor can be designed using the method described above.
A target nucleotide sequence of a nucleic acid such as miRNA refers to a nucleotide sequence consisting of several nucleotides recognized by a nucleic acid such as miRNA and the like of the present invention, wherein the expression of a mRNA having the nucleotide sequence is suppressed by a nucleic acid such as miRNA of the present invention. Because a mRNA having a nucleotide sequence complementary to the 2nd to 8th nucleotides on the 5′ terminal side of a miRNA undergoes suppression of the translation thereof by the miRNA [Current Biology, 15, R458-R460 (2005)], a nucleotide sequence complementary to the nucleotide sequence of the 2nd to 8th nucleotides on the 5′ terminal side of a nucleic acid such as miRNA of the present invention can be mentioned as a target nucleotide sequence. For example, by providing a target sequence complementary to the 2nd to 8th nucleotides on the 5′ terminal side of a miRNA, and selecting a mRNA comprising a sequence completely identical to a set of 3′ UTR nucleotide sequences of human mRNAs by a method such as character string search, the target nucleotide sequence can be determined. A set of 3′ UTR nucleotide sequences of human mRNAs can be prepared using information on genomic sequences and gene positions that can be acquired from “UCSC Human Genome Browser Gateway (http://genome.ucsc.edu/cgi-bin/hgGateway)”. As specific examples of genes having a target nucleotide sequence of miRNA consisting of the nucleotide sequence shown by any of SEQ ID NOs: 1-939, 2057-2132, 2212-2261 and 2315-2321, the genes shown in Table 4 (Table 4-1 to Table 4-157), represented by names (Official Symbols and Gene IDs) used in the EntreGene database (http://www.ncbi.nlm.nih.gov/Entrez/) of US National 30 Center for Biotechnology Information (NCBI), can be mentioned.
As a method of expressing a nucleic acid such as a miRNA in cells in the present invention, a method using a nucleic acid that causes the expression of a gene that encodes the miRNA and the like when introduced into the cells can be mentioned. As the nucleic acid, a DNA, an RNA, or a nucleotide analogue, as well as a chimeric molecule thereof, or a derivative of the nucleic acid can also be used. Specifically, the nucleic acid can be designed in the same way as with Pre-miR™ miRNA Precursor Molecules (manufactured by Ambion) or miRIDIAN microRNA Mimics (manufactured by GE Healthcare), and the nucleic acid such as the miRNA can be expressed in cells. Any method can be used to express a miRNA, as far as it allows the miRNA to be finally formed in the cells; for example, (1) a method wherein a single-stranded RNA that is a precursor of the miRNA is introduced, as well as (2) a method wherein an RNA consisting of a 100% matched double-strand consisting of the miRNA as it is and a complementary strand for the miRNA, is introduced, and (3) a method wherein a double-stranded RNA assumed to have resulted from cleavage of a miRNA with a Dicer is introduced, can be mentioned. As examples of commercial products based on these methods, miCENTURY OX Precursor (manufactured by B-Bridge), miCENTURY OX siMature (manufactured by B-Bridge), and miCENTURY OX miNatural (manufactured by B-Bridge), respectively, can be mentioned.
The method of synthesizing a nucleic acid to be used in the present invention is not particularly limited; the same can be produced by a method using a publicly known chemical synthesis, or an enzymatic transcription method and the like. As methods using a publicly known chemical synthesis, the phosphoroamidite method, the phosphorothioate method, the phosphotriester method and the like can be mentioned; for example, the same can be synthesized using the ABI3900 high throughput nucleic acid synthesizer (manufactured by Applied Biosystems). As an enzymatic transcription method, transcription with a plasmid or DNA having a desired nucleotide sequence as the template using a typical phage RNA polymerase, for example, T7, T3, or SP6RNA polymerase, can be mentioned.
As an example of a method of screening a substance that promotes or suppresses the expression or a function of a nucleic acid used in the present invention by means of the nucleic acid, a method can be mentioned wherein a vector that expresses the nucleic acid is introduced into cells, and a substance that promotes or suppresses the expression or a function of a mRNA having a target nucleotide sequence therefor is screened for.
A pharmaceutical with a nucleic acid to be used in the present invention as an active ingredient can be used to diagnose or treat a disease caused by an abnormality of cell proliferation and the like, tissue hyperplasia and the like. In addition, a nucleic acid can also be used as an agent for suppressing or promoting cell proliferation. Here, an abnormality of cell proliferation refers to a condition wherein cells are proliferating at a rate that is not a normal proliferation rate in a living organism.
As diseases caused by a cell proliferation abnormality, tissue hyperplasia and the like, specifically, cancers, arteriosclerosis, rheumatoid arthritis, prostatic hyperplasia, blood vessel restenosis after percutaneous transvascular coronary angioplasty, fibroid lung, glomerulonephritis, autoimmune diseases and the like, preferably cancer, can be mentioned.
Hereinafter, the present invention is described in detail.
1. Method of Detecting the Expression of a Nucleic Acid Such as miRNA or miRNA Precursor Expressed in Cancer Cells
(1-1) Identification of miRNA
Whether or not the small RNA sequence is a miRNA can be determined on the basis of whether or not the criteria described in RNA, 9, 277-279 (2003) are met. For example, in cases where the small RNA was newly acquired and the nucleotide sequence thereof was determined, this can be performed as described below.
A surrounding genomic sequence wherein a DNA sequence corresponding to the nucleotide sequence of the small RNA acquired is extended by about 50 nt toward the 5′ terminal side and the 3′ terminal side, respectively, is acquired, and the secondary structure of the RNA expected to be transcribed from the genomic sequence is predicted. If the result shows that a hairpin structure is present and the nucleotide sequence of the small RNA is located in one chain of the hairpin, the small RNA can be judged to be a miRNA. Genomic sequences are open to the general public, and are available from, for example, UCSC Genome Bioinformatics (http://genome.ucsc.edu/). For prediction of secondary structures, various programs are open; for example, RNAfold [Nucleic Acids Research 31, 3429-3431 (2003)], Mfold [Nucleic Acids Research 31, 3406-3415 (2003)] and the like can be used. Existing miRNA sequences are registered in the database called miRBase in Sanger Institute, UK; whether or not a miRNA is identical to an existing miRNA can be determined on the basis of whether or not the sequence thereof is identical to one of the sequences listed therein.
Total RNA is extracted from a human cancer cell line or a cancer patient-derived sample, and a small RNA comprising a miRNA expressed in cancer cells or cancer tissue can be acquired using the total RNA as described below.
As a method of acquiring a small RNA, specifically, a method wherein separation and cutting out of a small RNA by 15% polyacrylamide gel electrophoresis as described in Genes & Development 15, 188-200 (2000), can be mentioned. Then, 5′ terminal dephosphorylation, 3′-adapter ligation, phosphorylation, 5′-adapter ligation, reverse transcription, PCR amplification, concatemerization, and ligation to a vector are performed sequentially, thereafter the small RNA is cloned, and the nucleotide sequence of the clone is determined. Alternatively, for example, a method wherein 5′-adenylation 3′-adapter ligation, 5′-adapter ligation, reverse transcription, PCR amplification, concatemerization, and ligation to a vector are performed sequentially, thereafter the small RNA is cloned, and the nucleotide sequence of the clone is also determined, as described in Science 294, 858-862 (2001).
Alternatively, 5′ terminal dephosphorylation, 3′-adapter ligation, phosphorylation, 5′-adapter ligation, reverse transcription, PCR amplification, and ligation to a microbead vector are performed sequentially, thereafter the small RNA is cloned, and the nucleotide sequence of the microbeads is read to determine the nucleotide sequence, whereby nucleotide sequence information of the small RNA can also be acquired, as described in Nucleic Acids Research 34, 1765-1771 (2006).
As another method for acquiring a small RNA, a method using a small RNA Cloning Kit (manufactured by Takara Bio Inc.) can be mentioned.
(1-3) Method of Detecting the Expression Level of a Nucleic Acid Such as a miRNA
As examples of methods of detecting the expression level of a nucleic acid such as miRNA, a miRNA precursor and the like include (1) Northern hybridization, (2) dot blot hybridization, (3) in situ hybridization, (4) quantitative PCR, (5) differential hybridization, (6) microarray, (7) ribonuclease protection assay and the like.
The Northern blot method is a method wherein a sample-derived RNA is separated by gel electrophoresis, then transferred to a solid support such as a nylon filter, and an appropriately-labeled probe is prepared on the basis of the nucleotide sequence of a nucleic acid, and hybridization and washing are performed, whereby a band specifically bound to the nucleic acid is detected; specifically, for example, this method can be performed as described in Science 294, 853-858 (2001) and the like.
A labeled probe can be prepared by incorporating a radioisotope, biotin, digoxigenin, a fluorescent group, a chemiluminescent group and the like in a DNA, RNA, or LNA having a sequence complementary to the nucleotide sequence of a nucleic acid to be used in the present invention by a method, for example, nick translation, random priming or 5′-terminal phosphorylation. Because the amount of labeled probe bound reflects the expression level of the nucleic acid, the expression level of the nucleic acid can be quantified by quantifying the amount of labeled probe bound. Electrophoresis, membrane transfer, probe preparation, hybridization, and nucleic acid detection can be achieved by a method described in Molecular Cloning, 3rd edition [Cold Spring Harbor Press, (2001) Cold Spring Harbor, N.Y.].
Dot blot hybridization is a method wherein an RNA extracted from a tissue or a cell is spotted in dot forms and immobilized on a membrane, and hybridized with a labeled polynucleotide to be a probe, and an RNA that specifically hybridizes with the probe is detected. The probe used may be the same as that used for Northern hybridization. RNA preparation, RNA spotting, hybridization, and RNA detection can be achieved by a method described in Molecular Cloning, 3rd edition.
In situ hybridization is a method wherein a paraffin-embedded or cryostat-treated section of a tissue acquired from a living organism, or a cell fixed, is used as a sample and subjected to steps for hybridization with a labeled probe and washing, and the distribution and localization of a nucleic acid in the tissue or cell are examined by microscopic examination [Methods in Enzymology, 254, 419 (1995)]. The probe used may be the same as that used for Northern hybridization. Specifically, a miRNA can be detected in accordance with a method described in Nature Method 3, 27 (2006).
In quantitative PCR, a cDNA synthesized from a sample-derived RNA using a primer for reverse transcription and a reverse transcriptase (hereunder, this cDNA is referred to as a sample-derived cDNA) is used for the measurement. As a primer for reverse transcription to be supplied for cDNA synthesis, a random primer or a specific RT primer and the like can be used. A specific RT primer refers to a primer having a sequence complementary to a nucleotide sequence corresponding to a nucleic acid and a genomic sequence therearound.
For example, a sample-derived cDNA is synthesized, after which a PCR is performed with this cDNA as the template, using a template-specific primer designed from a nucleotide sequence corresponding to a nucleic acid such as miRNA or miRNA precursor and a genomic sequence therearound, or from a nucleotide sequence corresponding to a primer for reverse transcription, to amplify a cDNA fragment containing the nucleic acid, and the amount of the nucleic acid contained in the sample-derived RNA is detected from the number of cycles for reach to a given amount of the fragment. As the template-specific primer, an appropriate region corresponding to a nucleic acid and a genomic sequence therearound is selected, and a pair of a DNA or LNA consisting of a sequence of 15 to 40 nucleotides at the 5′ terminus, preferably 20 to 30 nucleotides of the nucleotide sequence of the region, and a DNA or LNA consisting of a sequence complementary to a sequence of 15 to 40 nucleotides at the 3′ terminus, preferably 20 to 30 nucleotides can be used. Specifically, this can be performed in accordance with a method described in Nucleic Acids Research, 32, e43 (2004) and the like.
Alternatively, as the primer for reverse transcription to be supplied for cDNA synthesis, a specific RT primer having a stem-loop structure can also be used. Specifically, this can be performed using a method described in Nucleic Acid Research, 33, e179 (2005), or TaqMan MiRNA Assays (manufactured by Applied Biosystems).
As still another method of synthesizing a sample-derived cDNA, a polyA sequence is added to a sample-derived RNA by means of polyA polymerase, and a nucleotide sequence comprising an oligodT sequence is used as a primer for reverse transcription, whereby a reverse transcription reaction can be performed. Specifically, this can be performed using the miScript System (manufactured by QIAGEN) or the QuantiMir RT Kit (manufactured by System Biosciences). Furthermore, by hybridizing a sample-derived RNA or cDNA to a DNA or LNA corresponding to a nucleotide sequence comprising at least one or more of a nucleic acid immobilized on a substrate such as a filter, glass slide, or silicone having, and performing washing, a change in the amount of the nucleic acid can be detected.
As such methods based on hybridization, methods using differential hybridization [Trends Genet., 7, 314 (1991)] or a microarray [Genome Res., 6, 639 (1996)] can be mentioned. Both methods enable accurate detection of a difference in the amount of a nucleic acid between a control sample and a target sample by immobilizing an internal control, such as a nucleotide sequence corresponding to U6RNA, on a filter or a substrate. Also, by synthesizing labeled cDNAs using differently labeled dNTPs (mixtures of dATP, dGTP, dCTP, and dTTP) on the basis of RNAs derived from a control sample and a target sample, and simultaneously hybridizing the two labeled cDNAs to a single filter or a single substrate, accurate quantitation of a nucleic acid can be performed. Furthermore, quantitation of a nucleic acid can also be performed by directly labeling and hybridizing an RNA derived from a control sample and/or a target sample. For example, a miRNA can be detected using a microarray described in Proc. Natl. Acad. Sci. USA, 101, 9740-9744 (2004), Nucleic Acid Research, 32, e188 (2004), RNA, 13, 151-159 (2007) and the like. Specifically, a miRNA can be detected or quantified in the same manner as mirVana miRNA Bioarray (manufactured by Ambion) and miRNA microarray kit (manufactured by Agilent Technologies).
In ribonuclease protection assay, first, a promoter sequence such as the T7 promoter or the SP6 promoter is bound to the 3′ terminus of a nucleotide sequence corresponding to a nucleic acid or a genomic sequence therearound, and a labeled antisense RNA is synthesized with an in vitro transcription system using a labeled NTP (a mixture of ATP, GTP, CTP, and UTP) and an RNA polymerase. The labeled antisense RNA is bound to a sample-derived RNA to form an RNA-RNA hybrid, after which the hybrid is digested with ribonuclease A, which degrades single-stranded RNAs only. The digest is subjected to gel electrophoresis to detect or quantify an RNA fragment protected against the digestion by forming the RNA-RNA hybrid, as a nucleic acid. Specifically, the fragment can be detected or quantified using the mirVana miRNA Detection Kit (manufactured by Ambion).
After a nucleic acid such as a miRNA or miRNA precursor expressed in cancer cells or cancer tissue, or exhibiting increased or decreased expression compared with normal tissue, has been identified as described in 1 above, not only an RNA, which is a polymer of a ribonucleotide, but also a DNA, which is a polymer of a deoxyribonucleotide, can be synthesized on the basis of the nucleotide sequences. For example, on the basis of the nucleotide sequence of the miRNA identified in 1 above, the nucleotide sequence of a DNA can be determined. The nucleotide sequence of a DNA corresponding to the nucleotide sequence of an RNA can be determined, without exception, by reading the U (uracil) contained in the sequence of the RNA as T (thymine). A polymer being a mixture of a ribonucleotide and a deoxyribonucleotide and a polymer comprising a nucleotide analogue and a derivative of a nucleic acid can also be synthesized in the same manner.
The method of synthesizing a nucleic acid is not particularly limited; the same can be produced by a method using a publicly known chemical synthesis, or an enzymatic transcription method and the like. As methods using a publicly known chemical synthesis, the phosphoroamidite method, the phosphorothioate method, the phosphotriester method and the like can be mentioned; for example, a nucleic acid can be synthesized using the ABI3900 high throughput nucleic acid synthesizer (manufactured by Applied Biosystems). As an enzymatic transcription method, a method by transcription with a plasmid or DNA having a desired nucleotide sequence as the template using a typical phage RNA polymerase, for example, T7, T3, or SP6RNA polymerase, can be mentioned.
3. Method of Detecting a Function of a Nucleic Acid Such as miRNA or miRNA Precursor
As a method of detecting a function of a nucleic acid such as a miRNA, a method can be mentioned wherein the function is detected on the basis of whether or not the translation of the mRNA having a target nucleotide sequence is suppressed.
MiRNAs are known to suppress the translation of a mRNA comprising a target nucleotide sequence thereof in the untranslated region on the 3′ side (3′UTR) [Current Biology, 15, R458-R460 (2005)]. Hence, a DNA wherein a target nucleotide sequence for the single-stranded RNA to be measured is inserted into the 3′UTR of an appropriate reporter gene expression vector is prepared and introduced into a host cell suitable for the expression vector, and the expression of the reporter gene is measured when the cell is allowed to express the single-stranded RNA, whereby whether or not a function of a miRNA is possessed can be determined.
The reporter gene expression vector may be any one, as far as it has a promoter upstream of a reporter gene, and is capable of expressing the reporter gene in the host cell. Any reporter gene can be used; for example, the firefly luciferase gene, the Renilla luciferase gene, the chloramphenicol acetyltransferase gene, the β-glucuronidase gene, the β-galactosidase gene, the β-lactamase gene, the aequorin gene, the green fluorescent protein gene, the DsRed fluorescence gene and the like can be utilized. As examples of reporter gene expression vectors having these properties, psiCHECK-1 (manufactured by Promega), psiCHECK-2 (manufactured by Promega), pGL3-Control (manufactured by Promega), pGL4 (manufactured by Promega), pRNAi-GL (manufactured by Takara Bio Inc.), pCMV-DsRed-Express (manufactured by CLONTECH) and the like can be mentioned. A single-stranded RNA can be expressed by the method described in 6 below.
As the function of a miRNA of a single-stranded RNA, specifically, detection can be achieved as described below. First, host cells are cultured on a multi-well plate and the like, and a reporter gene expression vector having a target sequence and a single-stranded RNA are expressed. Thereafter, reporter activity is measured both when the single-stranded RNA is and is not expressed, based on which the function as the miRNA of the single-stranded RNA can be detected.
4. Method of Detecting a Mutation of a Nucleic Acid Such as miRNA or miRNA Precursor
As a method of detecting a mutation of a nucleic acid such as a miRNA or miRNA precursor, a method can be used wherein a hetero-double-strand formed by hybridization of a normal type nucleic acid and a mutated type nucleic acid are detected.
As methods of detecting a hetero-double-strand, (1) detection of a hetero-double-strand by polyacrylamide gel electrophoresis [Trends genet., 7, 5 (1991)], (2) single-strand conformation polymorphism analysis [Genomics, 16, 325-332 (1993)], (3) chemical cleavage of mismatches (CCM) [Human Genetics (1996), Tom Strachan and Andrew P. Read, BIOS Scientific Publishers Limited], (4) enzymatic cleavage of mismatches [Nature Genetics, 9, 103-104 (1996)], (5) denatured gel electrophoresis [Mutat. Res., 288, 103-112 (1993)] and the like can be mentioned.
Detection of a hetero-double-strand by polyacrylamide gel electrophoresis is, for example, performed as described below. First, with a sample-derived DNA or a sample-derived cDNA as the template, and using a primer designed on the basis of a genomic nucleotide sequence comprising the nucleotide sequence of a nucleic acid, a fragment smaller than 200 by is amplified. Hetero-double-strands, if formed, are slower in mobility than mutation-free homo-double-strands, and can be detected as extra bands. In the case of search for a fragment smaller than 200 bp, almost any insertions, deletions, and substitutions of one or more nucleotides can be detected. It is desirable that hetero-double-strand analysis be performed using a single gel in combination with the single strand conformation analysis described below.
In single strand conformation polymorphism analysis (SSCP analysis), a DNA amplified as a fragment smaller than 200 by with a sample-derived DNA or sample-derived cDNA as the template, using a primer designed on the basis of a genomic nucleotide sequence comprising the nucleotide sequence of a nucleic acid, is denatured, after which it is electrophoresed in non-denatured polyacrylamide gel. By labeling the primer with an isotope or a fluorescent dye at the time of DNA amplification, or by silver-staining the non-labeled amplification product, the amplified DNA can be detected as a band. To clarify a difference from the wild type pattern, a control sample may be electrophoresed simultaneously, whereby a fragment with a mutation can be detected on the basis of a difference in mobility.
In chemical cleavage of mismatches (CCM method), a DNA fragment amplified with a sample-derived DNA or sample-derived cDNA as the template, using a primer designed on the basis of a genomic nucleotide sequence comprising the nucleotide sequence of a nucleic acid is hybridized to a labeled nucleic acid prepared by allowing a nucleic acid to incorporate an isotope or a fluorescent target, and treated with osmium tetraoxide to cleave one strand of the DNA at the mismatched portion, whereby a mutation can be detected. CCM is one of the most sensitive methods of detection, and can be applied to samples of kilobase length.
In place of osmium tetraoxide used above, T4 phage resolvase and an enzyme involved in mismatch repair in cells, such as endonuclease VII, and RNaseA may be used in combination to enzymatically cleave a mismatch. In denaturing gradient gel electrophoresis (DGGE method), a DNA amplified with a sample-derived DNA or sample-derived cDNA as the template, using a primer designed on the basis of a genomic nucleotide sequence comprising the nucleotide sequence of a nucleic acid is electrophoresed using a gel having a chemical denaturant density gradient or a temperature gradient. The DNA fragment amplified migrates in the gel to a position where it denatures to a single strand, and no longer migrates after the denaturation. Because the migration of the amplified DNA in the gel differs between the presence and absence of a mutation, the presence of the mutation can be detected. To increase the detection sensitivity, the addition of a poly (G:C) end to each primer is effective.
By directly determining and analyzing the nucleotide sequence of a sample-derived DNA or sample-derived cDNA, a mutation of a nucleic acid can also be detected.
5. Methods of Expressing a Nucleic Acid Such as miRNA or miRNA Precursor
Existing miRNA sequences and sequences of precursors thereof are registered with a database known as miRBase at the Sanger Institute in UK; nucleic acids such as miRNAs and miRNA precursors can be prepared by utilizing such sequences. Such nucleic acids can also be prepared using miRNA sequences acquired by the method described in 1.
A nucleic acid can be expressed by using a vector such that a nucleic acid is transcribed and hence biosynthesized when the vector is introduced into a cell. Specifically, by preparing a DNA fragment comprising a hairpin portion on the basis of the nucleotide sequence of a nucleic acid or a genomic nucleotide sequence comprising the foregoing nucleotide sequence, and inserting the fragment downstream of a promoter in the expression vector to construct an expression plasmids, and then introducing the expression plasmid into a host cell suitable for the expression vector, whereby a nucleic acid can be expressed.
As an expression vector, one capable of self-replication in the host cell, and capable of being incorporated in the chromosome, which comprises a promoter at a position enabling the transcription of a gene comprising the nucleotide sequence of a nucleic acid is used. The promoter may be any one, as far as it is capable of expressing in the host cell; for example, a RNA polymerase II (pol II) system promoter, a RNA polymerase III (pol III) system promoter being a U6RNA and H1RNA transcription system and the like can be mentioned. As examples of pol II system promoters, the promoter of the cytomegalovirus (human CMV) IE (immediate early) gene, the early promoter of SV40 and the like can be mentioned. As examples of expression vectors using them, pCDNA6.2-GW/miR (manufactured by Invitrogen), pSilencer 4.1-CMV (manufactured by Ambion) and the like can be mentioned. As pol III system promoters, U6RNA, H1RNA or tRNA gene promoters can be mentioned. As examples of expression vectors using them, pSINsi-hHl DNA (manufactured by Takara Bio Inc.), pSINsi-hU6 DNA (manufactured by Takara Bio Inc.), pENTR/U6 (manufactured by Invitrogen) and the like can be mentioned.
Alternatively, by inserting a gene comprising the nucleotide sequence of a nucleic acid into the downstream of the promoter in a viral vector to construct a recombinant viral vector, and introducing the vector into a packaging cell to produce a recombinant virus, the gene comprising the nucleic acid can be expressed.
The packaging cell may be any cell, as far as it is capable of supplementing a recombinant viral vector deficient in any one of the genes that encode the proteins necessary for the packaging of the virus with the lacked protein; for example, human kidney-derived HEK293 cells, mouse fibroblasts NIH3T3-derived cells and the like can be used. As the protein supplemented by the packaging cell, in the case of a retrovirus vector, proteins derived from a mouse retrovirus, such as gag, pol, and env, can be used; in the case of a lentivirus vector, proteins derived from a HIV virus, such as gag, pol, env, vpr, vpu, vif, tat, rev, and nef, can be used; in the case of an adenovirus vector, proteins derived from an adenovirus, such as E1A and E1B, can be used; in the case of an adeno-associated viral vector, proteins such as Rep (p5, p19, p40) and Vp(Cap), can be used.
In addition to using an expression vector, a nucleic acid to be used in the present invention can also be introduced directly into a cell, without using a vector. As the nucleic acid used in this technique, a DNA, an RNA, or a nucleotide analogue, as well as a chimeric molecule thereof, or a derivative of the nucleic acid can also be used. Specifically, in the same way as with Pre-miR™ miRNA Precursor Molecules (manufactured by Ambion) or miRIDIAN miRNA Mimics (manufactured by GE Healthcare), a nucleic acid such as miRNA or miRNA precursor can be expressed. Any method can be used to express a miRNA, as far as it allows the miRNA to be finally formed in the cells; for example, (1) a method wherein a single-stranded RNA that is a precursor of the miRNA is introduced, as well as (2) a method wherein an RNA consisting of a 100% matched double-strand consisting of the miRNA as it is and a complementary strand for the miRNA, is introduced, and (3) a method wherein a double-stranded RNA assumed to have resulted from cleavage of the miRNA with a Dicer is introduced, can be mentioned. As commercial products based on these methods, miCENTURY OX Precursor (manufactured by B-Bridge), miCENTURY OX siMature (manufactured by B-Bridge), and miCENTURY OX miNatural (manufactured by B-Bridge), respectively, can be mentioned.
6. Methods of Suppressing the Activity of a Nucleic Acid Such as miRNA or miRNA Precursor
The activation of a nucleic acid such as miRNA or miRNA precursor can be suppressed using an antisense technology [Bioscience and Industry, 50, 322 (1992); Kagaku, 46, 681 (1991), Biotechnology, 9, 358 (1992), Trends in Biotechnology, 10, 87 (1992), Trends in Biotechnology, 10, 152 (1992); Cell Technology, 16, 1463 (1997)], triple helix technology [Trends in Biotechnology, 10, 132 (1992)], ribozyme technology [Current Opinion in Chemical Biology, 3, 274 (1999), FEMS Microbiology Reviews, 23, 257 (1999), Frontiers in Bioscience, 4, D497 (1999), Chemistry & Biology, 6, R33 (1999), Nucleic Acids Research, 26, 5237 (1998), Trends In Biotechnology, 16, 438 (1998)], decoy DNA method [Nippon Rinsho—Japanese Journal of Clinical Medicine, 56, 563 (1998), Circulation Research, 82, 1023 (1998), Experimental Nephrology, 5, 429 (1997), Nippon Rinsho—Japanese Journal of Clinical Medicine, 54, 2583 (1996)], or a siRNA (short interfering RNA).
An antisense refers to one that allows nucleotide sequence-specific hybridization of a nucleic acid having a nucleotide sequence complementary to a certain target nucleic acid to enable the suppression of the expression of the target nucleic acid. As the nucleic acid used as the antisense, a DNA, an RNA or a nucleotide analogue, as well as a chimeric molecule thereof, or a derivative of the nucleic acid can also be used. Specifically, by preparing an antisense in accordance with a method described in Nature 432, 226 (2004) and the like, the expression can be suppressed. In addition, in the same way as with Anti-miR™ miRNA Inhibitors (manufactured by Ambion) and miRIDIAN miRNA Inhibitors (manufactured by GE Healthcare), the expression of a nucleic acid such as miRNA or miRNA precursor can be suppressed.
An siRNA refers to a short double-stranded RNA comprising the nucleotide sequence of a certain target nucleic acid, that is capable of suppressing the expression of the target nucleic acid by RNA interference (RNAi). The sequence of an siRNA can be designed as appropriate from the target nucleotide sequence on the basis of conditions shown in the literature [Genes Dev., 13, 3191 (1999)]. For example, by synthesizing two RNAs having a 19-nucleotide sequence selected and a complementary sequence, with TT added to the 3′ terminus of each thereof using a nucleic acid synthesizer, and performing annealing, an siRNA can be prepared. By inserting a DNA corresponding to the above-described 19-nucleotide sequence selected into an siRNA expression vector such as pSilencer 1.0-U6 (manufactured by Ambion) or pSUPER (manufactured by OligoEngine), a vector that expresses an siRNA capable of suppressing the expression of the gene can be prepared. Although any siRNA can be used to suppress a nucleic acid such as a miRNA used in the present invention, as far as it is capable of suppressing the activity of the nucleic acid, preference is given to siRNAs designed on the basis of continuous sequence information on the 9th nucleotide and beyond of each nucleotide sequence shown by any one of SEQ ID NO:1-939, 2057-2132, 2212-2261 and 2315-2321. The number of nucleotide residues constituting one strand of the siRNA is preferably 17 to 30 residues, more preferably 18 to 25 residues, still more preferably 19 to 23 residues.
Using an antisense or siRNA specific for a nucleic acid such as miRNA expressed in cancer cells, or a precursor of the miRNA, the expression of a miRNA expressed in cancer cells, a precursor of the miRNA and the like can be suppressed. For example, by administering an antisense DNA or siRNA specific for the miRNA or the miRNA precursor, the activation of the miRNA can be suppressed, and the action of the miRNA or miRNA precursor in cancer cells can be controlled.
In addition, in the case of a patient affected by an abnormality of the expression of a miRNA expressed in cancer cells or a precursor thereof, by administering an antisense oligonucleotide or siRNA specific for the miRNA or precursor thereof to the patient, it is possible to control a function of cancer cells to treat a disease that develops as a result of the above-described expression abnormality. Hence, an antisense oligonucleotide or siRNA that is specific for the miRNA or precursor thereof is useful as a therapeutic agent for a disease caused by a cell proliferation abnormality of cancer cells.
When an antisense oligonucleotide or siRNA specific for a nucleic acid such as a miRNA or a precursor thereof is used as the above-described therapeutic agent, the antisense oligonucleotide or siRNA alone, or a nucleic acid that encodes the same after being inserted into an appropriate vector such as a retrovirus vector, adenovirus vector, or adeno-associated virus vector, may be prepared as a pharmaceutical preparation according to the conventional method described in 9 below, and administered.
7. Methods of Suppressing Functions of Genes Using a Nucleic Acid Such as miRNA or miRNA Precursor
Any method can be used to suppress a function or the expression of the target gene for a nucleic acid, as far as it is based on an activity of a nucleic acid such as a miRNA to suppress the expression of the mRNA having the target nucleotide sequence. For example, a method can be mentioned wherein a nucleic acid such as a miRNA is expressed to increase the amount of the nucleic acid such as the miRNA in cells, whereby the translation of the mRNA having the target sequence is suppressed to suppress the expression of the gene. The expression of a nucleic acid can be achieved by the method described in 5 above. As examples of mRNAs having a target nucleotide sequence for a nucleic acid consisting of a nucleotide sequence shown by any one of SEQ ID NO:1-939, 2057-2132, 2212-2261 and 2315-2321, the respective sets of genes shown in the foregoing Table 4 can be mentioned.
Using an siRNA against a target gene shown in Table 4, it is possible to suppress a function of the target gene.
8. Methods of Screening for Substances that Promote or Suppress the Expression or a Function of a Nucleic Acid Such as a miRNA or miRNA Precursor Using the Nucleic Acid
A substance that promotes or suppresses the expression or a function of a nucleic acid such as a miRNA or precursor thereof can be screened for using a nucleic acid. For example, a nucleotide sequence to be targeted for screening is chosen from the nucleotide sequence of a nucleic acid, and by means of cells that express a nucleic acid having the nucleotide sequence, a substance that promotes or suppresses the expression or a function of the chosen miRNA or precursor thereof can be screened for.
As cells that express a miRNA or miRNA precursor and the like, used for screening, cancer cells, as well as transformant cells obtained by introducing a vector that expresses a nucleic acid having the nucleotide sequence into a host cell such as an animal cell or yeast, cells incorporating a nucleic acid having the nucleotide sequence introduced directly without using a vector and the like as described in 5 above can also be used.
As specific screening methods, a method comprising using a change in the expression level of a nucleic acid such as an miRNA or precursor thereof to be targeted in the screening as an index, as well as a method comprising using a change in the expression level of a mRNA having a target sequence for a nucleic acid such as a miRNA or a gene product encoded thereby as an index can be mentioned.
(a) Screening Method Comprising Using a Change in the Expression Level of a Nucleic Acid Such as a miRNA or Precursor Thereof to be Targeted in the Screening as an Index
A test substance is brought into contact with a cell that expresses a nucleic acid, and with a change in the expression level of the nucleic acid selected as an index, a substance that promotes or suppresses the nucleic acid of expression such as a miRNA and precursor thereof is obtained. The expression level of a nucleic acid can be detected by the method described in 3 above.
(b) Screening Method Comprising Using a Change in the Expression Level of a mRNA Having a Target Sequence for a Nucleic Acid Such as a miRNA to be Targeted in the Screening or a Gene Product Encoded Thereby as an Index
A test substance is brought into contact with a cell that expresses the mRNA, and with a change in the expression level of a mRNA having a target sequence of the nucleic acid selected or a gene product encoded thereby as an index, a substance that promotes or suppresses the expression or a function of a nucleic acid such as a miRNA and a precursor thereof is obtained. Alternatively, a DNA having a target sequence of a nucleic acid such as miRNA inserted into the 3′ UTR of an appropriate reporter gene expression vector is prepared and introduced into a host cell suitable for the expression vector, a test substance is brought into contact with the cell, and with a change in the expression level of the reporter gene as an index, a substance that promotes or suppresses the expression or a function of a nucleic acid such as a miRNA and a precursor thereof is obtained.
Choice of a target sequence can be achieved by the method described in 7 above; as examples of a mRNA having a target sequence of a nucleic acid such as a miRNA consisting of the nucleotide sequence of any one of SEQ ID NO:1 to 939, 2057 to 2132, 2212 to 2261 and 2315 to 2321, the above-described genes shown in Table 4 above can be exemplified.
9. Agent for Suppressing or Promoting Cell Proliferation Using a Nucleic Acid Such as miRNA or miRNA Precursor
Nucleic acids such as miRNAs and miRNA precursors, and nucleic acids having nucleotide sequences complementary to the nucleotide sequences thereof can be utilized as agents for suppressing or promoting cell proliferation because they control the expression of genes having target sequences.
As agents for suppressing cell proliferation, the nucleic acids (a) to (h) below can be mentioned.
As agents for promoting cell proliferation, the following nucleic acids (i)-(p) can be mentioned.
The above-described nucleic acids are suitably miRNAs or miRNA precursors.
As agents for promoting cell proliferation, nucleic acids consisting of a nucleotide sequence complementary to the nucleotide sequence of one of the nucleic acids (a) to (h) above can be mentioned; as agents for suppressing cell proliferation, nucleic acids consisting of a nucleotide sequence complementary to the nucleotide sequence of one of the nucleic acids (i) to (p) above can be mentioned. Vectors that express the above-described nucleic acids and nucleic acids that are complementary thereto can also be used as agents for suppressing or promoting cell proliferation.
Meanwhile, substances that suppress the expression of target genes for nucleic acids such as the above-described miRNAs, and substances that promote the expression of the target genes can also be used as agents for suppressing or promoting cell proliferation. As these substances, nucleic acids or vectors that express the same can also be used. As substances that suppress the expression of a target gene, an siRNA against the mRNA of the target gene and an antisense against the target gene can be mentioned; as substances that promote the expression of the target gene, an siRNA against a miRNA specific for the target gene and an antisense against the target gene can be mentioned.
Regarding the preparation forms, methods of administration and the like for an agent for suppressing or promoting cell proliferation, the same applies as with the diagnostic reagent and therapeutic drug containing a nucleic acid such as a miRNA or miRNA precursor described in 10 below.
10. Diagnostic Reagent and Therapeutic Drug Containing a Nucleic Acid Such as a miRNA or miRNA Precursor
Nucleic acids such as miRNAs and miRNA precursors can be utilized as drugs for treating diseases resulting from a cell proliferation abnormality and the like, such as cancers, because they control the expression of genes having a target sequence, or control the expression of nucleic acids such as miRNAs. Furthermore, siRNAs against the target gene for the nucleic acid can be utilized as drugs for treating diseases resulting from a cell proliferation or differentiation abnormality and the like because they control the expression of the gene. As diseases resulting from a cell proliferation abnormality and the like, cancers, arteriosclerosis, rheumatoid arthritis, prostatic hyperplasia, blood vessel restenosis after percutaneous transvascular coronary angioplasty, fibroid lung, glomerulonephritis, autoimmune diseases and the like can be mentioned.
By quantifying a nucleic acid or detecting a mutation therein, a disease resulting from a cell proliferation or differentiation abnormality and the like, such as a cancer, can be diagnosed.
A diagnostic reagent comprising a nucleic acid, according to the desired diagnostic method, may comprise reagents necessary for quantitation or detection of mutation of a nucleic acid, for example, buffering agents, salts, reaction enzymes, labeled proteins that bind to a nucleic acid, and a color developer for detection and the like.
Although a therapeutic agent containing a nucleic acid as an active ingredient can be administered alone, the same is normally desirably administered as a pharmaceutical preparation produced by an optionally chosen method known well in the technical field of pharmaceutical making with one or more pharmacologically acceptable carriers blended therein.
The route of administration used is desirably the most effective one in treatment; oral administration, or parenteral administration such as intraoral administration, airway administration, intrarectal administration, subcutaneous administration, intramuscular administration and intravenous administration can be mentioned, and desirably intravenous administration can be mentioned.
As dosage forms, sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injection formulations, ointments, tapes and the like can be mentioned. As preparations appropriate for oral administration, emulsions, syrups, capsules, tablets, powders, granules and the like can be mentioned.
Liquid preparations like emulsions and syrups can be produced using water, saccharides such as sucrose, sorbitol, and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil, and soybean oil, antiseptics such as p-hydroxybenzoic acid esters, flavors such as strawberry flavor and peppermint and the like as additives.
Capsules, tablets, powders, granules and the like can be produced using excipients such as lactose, glucose, sucrose, and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, binders such as polyvinyl alcohol, hydroxypropylcellulose, and gelatin, surfactants such as fatty acid esters, plasticizers such as glycerin and the like as additives.
As appropriate preparations for parenteral administration, injection formulations, suppositories, sprays and the like can be mentioned.
An injection formulation is prepared using a carrier consisting of a salt solution, a glucose solution or a mixture of both and the like. A suppository is prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid. A spray is prepared using a carrier that does not stimulate the recipient's oral cavity and airway mucosa, and that disperses the active ingredients as fine particles to facilitate the absorption thereof, and the like.
As examples of the carrier, specifically, lactose, glycerin and the like can be exemplified. Depending on the nature of the nucleic acid or miRNA precursor, and of the carrier used, preparations such as aerosols and dry powders are possible. In these parenteral preparations, components exemplified as additives for oral preparations can also be added.
The dose or frequency of administration varies depending on desired therapeutic effect, method of administration, duration of treatment, age, body weight and the like, and is normally 10 μg/kg to 20 mg/kg per day for an adult.
A therapeutic drug comprising a nucleic acid as an active ingredient can also be produced by blending a vector that expresses a nucleic acid and a nucleotide used in a nucleic acid therapeutic drug [Nature Genet., 8, 42(1994)].
The base used in the therapeutic drug may be any base for ordinary use in injection formulations; distilled water, solutions of salts such as sodium chloride or a mixture of sodium chloride and an inorganic salt, solutions of mannitol, lactose, dextran, glucose and the like, solutions of amino acids such as glycine and arginine, mixed solutions of organic acid solutions or salt solutions and glucose solution and the like can be mentioned. In accordance with a conventional method, using auxiliary agents such as an osmoregulator, a pH adjuster, a vegetable oil such as sesame oil or soybean oil, lecithin, and a surfactant such as a non-ionic surfactant in these bases, an injection formulation may be prepared as a solution, suspension, or dispersion. These injection formulations can also be prepared as preparations for dissolution before use, by procedures such as powdering and lyophilization. A therapeutic agent can be used for treatment as is in the case of a liquid, or after being dissolved in a base described above, optionally sterilized, in the case of a solid, just before treatment.
As a vector expressing a nucleic acid, the recombinant viral vector prepared in the method of the above-mentioned 5 can be mentioned, more specifically, retrovirus vector and lentivirus vector and the like can be mentioned.
For example, by combining a nucleic acid with a polylysine-conjugated antibody that is specific for adenovirus hexon protein to prepare a complex, and binding the complex obtained to an adenovirus vector, a viral vector can be prepared. The viral vector is capable of stably reaching the desired cell, being incorporated into cells by endosome, being decomposed in the cells, and efficiently expressing the nucleic acid.
A viral vector based on Sendai virus, which is a (−) strand RNA virus, has been developed (WO97/16538, WO97/16539), a Sendai virus incorporating a nucleic acid can be prepared using the Sendai virus.
A nucleic acid can also be transferred by a non-viral nucleic acid transfer method. The same can be transferred by, for example, calcium phosphate co-precipitation [Virology, 52, 456-467 (1973); Science, 209, 1414-1422 (1980)], microinjection method [Proc. Natl. Acad. Sci. USA, 77, 5399-5403 (1980); Proc. Natl. Acad. Sci. USA, 77, 7380-7384 (1980); Cell, 27, 223-231 (1981); Nature, 294, 92-94 (1981)], membrane fusion-mediated transfer mediated by liposome [Proc. Natl. Acad. Sci. USA, 84, 7413-7417 (1987); Biochemistry, 28, 9508-9514 (1989); J. Biol. Chem., 264, 12126-12129 (1989); Hum. Gene Ther., 3, 267-275, (1992); Science, 249, 1285-1288 (1990); Circulation, 83, 2007-2011 (1992)] or direct DNA uptake and receptor-mediated DNA transfer method [Science, 247, 1465-1468 (1990); J. Biol. Chem., 266, 14338-14342 (1991); Proc. Natl. Acad. Sci. USA, 87, 3655-3659 (1991); J. Biol. Chem., 264, 16985-16987 (1989); BioTechniques, 11, 474-485 (1991); Proc. Natl. Acad. Sci. USA, 87, 3410-3414 (1990); Proc. Natl. Acad. Sci. USA, 88, 4255-4259 (1991); Proc. Natl. Acad. Sci. USA, 87, 4033-4037 (1990); Proc. Natl. Acad. Sci. USA, 88, 8850-8854 (1991); Hum. Gene Ther., 3, 147-154 (1991)] and the like.
Membrane fusion-mediated transfer mediated by liposome allows a nucleic acid to be incorporated locally in the tissue, and to be expressed, by administering a liposome preparation directly to the target tissue [Hum. Gene Ther., 3, 399 (1992)]. For direct targeting of a DNA to a focus, direct DNA uptake technology is preferable.
For receptor-mediated DNA transfer, for example, a method performed by binding a DNA (usually assuming the form of a covalently cyclized supercoiled plasmid) to a protein ligand via polylysine can be mentioned. A ligand is chosen on the basis of the presence of a corresponding ligand receptor on the cell surface of the desired cell or tissue. The ligand-DNA conjugate can be injected directly into a blood vessel as desired, and can be directed to a target tissue wherein receptor binding and DNA-protein complex internalization occur. To prevent the destruction of the DNA in a cell, an adenovirus may be infected simultaneously to destroy the endosome function.
The method of measuring cell proliferation is not particularly limited, as far as it enables a measurement of an index that reflects cell count or cell proliferation rate. Viable cell counting, DNA synthesis rate measurements, total protein content measurements and the like can be used. As a method of evaluating viable cell counts, a method wherein the ATP content in cells is measured can be mentioned. It is known that the ATP content in cells is proportional to the number of cells in culture (J. Immunol. Meth. 160, 81-88 (1993)). As more specific methods of measuring the ATP content in cells, the MTT method, the XTT method and the like can be mentioned (J. Immunol. Meth. 65, 55-63 (1983)). A method can also be mentioned wherein ATP is measured by luminescence of a luciferin substrate by the ATP-dependent enzyme luciferase. As a kit for measuring the ATP content in cells, for example, CellTite-Glo® Luminescent Cell viability Assay (manufactured by Promega) and the like may be used.
As methods of measuring the degree of cell death, a method wherein dead cells are stained with a dye such as Propium Iodide, a method wherein the activity of an enzyme leaked extracellularly as a result of cell death is measured, and the like can be used. For the latter, for example, a method wherein the enzyme activity of extracellularly leaked adenylate kinase is measured can be utilized. More specifically, ToxiLight(R) Non-Destructive Cytotoxicity BioAssay Kit (manufactured by Lonza) and the like may be used.
It is also possible to measure the degree of apoptosis (programmed cell death), which is important in relation to cancers, out of the various types of cell death. As methods of evaluating cell apoptosis, a method wherein the degree of DNA fragmentation is measured, a method wherein changes in cell membrane constituent lipids are measured, and a method wherein the activity of caspase 3/7, an intracellular protease induced upon apoptosis, is measured, can be mentioned. As a more specific method of measuring caspase 3/7 activity in cells, a method wherein a luciferin substrate liberated by caspase 3/7 activity is measured by luciferin luminescence by a luciferase enzyme reaction can be mentioned. As kits for measuring caspase 3/7 activity in cells, for example, Caspase-Glo® 3/7 Assay (manufactured by Promega) and the like may be used.
The present invention is hereinafter described specifically by means of the following Examples. However, the present invention is not limited to these Examples.
Cell Viability and Apoptotic Activity in Colorectal Cancer-Derived Cell Line with a miRNA Expressed Forcedly Therein
Pre-miR™ miRNA Precursor Molecules (manufactured by Ambion) were introduced into a colorectal cancer-derived cell line, and the influences of the miRNAs produced from the molecules on cell viability and apoptotic activity were examined.
The DLD-1 human colorectal cancer-derived cell line (hereinafter referred to as DLD-1) was obtained from the American Type Culture Collection (hereinafter referred to as ATCC). Cells were cultured with an RPMI1640 medium (manufactured by Invitrogen) containing 10% fetal bovine serum (FBS) (manufactured by JRH Biosciences) in a 37° C. 5% CO2 concentration incubator.
DLD-1 was sown to a 96-well plate at 2500 cells per well, and cultured in an RPMI medium containing 10% FBS overnight. One day later, Pre-miR™miRNA Precursor Molecules that produce hsa-miR-7 (SEQ ID NO:3), 15a, 15b, 16, 18a (SEQ ID NO:5), 18a* (SEQ ID NO:7), 28 (SEQ ID NO:17), 95 (SEQ ID NO:24), 124a, 132 (SEQ ID NO:29), 133a (SEQ ID NO:31), 133b (SEQ ID NO:32), 147 (SEQ ID NO:37), 181b, 181c (SEQ ID NO:39), 181d (SEQ ID NO:40), 184 (SEQ ID NO:43), 192 (SEQ ID NO:44), 193a (SEQ ID NO:46), 193b (SEQ ID NO:47), 195 (SEQ ID NO:48), 196a (SEQ ID NO:51), 196b (SEQ ID NO:52), 197 (SEQ ID NO:53), 202 (SEQ ID NO:56), 204 (SEQ ID NO:57), 211 (SEQ ID NO:58), 212 (SEQ ID NO:30), 215 (SEQ ID NO:45), 299-3p (SEQ ID NO:62), 299-5p (SEQ ID NO:63), 324-3p (SEQ ID NO:66), 337 (SEQ ID NO:70), 342 (SEQ ID NO:72), 362 (SEQ ID NO:74), 368 (SEQ ID NO:75), 376a (SEQ ID NO:77), 376b (SEQ ID NO:78), 380-5p (SEQ ID NO:81), 422a (SEQ ID NO:84), 422b (SEQ ID NO:85), 424 (SEQ ID NO:49), 432* (SEQ ID NO:88), 452 (SEQ ID NO:92), 452* (SEQ ID NO:93), 455 (SEQ ID NO:94), 483 (SEQ ID NO:95), 489 (SEQ ID NO:98), 491 (SEQ ID NO:99), 493-5p (SEQ ID NO:100), 494 (SEQ ID NO:101), 497 (SEQ ID NO:50), 501 (SEQ ID NO:102), 504 (SEQ ID NO:103), 506 (SEQ ID NO:105), 512-3p (SEQ ID NO:110), 512-5p (SEQ ID NO:111), 517* (SEQ ID NO:114), 517b (SEQ ID NO:117), 517c (SEQ ID NO:116), 518b (SEQ ID NO:118), 518c* (SEQ ID NO:121), 518d (SEQ ID NO:120), 523 (SEQ ID NO:125), 527 (SEQ ID NO:129), let-7g, and let-7i were introduced into the DLD-1 to obtain a final concentration of 5 nM or 25 nM by a lipofection method, specifically a method using Lipofectamine2000 (manufactured by Invitrogen). Pre-miR™miRNA Precursor Molecules-Negative Control #2 (hereinafter, referred to as miR-negacon#2) (manufactured by Ambion) was also introduced into DLD-1, and this was used as the negative control. The lipofection was performed per the directions attached to the product.
Three days after introduction of the molecules by a lipofection method, cell viability was measured using CellTiter-Glo™luminescent Cell Viability Assay (manufactured by Promega) according to the method described in the protocol attached to the product. Taking the cell viability of DLD-1 in a control plot (Lipofectamine2000 only) as 1.0, the relative cell viability of each was calculated. As a result, as shown in Table 5, with introduction of molecules that produce hsa-miR-7, 28, 95, 147, 181d, 192, 193a, 193b, 195, 196a, 197, 211, 215, 299-3p, 299-5p, 337, 342, 362, 368, 376a, 376b, 380-5p, 422b, 432*, 452, 483, 489, 491, 493-5p, 494, 501, 506, 517c, 518b, and 518d, decreases of 40% or more in cell viability were observed. With introduction of molecules that produce hsa-miR-15b and 124a, which are known to be associated with cancers, decreases of 40% or more in cell viability were observed.
Two days after introduction of the molecules (final concentration 25 nM) by a lipofection method, caspase 3/7 activity was measured using Caspase-GloR 3/7 assay (manufactured by Promega) per the directions described in the protocol attached to the product. Taking the caspase 3/7 activity value of DLD-1 in a control plot (Lipofectamine2000 only) as 1.0, the relative caspase 3/7 activity value of each was calculated. As a result, as shown in Table 6, with introduction of molecules that produce hsa-miR-18a, 18a*, 132, 133a, 133b, 181c, 181d, 184, 192, 193a, 195, 196a, 196b, 197, 202, 204, 211, 212, 215, 299-3p, 299-5p, 324-3p, 337, 342, 362, 368, 376a, 380-5p, 422a, 422b, 424, 432*, 452*, 455, 483, 491, 493-5p, 497, 501, 504, 512-3p, 512-5p, 517*, 517b, 517c, 518c*, 523, and 527, increases of 50% or more in caspase 3/7 activity were observed. With introduction of molecules that produce hsa-miR-15a, 15b, 16, 181b, let-7g, and let-7i, which are known to be associated with cancers, increases of 50% or more in caspase 3/7 activity were observed.
In another test, DLD-1 was sown to a 96-well plate at 2500 cells per well, and cultured in an RPMI medium containing 10% FBS overnight. One day later, Pre-miR™miRNA Precursor Molecules (manufactured by Ambion) that produce hsa-miR-7 (SEQ ID NO:3), 24 (SEQ ID NO:11), 28 (SEQ ID NO:17), 34c, 95 (SEQ ID NO:24), 124a, 132 (SEQ ID NO:29), 134 (SEQ ID NO:33), 142-3p (SEQ ID NO:35), 147 (SEQ ID NO:37), 181a, 182* (SEQ ID NO:41), 183 (SEQ ID NO:42), 192 (SEQ ID NO:44), 197 (SEQ ID NO:53), 211 (SEQ ID NO:58), 222 (SEQ ID NO:61), 299-3p (SEQ ID NO:62), 329 (SEQ ID NO:68), 337 (SEQ ID NO:70), 342 (SEQ ID NO:72), 362 (SEQ ID NO:74), 368 (SEQ ID NO:75), 376a (SEQ ID NO:77), 376b (SEQ ID NO:78), 377 (SEQ ID NO:79), 380-5p (SEQ ID NO:81), 422b (SEQ ID NO:85), 424 (SEQ ID NO:49), 431 (SEQ ID NO:87), 432* (SEQ ID NO:88), 452 (SEQ ID NO:92), 452* (SEQ ID NO:93), 455 (SEQ ID NO:94), 483 (SEQ ID NO:95), 485-5p (SEQ ID NO:96), 489 (SEQ ID NO:98), 491 (SEQ ID NO:99), 493-5p (SEQ ID NO:100), 494 (SEQ ID NO:101), 505 (SEQ ID NO:104), 506 (SEQ ID NO:105), 509 (SEQ ID NO:107), 510 (SEQ ID NO:108), 517a (SEQ ID NO:115), and 517c (SEQ ID NO:116) were introduced into the DLD-1 to obtain a final concentration of 50 nM by a lipofection method, specifically a method using Lipofectamine2000 (manufactured by Invitrogen). miR-negacon#2 (manufactured by Ambion) was also introduced into DLD-1, and this was used as the negative control. The lipofection was performed per the directions attached to the product.
Three or five days after introduction of the molecules by the lipofection method, cell viability was measured using CellTiter-Glo™Luminescent Cell Viability Assay (manufactured by Promega) per the directions attached to the product. Taking the cell viability for the DLD-1 in the miR-negacon#2 introduction plot as 1.0, respective relative cell viability was calculated. As a result, as shown in Table 7, with introduction of molecules that produce hsa-miR-7, 24, 28, 95, 132, 134, 142-3p, 147, 182*, 183, 192, 197, 211, 222, 299-3p, 329, 337, 342, 362, 368, 376a, 376b, 377, 380-5p, 422b, 424, 431, 432*, 452, 452*, 455, 483, 485-5p, 489, 491, 493-5p, 494, 505, 506, 509, 510, 517a, and 517c, decreases of 50% or more in viable cell activity were observed. With introduction of molecules that produce hsa-miR-34c, 181a, and 124a, which are known to be associated with cancers, a decrease of 50% or more in cell viability was observed.
Antisense oligonucleotides that are complementary to miRNAs were introduced into DLD-1 to obtain a final concentration of 50 nM using a lipofection method. As the antisense oligonucleotides that are complementary to the miRNAs, Anti-miR™miRNA Inhibitors of hsa-miR-197 (SEQ ID NO:53) and 483 (SEQ ID NO:95) (manufactured by Ambion) were used. Anti-miR™miRNA Inhibitors-Negative Control#1 (hereinafter, referred to as anti-miR-negacon#1) (manufactured by Ambion) was also introduced into DLD-1, and this was used as the negative control. The lipofection was performed per the directions attached to the product.
Three days after introduction of the antisense oligonucleotides by the lipofection method, cell viability was measured using CellTiter-Glo™Luminescent Cell Viability Assay (manufactured by Promega) per the directions attached to the product. Taking the cell viability for the DLD-1 in the anti-miR-negacon#1 introduction plot as 1.0, respective relative cell viability was calculated. As a result, as shown in Table 8, with introduction of antisense oligonucleotides that are complementary to hsa-miR-197 and 483, decreases of 50% or more in cell viability were observed.
Cell Viability and Apoptotic Activity in Ovarian Cancer-Derived Cell Line with miRNA Expressed Forcedly Therein
Pre-miR™miRNA Precursor Molecules (manufactured by Ambion) were introduced into an ovarian cancer-derived cell line, and the influences of the miRNAs produced from the molecules on cell viability and apoptotic activity were examined.
The A2780 human ovarian cancer-derived cell line (Nature, 295, 116-119 (1982); Science, 224, 994-996 (1984); Semin. Oncol., 11, 285-298 (1984); hereinafter to be referred to as A2780) was cultured with an RPMI1640 medium (manufactured by Invitrogen) containing 5% FBS (manufactured by JRH Biosciences) in a 37° C. 5% CO2 concentration incubator.
The A2780 was sown to a 96-well plate at 2500 cells per well, and cultured with an RPMI medium containing 10% FBS overnight. One day later, Pre-miR™ miRNA Precursor Molecules that generate hsa-miR-1 (SEQ ID NO:1), 7 (SEQ ID NO:3), 15a, 15b, 16, 18a (SEQ ID NO:5), 18a* (SEQ ID NO:7), 18b (SEQ ID NO:6), 28 (SEQ ID NO:17), 34b, 95 (SEQ ID NO:24), 98 (SEQ ID NO:25), 124a, 133a (SEQ ID NO:31), 133b (SEQ ID NO:32), 143, 145, 147 (SEQ ID NO:37), 181c (SEQ ID NO:39), 192 (SEQ ID NO:44), 193a (SEQ ID NO:46), 193b (SEQ ID NO:47), 195 (SEQ ID NO:48), 196a (SEQ ID NO:51), 196b (SEQ ID NO:52), 197 (SEQ ID NO:53), 199a* (SEQ ID NO:54), 202 (SEQ ID NO:56), 204 (SEQ ID NO:57), 211 (SEQ ID NO:58), 212 (SEQ ID NO:30), 215 (SEQ ID NO:45), 299-3p (SEQ ID NO:62), 324-3p (SEQ ID NO:66), 324-5p (SEQ ID NO:67), 337 (SEQ ID NO:70), 342 (SEQ ID NO:72), 346 (SEQ ID NO:73), 362 (SEQ ID NO:74), 368 (SEQ ID NO:75), 376a (SEQ ID NO:77), 376b (SEQ ID NO:78), 378 (SEQ ID NO:80), 380-5p (SEQ ID NO:81), 382 (SEQ ID NO:82), 422a (SEQ ID NO:84), 422b (SEQ ID NO:85), 424 (SEQ ID NO:49), 432* (SEQ ID NO:88), 451 (SEQ ID NO:91), 452 (SEQ ID NO:92), 452* (SEQ ID NO:93), 455 (SEQ ID NO:94), 483 (SEQ ID NO:95), 489 (SEQ ID NO:98), 491 (SEQ ID NO:99), 493-5p (SEQ ID NO:100), 494 (SEQ ID NO:101), 497 (SEQ ID NO:50), 501 (SEQ ID NO:102), 504 (SEQ ID NO:103), 506 (SEQ ID NO:105), 511 (SEQ ID NO:109), 512-5p (SEQ ID NO:111), 517a (SEQ ID NO:115), 517c (SEQ ID NO:116), 518b (SEQ ID NO:118), 518c (SEQ ID NO:119), 518c* (SEQ ID NO:121), 518d (SEQ ID NO:120), 527 (SEQ ID NO:129), let-7a, let-7b, let-7d, let-7d, let-7e, let-7f, let-7g, and let-7i were introduced into the A2780 to obtain a final concentration of 5 nM or 25 nM using a lipofection method, specifically Lipofectamine2000 (manufactured by Invitrogen). miR-negacon#2 (manufactured by Ambion) was also introduced into A2780, and this was used as the negative control. The lipofection was performed per the directions attached to the product.
Three days after introduction of the molecules by the lipofection method, cell viability was measured using CellTiter-Glo™Luminescent Cell Viability Assay (manufactured by Promega) per the directions attached to the product. Taking the cell viability for the A2780 in the control plot (Lipofectamine2000 only) as 1.0, respective relative cell viability was calculated. As a result, as shown in Table 9 (Table 9-1 and Table 9-2), with introduction of molecules that produce hsa-miR-1, 7, 18a, 18a*, 18b, 28, 95, 98, 133a, 133b, 147, 181c, 192, 193a, 193b, 195, 196a, 196b, 197, 199a*, 202, 204, 211, 212, 215, 299-3p, 324-3p, 324-5p, 342, 346, 362, 368, 376a, 376b, 378, 380-5p, 382, 422a, 422b, 424, 432*, 451, 452, 452*, 455, 483, 489, 491, 493-5p, 494, 497, 501, 504, 506, 511, 512-5p, 517a, 517c, 518b, 518c, 518c*, 518d, and 527, decreases of 40% or more in viable cell activity were observed. With introduction of molecules that produce hsa-miR-15a, 15b, 16, 34b, 124a, 143, 145, let-7a, let-7b, let-7d, let-7e, let-7f, let-7g, and let-7i, which are known to be associated with cancers, decreases of 40% or more in cell viability were observed.
Two days after introduction of the molecules (final concentration 25 nM) by the lipofection method, caspase 3/7 activity was measured using Caspase-Glo®3/7 assay (manufactured by Promega) per the directions attached to the product. Taking the caspase 3/7 activity value for the A2780 in the control plot (Lipofectamine2000 only) as 1.0, respective relative caspase 3/7 activity values were calculated. As a result, as shown in Table 10, with introduction of molecules that produce hsa-miR-18a*, 133a, 193a, 193b, 197, 199a*, 212, 324-3p, 337, 432*, 452*, 489, 491, 493-5p, 512-5p, 517a, 517c, and 527, increases of 50% or more in caspase 3/7 activity were observed. With introduction of a molecule that produces hsa-miR-124a, which is known to be associated with cancers, an increase of 50% or more in caspase 3/7 activity was observed.
In another test, A2780 was sown to a 96-well plate at 2500 cells per well, and cultured in an RPMI medium containing 10% FBS overnight. One day later, Pre-miR™miRNA Precursor Molecules of hsa-miR-7 (SEQ ID NO:3), 15a, 15b, 18a* (SEQ ID NO:7), 24 (SEQ ID NO:11), 26a (SEQ ID NO:14), 26b (SEQ ID NO:15), 34a, 34b, 34c, 124a, 130a (SEQ ID NO:26), 130b (SEQ ID NO:27), 134 (SEQ ID NO:33), 147 (SEQ ID NO:37), 192 (SEQ ID NO:44), 193a (SEQ ID NO:46), 193b (SEQ ID NO:47), 196a (SEQ ID NO:51), 196b (SEQ ID NO:52), 197 (SEQ ID NO:53), 199a* (SEQ ID NO:54), 204 (SEQ ID NO:57), 206 (SEQ ID NO:2), 211 (SEQ ID NO:58), 212 (SEQ ID NO:30), 215 (SEQ ID NO:45), 301 (SEQ ID NO:28), 346 (SEQ ID NO:73), 368 (SEQ ID NO:75), 371 (SEQ ID NO:76), 376b (SEQ ID NO:78), 378 (SEQ ID NO:80), 422a (SEQ ID NO:84), 424 (SEQ ID NO:49), 431 (SEQ ID NO:87), 432* (SEQ ID NO:88), 433 (SEQ ID NO:89), 449 (SEQ ID NO:90), 452 (SEQ ID NO:92), 483 (SEQ ID NO:95), 485-5p (SEQ ID NO:96), 488 (SEQ ID NO:97), 489 (SEQ ID NO:98), 491 (SEQ ID NO:99), 493-5p (SEQ ID NO:100), 497 (SEQ ID NO:50), 504 (SEQ ID NO:103), 505 (SEQ ID NO:104), 506 (SEQ ID NO:105), 507 (SEQ ID NO:106), 510 (SEQ ID NO:108), 511 (SEQ ID NO:109), 512-5p (SEQ ID NO:111), 513 (SEQ ID NO:112), 517a (SEQ ID NO:115), 517c (SEQ ID NO:116), 518b (SEQ ID NO:118), 518c (SEQ ID NO:119), 518d (SEQ ID NO:120), 526a (SEQ ID NO:128), 527 (SEQ ID NO:129), let-7b, let-7c, and let-7i were introduced into the A2780 to obtain a final concentration of 50 nM using a lipofection method, specifically Lipofectamine2000 (manufactured by Invitrogen). miR-negacon#2 (manufactured by Ambion) was also introduced into A2780, and this was used as the negative control. The lipofection was performed per the directions attached to the product.
Three or five days after introduction of the molecules by the lipofection method, cell viability was measured using CellTiter-Glo™Luminescent Cell Viability Assay (manufactured by Promega) per the directions attached to the product. Taking the cell viability for the A2780 in the miR-negacon#2 introduction plot as 1.0, respective relative cell viability was calculated. As a result, as shown in Table 11 (Table 11-1 and Table 11-2), with introduction of molecules that produce hsa-miR-7, 18a*, 24, 26a, 26b, 134, 147, 192, 193a, 193b, 196a, 196b, 197, 199a*, 204, 206, 211, 212, 215, 346, 368, 371, 376b, 378, 422a, 424, 431, 432*, 433, 449, 452, 483, 485-5p, 488, 489, 491, 493-5p, 497, 504, 505, 506, 507, 510, 511, 512-5p, 513, 517a, 517c, 518b, 518c, 518d, 526a, and 527, decreases of 50% or more in cell viability were observed. With introduction of miRNA precursors of molecules that produce hsa-miR-15a, 15b, 34a, 34b, 34c, 124a, let-7b, let-7c, and let-7i, which are known to be associated with cancers, decreases of 50% or more in cell viability were observed. Conversely, with introduction of molecules that produce hsa-miR-130a, 130b, and 301, increases in cell viability were observed.
Antisense oligonucleotides that are complementary to miRNAs were introduced into A2780 to obtain a final concentration of 50 nM using a lipofection method. As the antisense oligonucleotides that are complementary to the miRNAs, Anti-miR™miRNA Inhibitors (manufactured by Ambion) of hsa-miR-10a (SEQ ID NO:4), 20b (SEQ ID NO:8), 25 (SEQ ID NO:12), 27b (SEQ ID NO:16), 29a (SEQ ID NO:18), 29c (SEQ ID NO:19), 30b (SEQ ID NO:20), 30c (SEQ ID NO:21), 32 (SEQ ID NO:13), 93 (SEQ ID NO:22), 106a (SEQ ID NO:9), 106b (SEQ ID NO:10), 137 (SEQ ID NO:34), 146a (SEQ ID NO:36), 149 (SEQ ID NO:38), 191, 195 (SEQ ID NO:48), 200a (SEQ ID NO:55), 204 (SEQ ID NO:57), 205 (SEQ ID NO:59), 208 (SEQ ID NO:60), 212 (SEQ ID NO:30), 215 (SEQ ID NO:45), 301 (SEQ ID NO:28), 302a* (SEQ ID NO:64), 302b* (SEQ ID NO:65), 302d (SEQ ID NO:23), 324-3p (SEQ ID NO:66), 331 (SEQ ID NO:69), 340 (SEQ ID NO:71), 362 (SEQ ID NO:74), 412 (SEQ ID NO:83), 423 (SEQ ID NO:86), 451 (SEQ ID NO:91), 452 (SEQ ID NO:92), 483 (SEQ ID NO:95), 516-5p (SEQ ID NO:113), 518e (SEQ ID NO:122), 518f (SEQ ID NO:123), 520h (SEQ ID NO:124), 524* (SEQ ID NO:126), and 525 (SEQ ID NO:127) were used. Anti-miR-negacon#1 (manufactured by Ambion) was also introduced into A2780, and this was used as the negative control. The lipofection was performed per the directions attached to the product.
Three days after introduction of the antisense oligonucleotides by the lipofection method, cell viability was measured using CellTiter-Glo™luminescent Cell Viability Assay (manufactured by Promega) per the directions attached to the product. Taking the cell viability for the A2780 in the anti-miR-negacon#1 introduction plot as 1.0, respective relative cell viability was calculated. As a result, as shown in Table 12, with introduction of the antisense oligonucleotides that are complementary to hsa-miR-10a, 20b, 25, 27b, 29a, 29c, 30b, 30c, 32, 93, 106a, 106b, 137, 146a, 149, 191, 195, 200a, 204, 205, 208, 212, 215, 301, 302a*, 302b*, 302d, 324-3p, 331, 340, 362, 412, 423, 451, 452, 483, 516-5p, 518e, 518f, 520h, 524*, and 525, decreases of 40% or more in cell viability were observed.
Cell Viability and Apoptotic Activity in Prostatic Cancer-Derived Cell Line with miRNA Expressed Forcedly Therein
Pre-miR™ miRNA Precursor Molecules (manufactured by Ambion) were introduced into a prostatic cancer-derived cell line, and the influences of the miRNAs produced from the molecules on cell viability and apoptotic activity were examined.
The PC-3 human prostatic cancer-derived cell line (hereinafter referred to as PC-3) was obtained from ATCC, and cultured with F-12 Kaighn's medium (manufactured by Invitrogen) containing 10% fetal bovine serum (FBS) (manufactured by JRH Biosciences) in an incubator at 37° C. and a 5% CO2 concentration.
The PC-3 was sown to a 96-well plate at 3000 cells per well, and cultured in F-12 Kaighn's containing 10% FBS overnight. One day later, Pre-miR™ miRNA Precursor Molecules that produce hsa-miR-15a, 15b, 16, 18a* (SEQ ID NO:7), 34b, 124a, 133b (SEQ ID NO:32), 181b, 192 (SEQ ID NO:44), 195 (SEQ ID NO:48), 196b (SEQ ID NO:52), 324-3p (SEQ ID NO:66), 337 (SEQ ID NO:70), 362 (SEQ ID NO:74), 368 (SEQ ID NO:75), 376b (SEQ ID NO:78), 380-5p (SEQ ID NO:81), 432* (SEQ ID NO:88), 452 (SEQ ID NO:92), 452* (SEQ ID NO:93), 489 (SEQ ID NO:98), 491 (SEQ ID NO:99), 493-5p (SEQ ID NO:100), and 494 (SEQ ID NO:101) were introduced into the PC-3 to obtain a final concentration of 25 nM using a lipofection method, specifically Lipofectamine2000 (manufactured by Invitrogen). miR-negacon#2 (manufactured by Ambion) was also introduced into PC-3, and this was used as the negative control. The lipofection was performed per the directions attached to the product.
Three days after introduction of the molecules by the lipofection method, cell viability was measured using CellTiter-Glo™Luminescent Cell Viability Assay (manufactured by Promega) per the directions attached to the product. Taking the cell viability for the PC-3 in the control plot (Lipofectamine2000 only) as 1.0, respective relative cell viability was calculated. As a result, as shown in Table 13, with introduction of the molecules that produce hsa-miR-18a*, 133b, 192, 195, 196b, 324-3p, 337, 362, 368, 376b, 380-5p, 432*, 452, 452*, 489, 491, 493-5p, and 494, decreases of 40% or more in cell viability were observed. With the molecules that produce hsa-miR-15a, 15b, 16, 34b, 124a, and 181b, which are known to be associated with cancers, decreases of 40% or more in cell viability were observed.
Two days after introduction of the molecules by the lipofection method, caspase 3/7 activity was measured using Caspase-Glo® 3/7 assay (manufactured by Promega) per the directions attached to the product. Taking the caspase 3/7 activity value for the PC-3 in the control plot (Lipofectamine2000 only) as 1.0, respective relative caspase 3/7 activity values were calculated. As a result, as shown in Table 14, with introduction of the molecules that produce hsa-miR-133b, 337, 362, 432*, 452*, 491, and 493-5p, increases of 50% or more in caspase 3/7 activity were observed. With introduction of the molecule that produces hsa-miR-181b, which is known to be associated with cancers, an increase of 50% or more in caspase 3/7 activity was observed.
BrdU Uptake Activity in Colorectal Cancer-Derived Cell Line (DLD-1) with miRNAs Expressed Forcedly Therein
Pre-miR™ miRNA Precursor Molecules (manufactured by Ambion) were introduced into DLD-1, and the influences of the miRNAs produced from the molecules on BrdU uptake activity were examined.
The DLD-1 was sown to a 96-well plate at 2500 cells per well, and cultured in an RPMI medium containing 10% FBS overnight. One day later, Pre-miR miRNA Precursor Molecules that produce hsa-miR-147 (SEQ ID NO:37), 342 (SEQ ID NO:72), 362 (SEQ ID NO:74), 376b (SEQ ID NO:78), 483 (SEQ ID NO:95), and 494 (SEQ ID NO:101) were introduced into the DLD-1 to obtain a final concentration of 25 nM using a lipofection method, specifically Lipofectamine2000 (manufactured by Invitrogen). miR-negacon#2 (manufactured by Ambion) was also introduced into DLD-1, and this was used as the negative control. The lipofection was performed per the directions attached to the product.
One day after introduction of the molecules by the lipofection method, BrdU uptake activity was measured using a BrdU labeling & detection kit III (manufactured by Roche) per the directions attached to the product. Taking the BrdU uptake activity for the DLD-1 in the miR-negacon#2 introduction plot as 1.0, respective relative BrdU uptake activities were calculated. As a result, as shown in Table 15, with introduction of the molecules that produce hsa-miR-147, 342, 362, 376b, 483, and 494, decreases of 40% or more in BrdU uptake activity were observed.
BrdU Uptake Activity in Ovarian Cancer-Derived Cell Line (A2780) with miRNAs Expressed Forcedly Therein
Pre-miR™miRNA Precursor Molecules (manufactured by Ambion) were introduced into A2780, and the influences of the miRNAs produced from the molecules on BrdU uptake activity were examined.
A2780 was sown to a 96-well plate at 4000 cells per well, 25 and cultured in an RPMI medium containing 10% FBS overnight. One day later, the Pre-miR™ miRNA Precursor Molecules of hsa-miR-15a, 16, 124a, 147 (SEQ ID NO:37), 192 (SEQ ID NO:44), 193a (SEQ ID NO:46), 193b (SEQ ID NO:47), 215 (SEQ ID NO:45), 378 (SEQ ID NO:80), 432* (SEQ ID NO:88), 506 (SEQ ID NO:105), and 518c (SEQ ID NO:119) were introduced into the A2780 to obtain a final concentration of 25 nM using a lipofection method, specifically Lipofectamine2000 (manufactured by Invitrogen). miR-negacon#2 (manufactured by Ambion) was also introduced into A2780, and this was used as the negative control. The lipofection was performed per the directions attached to the product.
One day after introduction of the molecules by the lipofection method, BrdU uptake activity was measured using the BrdU labeling & detection kit III (manufactured by Roche) per the directions attached to the product. Taking the BrdU uptake activity for the A2780 in the miR-negacon#2 introduction plot as 1.0, respective relative BrdU uptake activities were calculated. As a result, as shown in Table 16, with introduction of the molecules that produce hsa-miR-hsa-miR-147, 192, 193a, 193b, 215, 378, 432*, 506, and 518c, decreases of 40% or more in BrdU uptake activity were observed. With introduction of the molecules that produce hsa-miR-15a, 16, and 124a, which are known to be associated with cancers, decreases of 40% or more in BrdU uptake activity were observed.
Analysis of the Expression Level of Proteins Encoded by Target Genes for hsa-miR-337, 342, 432*, 491, and 518b
Molecules that produce hsa-miR-337, 342, 432*, 491, and 518b, which exhibited DLD-1 proliferation suppression or caspase activity in Example 1, were introduced into DLD-1, and changes in the amounts of proteins encoded by the target genes were examined.
Pre-miR™miRNA Precursor Molecules (manufactured by Ambion) that produce hsa-miR-16, 147, 337, 342, 432*, 491, and 518b were introduced into previously sown 1×105 cells of DLD-1 to obtain a final concentration of 25 nM, using Lipofectamine2000. miR-negacon#2 was also introduced, and this was used as the negative control. Also, si-Bc1211, si-Birc5, and si-Bc191 (manufactured by QIAGEN) were introduced to obtain a final concentration of 25 nM, and these were used as the positive controls. Two days after the introduction, the cells were recovered in solution in an RIPA buffer [50 mM Tris-HCL (pH 8.0), 150 mM NaCl, 1% Nonident P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1% Protease Inhibitor Cocktail SetIII (manufactured by Calbiochem)]. After separation by a conventional method of SDS-PAGE, the amounts of the proteins of Bc1211, Birc5 and Bc191 were detected by a Western blotting method. For detection of Bc1211, an anti-Bc1211 antibody (manufactured by BD Biosciences) was used; for detection of Birc5, an anti-Birc5 antibody (manufactured by R&D Systems) was used; for detection of Bc191, an anti-Bc191 antibody (manufactured by Abnova) was used. Also detected was the amount of the alpha-tubulin protein; this was used as the control for the amount of sample supplied. To detect alpha-tubulin, an anti-alpha-tubulin antibody (manufactured by Sigma) was used.
As a result, as shown in
Cell Viability and Apoptotic Activity in Colorectal Cancer-Derived Cell Line with miRNAs Expressed Forcedly Therein
DLD-1 was sown to a 96-well plate at 2500 cells per well, and cultured in an RPMI medium containing 10% FBS overnight. One day later, Pre-miR™miRNA Precursor Molecules that produce hsa-miR-493-3p (SEQ ID NO:2061), 542-3p (SEQ ID NO:2063), 571 (SEQ ID NO:2076), 579 (SEQ ID NO:2077), 625 (SEQ ID N0:2099), 630 (SEQ ID NO:2101), 634 (SEQ ID NO:2103), 650 (SEQ ID NO:2108), and 653 (SEQ ID NO:2110) were introduced into the DLD-1 to obtain a final concentration of 50 nM using a lipofection method, specifically Lipofectamine2000 (manufactured by Invitrogen). miR-negacon#2 (manufactured by Ambion) was also introduced into DLD-1, and this was used as the negative control. The lipofection was performed per the directions attached to the product.
Three days after introduction of the miRNA precursors by the lipofection method, cell viability was measured using CellTiter-Glo™Luminescent Cell Viability Assay (manufactured by Promega) per the directions attached to the product. Taking the cell viability for the DLD-1 in the miR-negacon#2 introduction plot as 1.0, respective relative cell viability was calculated. As a result, as shown in Table 17, with introduction of the molecules that produce hsa-miR-493-3p, 542-3p, 571, 579, 625, 630, 634, 650, and 653, decreases of 50% or more in cell viability were observed.
In another test, two days after introduction of the Pre-miR™ miRNA Precursor Molecules that produces hsa-miR-634 by the lipofection method, caspase 3/7 activity was measured using Caspase-GloR 3/7 assay (manufactured by Promega) per the directions attached to the product. Taking the caspase activity for the DLD-1 in the miR-negacon#2 introduction plot as 1.0, relative caspase activity was calculated. As a result, with introduction of the molecule that produces hsa-miR-634, an increase of 50% or more in caspase activity was observed.
Antisense oligonucleotides that are complementary to miRNAs were introduced into DLD-1 to obtain a final concentration of 50 nM using a lipofection method. As the antisense oligonucleotides that are complementary to the miRNAs, Anti-miR™ miRNA Inhibitors (manufactured by Ambion) of hsa-miR-411 (SEQ ID NO:2057), 425-5p (SEQ ID NO:2059), 449b (SEQ ID NO:917), 532 (SEQ ID NO:2062), 548b (SEQ ID NO:2066), 558 (SEQ ID NO:2072), 589 (SEQ ID NO:2079), 593 (SEQ ID NO:2081), 599 (SEQ ID NO:2083), 615 (SEQ ID NO:2093), 637 (SEQ ID NO:2104), 657 (SEQ ID NO:2112), 769-5p (SEQ ID NO:2114), and 92b (SEQ ID NO:819) were used. Anti-miR-negacon#1 (manufactured by Ambion) was also introduced into DLD-1, and this was used as the negative control. The lipofection was performed per the directions attached to the product.
Three days after introduction of the antisense oligonucleotides by the lipofection method, cell viability was measured using CellTiter-Glo™Luminescent Cell Viability Assay (manufactured by Promega) per the directions attached to the product. Taking the cell viability for the DLD-1 in the anti-miR-negacon#1 introduction plot as 1.0, respective relative cell viability was calculated. As a result, as shown in Table 18, with introduction of the antisense oligonucleotides complementary to hsa-miR-411, 425-5p, 449b, 532, 548b, 558, 589, 593, 599, 615, 637, 657, 769-5p, and 92b, decreases of 40% or more in cell viability were observed.
Cell Viability in Ovarian Cancer-Derived Cell Line with miRNAs Expressed Forcedly Therein
A2780 was sown to a 96-well plate at 2500 cells per well, and cultured in an RPMI medium containing 10% FBS overnight. One day later, Pre-miR™ miRNA Precursor Molecules that produce hsa-miR-421 (SEQ ID NO:2058), 454-5p (SEQ ID NO:2060), 542-3p (SEQ ID NO:2063), 545 (SEQ ID NO:2064), 548a (SEQ ID NO:2065), 548d (SEQ ID NO:2067), 550 (SEQ ID NO:2068), 551b (SEQ ID NO:2069), 552 (SEQ ID NO:2070), 553 (SEQ ID NO:2071), 557 (SEQ ID NO:925), 561 (SEQ ID NO:2073), 563 (SEQ ID NO:916), 565 (SEQ ID NO:2074), 570 (SEQ ID NO:2075), 571 (SEQ ID NO:2076), 584 (SEQ ID NO:2078), 591 (SEQ ID NO:2080), 595 (SEQ ID NO:2082), 600 (SEQ ID NO:2084), 601 (SEQ ID NO:2085), 604 (SEQ ID NO:2086), 605 (SEQ ID NO:2087), 606 (SEQ ID NO:2088), 609 (SEQ ID NO:2089), 610 (SEQ ID NO:2090), 611 (SEQ ID NO:2091), 613 (SEQ ID NO:803), 614 (SEQ ID NO:2092), 618 (SEQ ID NO:2096), 621 (SEQ ID NO:2097), 624 (SEQ ID NO:2098), 625 (SEQ ID NO:2099), 627 (SEQ ID NO:2100), 630 (SEQ ID NO:2101), 631 (SEQ ID NO:2102), 634 (SEQ ID NO:2103), 637 (SEQ ID NO:2104), 646 (SEQ ID NO:2105), 647 (SEQ ID NO:2106), 648 (SEQ ID NO:2107), 653 (SEQ ID NO:2110), 654 (SEQ ID NO:2111), and 661 (SEQ ID NO:2113) were introduced into the A2780 to obtain a final concentration of 50 nM using a lipofection method, specifically Lipofectamine2000 (manufactured by Invitrogen). miR-negacon#2 (manufactured by Ambion) was also introduced into A2780, and this was used as the negative control. The lipofection was performed per the directions attached to the product.
Three days after introduction of the molecules by the lipofection method, cell viability was measured using CellTiter-Glo™luminescent Cell Viability Assay (manufactured by Promega) per the directions attached to the product. Taking the cell viability for the A2780 in the miR-negacon#2 introduction plot as 1.0, respective relative cell viability was calculated. As a result, as shown in Table 19, with introduction of the molecules that produce hsa-miR-421, 454-5p, 542-3p, 545, 548a, 548d, 550, 551b, 552, 553, 557, 561, 563, 565, 570, 571, 584, 591, 595, 600, 601, 604, 605, 606, 609, 610, 611, 613, 614, 618, 621, 624, 625, 627, 630, 631, 634, 637, 646, 647, 648, 653, 654, and 661, decreases of 50% or more in cell viability were observed.
Antisense oligonucleotides that are complementary to miRNAs were introduced into A2780 to obtain a final concentration of 50 nM using a lipofection method. As the antisense oligonucleotides that are complementary to the miRNAs, Anti-miR™miRNA Inhibitors (manufactured by Ambion) of hsa-miR-611 (SEQ ID NO:2091), 616 (SEQ ID NO:2094), 617 (SEQ ID NO:2095), 651 (SEQ ID NO:2109), and 657 (SEQ ID NO:2112) were used. Anti-miR-negacon#1 (manufactured by Ambion) was also introduced into A2780, and this was used as the negative control. The lipofection was performed per the directions attached to the product.
Three days after introduction of the antisense oligonucleotides by the lipofection method, cell viability was measured using CellTiter-Glo™Luminescent Cell Viability Assay (manufactured by Promega) per the directions attached to the product. Taking the cell viability for the A2780 in the anti-miR-negacon#1 introduction plot as 1.0, respective relative cell viability was calculated. As a result, as shown in Table 20, with introduction of the antisense oligonucleotides that are complementary to hsa-miR-611, 616, 617, 651, and 657, decreases of 40% or more in cell viability were observed.
Tumorigenesis by Transplantation of Colorectal Cancer-Derived Cell Line with miRNAs Expressed Forcedly Therein to Nude Mice
DLD-1 incorporating molecules that produce hsa-miR-147, 342, and 491, which exhibited DLD-1 proliferation suppression or caspase activity in Example 1, was transplanted to nude mice, and the influences of miRNAs on tumorigenesis were examined.
Pre-miR™ miRNA Precursor Molecules (manufactured by Ambion) that produce hsa-miR-147, 342, and 491 were introduced into previously sown 1×106 cells of DLD-1 to obtain a final concentration of 50 nM using Lipofectoamine 2000. miR-negacon#2 was also introduced, and this was used as the negative control. One day after the introduction, cells were detached using 0.05% trypsin solution (manufactured by Gibco). The detached cells were mixed with Matrigel (manufactured by BD Bioscience), and 2.5×105 cells were subcutaneously transplanted to the flanks of nude mice at 6 weeks of age. The major diameters and minor diameters of the tumors formed after 7, 10, 13, and 16 days of transplantation were measured. Tumor size was calculated as major diameterx(minor diameter)2×0.5. As shown in
As shown in
An agent for suppressing or promoting cell proliferation, a diagnostic reagent or therapeutic drug for a disease resulting from a cell proliferation abnormality, an agent for inducing apoptosis, an agent for suppressing or promoting the expression of a target gene for a nucleic acid such as a miRNA, and a method of suppressing or promoting cell proliferation can be provided.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2007-260393 | Oct 2007 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2008/068109 | 10/3/2008 | WO | 00 | 7/6/2010 |
