WO2011111715A1

WO2011111715A1 - Nucleic acid capable of regulating cell cycle

Info

Publication number: WO2011111715A1
Application number: PCT/JP2011/055412
Authority: WO
Inventors: 圭太木下; 哲郎吉田; 陽史山田
Original assignee: 協和発酵キリン株式会社
Priority date: 2010-03-09
Filing date: 2011-03-08
Publication date: 2011-09-15

Abstract

A cell growth regulator, a diagnostic or therapeutic agent for diseases associated with abnormal progression of cell cycle, an inhibitor, a promoter or the like for the expression of a target gene for a nucleic acid such as micro-RNA, and a method for screening for a cell growth regulator, each of which utilizes a nucleic acid comprising a nucleotide sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 920, a nucleic acid having a 90% or higher identity with the aforementioned nucleic acid or a nucleic acid capable of hybridizing with a complementary strand of the aforementioned nucleic acid under stringent conditions, or a nucleic acid comprising a nucleotide sequence lying between position-2 to position-8 in a nucleotide sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 407, a nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence for the aforementioned nucleic acid or the like.

Description

Nucleic acids that control the cell cycle

The present invention relates to a cell growth regulator using a nucleic acid, a diagnostic or therapeutic agent for a disease caused by an abnormal cell cycle, a cell cycle variation method, a cell growth control method, a nucleic acid target gene expression suppression method or expression promotion method, The present invention relates to a screening method for a cell growth regulator.

MicroRNA (miRNA), which is a kind of nucleic acid, is a small non-coding single-stranded RNA consisting of about 22 nucleotides that are not translated into protein, and is known to exist in many organisms including humans (non-patent literature). 1, 2). MicroRNAs are generated from genes that are transcribed into single or clustered microRNA precursors. That is, the primary transcript, primary-microRNA (pri-miRNA), is first transcribed from the gene, and then in a stepwise process from pri-miRNA to mature microRNA, a precursor of about 70 bases with a characteristic hairpin structure. -microRNA (pre-miRNA) is generated from pri-miRNA. Furthermore, mature microRNA is generated from pre-miRNA by Dicer-mediated processing (Non-patent Document 3).

Mature microRNAs are thought to be involved in post-transcriptional control of gene expression by binding complementarily to the target mRNA and suppressing translation of the mRNA, or by degrading the mRNA.
Although the mechanism by which microRNA suppresses the expression of target mRNA is not completely clarified, an outline has been elucidated by recent studies. MicroRNA binds to a partially complementary sequence in the 3 'untranslated region (3'-UTR) of the target mRNA, suppresses its translation, or suppresses expression by degrading the target mRNA . Although the above complementarity may not be perfect, it has been shown that complementarity of the 2nd to 8th bases from the 5 ′ end of the microRNA is particularly important. Sometimes referred to as a “seed sequence” (Non-Patent Document 4). It has been shown that microRNAs with a common seed sequence suppress the expression of a common target mRNA even if other sequences are different. Therefore, RNA having microRNA-like activity can be designed by using a sequence complementary to the sequence present at the 3 ′ end of any mRNA as a seed sequence. Unlike siRNA, microRNA usually refers only to RNA that is “naturally present in cells”. Therefore, a microRNA-like sequence designed in this way may be particularly referred to as “artificial microRNA”.

As of July 2009, the microRNA database miRBase (http://microrna.sanger.ac.uk/) contains 885 microRNAs for humans and 10,097 types for all biological species. Among the microRNAs expressed in mammals including humans, those known for their physiological functions include miR-181 (Non-patent Document 5) involved in blood cell differentiation and miR-375 (involved in insulin secretion). Non-patent document 6) is only a small part, and many of them have unclear physiological activity. However, studies using nematodes and Drosophila have revealed that microRNAs play various important roles in the development and differentiation of living organisms. There has been a report suggesting a relationship (Non-patent Document 7).

Since microRNA is involved in the control of the expression of various genes, abnormalities in microRNA are expected to be involved in various human diseases. Research is progressing especially in cancer, and it has been reported that the expression of microRNA is different from that in normal tissues in many cancers, and that cancer can be classified by analyzing the expression profile of microRNA ( Non-patent document 8). It is also known that about half of the human microRNAs found so far are present at chromosomal abnormalities or fragile sites of chromosomes known in human cancer (Non-patent Document 9).

Examples of cancer-microRNA relationships reported so far include the miR-15a / miR-16 cluster on chromosome 13q14, which is deleted in B-cell chronic lymphocytic leukemia (B-CLL), It is predicted that the deletion is one of the causes of B-CLL (Non-patent Document 10). In B-CLL, the expression of miR-29 and miR-181 is further reduced. One of the targets is known to be Tcl1 known as a proto-oncogene (Non-patent Document 11). In lung cancer, expression of Let-7, which is one of microRNAs, is decreased, and one of its targets is Ras known as a proto-oncogene (Non-patent Documents 12 and 13). In addition, the expression of microRNAs such as miR-127 and miR-124a is reduced by hypermethylation modification of genes in cancer, and the target is Bcl6 or Cdk6 known as proto-oncogenes (Non-Patent Document 14, 15) etc. are known. Many microRNAs have decreased expression in cancer cells, but there are also microRNAs in which gene amplification and overexpression are seen in cancer cells. For example, in a region where gene amplification is observed in malignant lymphoma, there are 6 types of microRNA clusters (miR-17-92). When this miRNA cluster gene is forcibly expressed in a model mouse of human B cell lymphoma, lymphoma It is known that the occurrence of the above is promoted (Non-patent Document 16). It has also been clarified that a gene called BIC, which has been regarded as a candidate oncogene that does not encode a protein overexpressed in Hodgkin lymphoma, encodes miR-155 (Non-patent Document 17).

As described above, many relations between cancer and microRNA have been reported in recent years, but many of them show abnormal expression in cancer cells, and reports showing the function of microRNA include Let-7 and miR-143. Report that the growth of cancer cell lines was inhibited by forced expression of miR-145 and cancer cells (Non-Patent Documents 18, 19, and 20), and cancer cells by forced expression of miR-16 and miR-34 There are few reports such as reports that the cell cycle of the strain was stopped (Non-Patent Documents 21 and 22), and reports that cell transformation was caused by forced expression of miR-372 and miR-373 (Non-Patent Document 23). . Cancer growth in an animal model by increasing microRNA expression by administering microRNA or its precursor from outside the body, or by reducing its expression by administering its antisense oligonucleotide Reported that tumor formation of colon cancer cell lines transplanted by miR-34a administration was suppressed (Non-patent Document 24), tumors of breast cancer cell lines transplanted by miR-21 antisense administration There are only a few reports (Non-patent Document 25) that formation was suppressed.

In general, it is widely known that abnormalities in the cell cycle are one of the causes of cancer. In normal cell cycle, there is a mechanism called checkpoint in G1 / S phase, G2 / M phase, M phase, etc. and stops cell cycle in response to adverse growth conditions. . On the other hand, it has been reported that in many cancer cells, these cell cycle checkpoints are broken as a result of gene abnormalities and the like, resulting in abnormal cell proliferation. It is also known that many genes that control the cell cycle function as oncogenes and tumor suppressor genes. Therefore, anticancer agents aimed at selectively causing cancer cell death by acting on these cell cycles have also been developed, especially the taxane system that disrupts microtubule spindle formation, which is important for M-phase progression Many compounds and vinca alkaloid compounds are already on the market. In addition, CDK inhibitors, M phase kinase (PLK, Aurora) inhibitors and the like are expected as anticancer agents as low molecular weight compounds that act on cell cycle regulators and stop the cell cycle at a certain place.

Although the relationship between microRNA and cell cycle is still unknown, several microRNAs have recently been reported to be involved in the cell cycle. Examples of microRNAs involved in the cell cycle include Let-7 family targeting Cdc34 and accumulating cells in G2 / M phase (Non-patent Document 26), miR-107 and miR-185 are non-small cell lung cancer The cell line is arrested in the G1 phase (Non-patent Document 27), and miR-21 and miR-221 antisense bodies arrest pancreatic cancer cells in the G1 phase, and then cause apoptosis (Non-patent Document 28). )and so on. There is also a report that found a microRNA that acts on the cell cycle of mouse ES cells (Non-patent Document 29). However, there has been no report to date that microRNAs that affect the cell cycle of cancer cells have been exhaustively searched, and as a result, microRNAs that cause cancer cell growth suppression and cell death have been found.

It is expected that new therapeutic agents and diagnostic agents will be developed by identifying microRNAs expressed in various human organs, analyzing their functions, and elucidating the relationship with diseases.
In particular, the discovery of microRNAs that cause cell cycle fluctuations in cancer cells and, as a result, suppress cell growth of cancer cells and cause cell death, not only helps understand the mechanism of carcinogenesis, It is expected that it will lead to the development of therapeutic drugs, and to new diagnostic and therapeutic methods for cancer using them. In addition to diseases other than cancer, cell proliferation abnormalities such as arteriosclerosis, rheumatoid arthritis, prostatic hypertrophy, vascular restenosis after percutaneous transvascular coronary angioplasty, pulmonary fibrosis, glomerulonephritis, autoimmune diseases, It is expected to contribute to the development of diagnostic and therapeutic agents for diseases caused by tissue hyperplasia and the like, and diagnostic and therapeutic methods using them.

An object of the present invention is to provide nucleic acids useful for cell cycle control and methods for using them.

The present invention relates to the following [1] to [29].
[1] A cell growth regulator comprising as an active ingredient any of the following nucleic acids (a) to (h):
(A) Nucleic acid comprising a base sequence represented by any of SEQ ID NOs: 1 to 407 (b) Nucleic acid comprising 17 to 28 bases comprising a nucleic acid comprising a base sequence represented by any of SEQ ID NOs: 1 to 407 (C) a nucleic acid comprising a base sequence represented by any one of SEQ ID NOs: 1 to 407 and having a nucleotide sequence having 90% or more identity (d) comprising a base sequence represented by any of SEQ ID NOs: 1 to 407 Nucleic acid that hybridizes with a complementary strand of nucleic acid under stringent conditions (e) A nucleic acid comprising the second to eighth base sequences of the base sequence represented by any one of SEQ ID NOs: 1 to 407 (f) SEQ ID NOs: 408 to 920 A nucleic acid consisting of a base sequence represented by any of (g) a nucleic acid comprising a base sequence represented by any one of SEQ ID NOs: 408 to 920 and a base sequence having 90% or more identity (h) SEQ ID NOs: 408 to Any of 920 A nucleic acid that hybridizes to a complementary strand under stringent conditions nucleic acid consisting of the nucleotide sequence.
[2] The cell growth regulator according to [1], wherein the nucleic acid is microRNA or a microRNA precursor.
[3] A cell growth regulator comprising, as an active ingredient, a nucleic acid having a base sequence complementary to the base sequence of the nucleic acid according to [1].
[4] A cell growth regulator comprising the vector that expresses the nucleic acid according to any one of [1] to [3] as an active ingredient.
[5] A cell growth regulator comprising, as an active ingredient, a substance that suppresses the expression of the gene having the target base sequence of the nucleic acid according to [1].
[6] A cell growth regulator comprising, as an active ingredient, a substance that promotes the expression of the gene having the target base sequence of the nucleic acid according to [1].
[7] The cell growth regulator according to [5] or [6], wherein the substance that suppresses or promotes expression is a nucleic acid.
[8] The cell growth regulator according to [7], wherein the nucleic acid is siRNA.
[9] A cell growth regulator comprising the vector expressing the nucleic acid according to [7] as an active ingredient.
[10] A cell cycle containing the nucleic acid according to any one of [1] to [3], the vector according to [4], or the substance according to any one of [5] to [8] as an active ingredient A therapeutic or diagnostic agent for a disease caused by an abnormality.
[11] A diagnostic agent for a disease caused by abnormal cell cycle, comprising as an active ingredient a reagent for detecting the expression level of the nucleic acid according to [1], mutation of the nucleic acid, and mutation of the genome encoding the nucleic acid.
[12] Diseases caused by abnormal cell cycle are cancer, arteriosclerosis, rheumatoid arthritis, benign prostatic hyperplasia, vascular restenosis after percutaneous transvascular coronary angioplasty, pulmonary fibrosis, glomerulonephritis and autoimmune disease The therapeutic or diagnostic agent according to [10] or [11], which is a disease selected from the group consisting of:
[13] An effective amount of the nucleic acid according to any one of [1] to [3], the vector according to [4], or the substance according to any of [5] to [8], A method for treating a disease caused by an abnormal cell cycle.
[14] A method for diagnosing a disease caused by an abnormal cell cycle, comprising detecting the expression level of the nucleic acid according to [1], a mutation of the nucleic acid, and a mutation of a genome encoding the nucleic acid.
[15] Use of the nucleic acid according to any one of [1] to [3] and [7] for the manufacture of a therapeutic agent for a disease caused by cell cycle abnormality.
[16] An agent for controlling expression of a target gene of the nucleic acid, comprising the nucleic acid according to [1] or [3] as an active ingredient.
[17] The agent for controlling expression of a target gene of nucleic acid according to [1], comprising the vector according to [4] as an active ingredient.
[18] A cell cycle fluctuation method, characterized by using the nucleic acid according to [1] or [3].
[19] A cell cycle variation method comprising using the vector according to [4].
[20] A method for changing the cell cycle, comprising using a substance such as an antisense nucleic acid, siRNA, decoy nucleic acid or the like that suppresses or promotes the expression of the target gene of the nucleic acid according to [1].
[21] A method for controlling cell proliferation, comprising using the nucleic acid according to [1] or [3].
[22] A method for controlling cell proliferation, comprising using the vector according to [4].
[23] A method for controlling cell proliferation, comprising using a substance such as an antisense nucleic acid, siRNA, or decoy nucleic acid that suppresses or promotes expression of a target gene of the nucleic acid according to [1].
[24] The method for inhibiting or promoting the expression of a target gene of nucleic acid according to [1], wherein the nucleic acid according to [1] or [3] is used.
[25] The method for suppressing expression or promoting expression of a target gene of a nucleic acid according to [1], wherein the vector according to [4] is used.
[26] A method for screening a cell growth inhibitor, characterized by using the promotion of the expression of the nucleic acid according to [1] as an index.
[27] A screening method for a cell growth inhibitor, comprising suppressing the expression of a target gene of the nucleic acid according to [1] as an index.
[28] A screening method for a cell growth promoter, wherein suppression of the expression of the nucleic acid according to [1] is used as an index.
[29] A screening method for a cell growth promoting agent, characterized by promoting expression of a target gene of the nucleic acid according to [1].

According to the present invention, a cell growth inhibitor or a cell growth promoter, a diagnostic or therapeutic agent for a disease caused by an abnormality in the cell cycle, an expression inhibitor or promoter for a target gene of a nucleic acid such as microRNA, a cell cycle variation method In addition, a method for inhibiting cell growth or a method for promoting cell growth can be provided.

Figure 1 shows miR-148a, miR-148b, and miR-152, and, as a control, Pre-miR ^™ miRNA Precursor Molecules-Negative Control # 2 and Dnmt1 siRNA at a final concentration of 50 nM in HeLa cells or AGS cells, respectively. The results of quantification and evaluation of Dnmt1 mRNA expression by RT-PCR after 24 hours or 48 hours of transfection and culturing are shown. When the amount of Dnmt1 mRNA was semi-quantified, the amount of mRNA of gapdh (D-glyceraldehyde-3-phosphate dehydrogenase) of the corresponding sample was calculated as an internal control. Figure 2 shows miR-148a, miR-148b, and miR-152, and Pre-miR ^™ miRNA Precursor Molecules-Negative Control # 2 and Dnmt1 siRNA at a final concentration of 50 nM as controls, respectively, in HeLa cells or AGS cells. The result of having evaluated the expression of the protein of Dnmt1 by immunoblotting after transfection for 72 hours is shown.

The nucleic acid used in the present invention may be any molecule in which nucleotides and molecules having functions equivalent to those of nucleotides are polymerized, such as RNA that is a polymer of ribonucleotides and polymers of deoxyribonucleotides. Examples thereof include a polymer in which DNA, RNA and DNA are mixed, and a nucleotide polymer containing a nucleotide analog, and may be a nucleotide polymer containing a nucleic acid derivative. The nucleic acid in the present invention may be a single-stranded nucleic acid or a double-stranded nucleic acid. The double-stranded nucleic acid also includes a double-stranded nucleic acid in which one strand is hybridized under stringent conditions to the other strand.

In the present invention, nucleotide analogues are used to improve or stabilize nuclease resistance, to increase affinity with complementary strand nucleic acids, to increase cell permeability, or to be visualized as compared to RNA or DNA. In addition, any molecule may be used as long as it is a modification of ribonucleotide, deoxyribonucleotide, RNA or DNA, and it may be a naturally occurring molecule or a non-natural molecule such as a sugar-modified nucleotide analog or phosphodiester. Examples thereof include binding modified nucleotide analogs.

The sugar moiety-modified nucleotide analog may be any one obtained by adding or substituting any chemical structural substance to a part or all of the chemical structure of the sugar of the nucleotide. For example, 2'-O-methyl Nucleotide analogues substituted with ribose, nucleotide analogues substituted with 2'-O-propylribose, nucleotide analogues substituted with 2'-methoxyethoxyribose, substituted with 2'-O-methoxyethylribose Nucleotide analogues, nucleotide analogues substituted with 2'-O- [2- (guanidinium) ethyl] ribose, nucleotide analogues substituted with 2'-O-fluororibose, introducing a bridging structure into the sugar moiety Bridged Nucleic Acid (BNA) having two circular structures, more specifically, 2′-position oxygen atom and 4′-position charcoal Locked artificial nucleic acid in which atoms are bridged via methylene (Locked Nucleic Acid) (LNA) , and ethylene bridged structure type artificial nucleic acid (Ethylene bridged nucleic acid) (ENA ) [Nucleic Acid Research, 32, e175 (2004)] is mentioned Furthermore, peptide nucleic acids (PNA) [Acc. Chem. Res., 32 , 624 (1999)], oxypeptide nucleic acids (OPNA) [J. Am. Chem. Soc., 123 , 4653 (2001)], and peptides Ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122 , 6900 (2000)] and the like can be mentioned.

The phosphodiester bond-modified nucleotide analog may be any one obtained by adding or substituting an arbitrary chemical substance to a part or all of the chemical structure of a phosphodiester bond of a nucleotide. Examples include nucleotide analogues substituted with thioate linkages, nucleotide analogues substituted with N3'-P5 'phosphoramidate linkages [Cell engineering, 16 , 1463-1473 (1997)] [RNAi method And Antisense, Kodansha (2005)].

In the present invention, as a nucleic acid derivative, in order to improve nuclease resistance, to stabilize, to increase affinity with a complementary strand nucleic acid, to increase cell permeability, or to be visualized as compared with nucleic acid, Any molecule added with another chemical substance may be used, for example, 5′-polyamine addition derivative, cholesterol addition derivative, steroid addition derivative, bile acid addition derivative, vitamin addition derivative, Cy5 addition derivative, Cy3 addition derivative, Examples thereof include 6-FAM addition derivatives and biotin addition derivatives.

Examples of the nucleic acid of the present invention include nucleic acids represented by the following (a) to (k).
(A) Nucleic acid comprising a base sequence represented by any of SEQ ID NOs: 1 to 407 (b) Nucleic acid comprising 17 to 28 bases comprising a nucleic acid comprising a base sequence represented by any of SEQ ID NOs: 1 to 407 (C) a nucleic acid comprising a base sequence represented by any one of SEQ ID NOs: 1 to 407 and having a nucleotide sequence having 90% or more identity (d) comprising a base sequence represented by any of SEQ ID NOs: 1 to 407 Nucleic acid that hybridizes with a complementary strand of nucleic acid under stringent conditions (e) A nucleic acid comprising the second to eighth base sequences of the base sequence represented by any one of SEQ ID NOs: 1 to 407 (f) (a) to ( a double-stranded nucleic acid comprising the nucleic acid of e) and a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid, or a nucleic acid containing the double-stranded nucleic acid (g) (a) to (e) Nucleic acids that hybridize with these nucleic acids under stringent conditions Or a hairpin structure in which a double-stranded nucleic acid (h), (f) or (g) containing the double-stranded nucleic acid is linked with a spacer oligonucleotide consisting of 8 to 28 bases. A single-stranded nucleic acid or a nucleic acid containing the single-stranded nucleic acid (i) a nucleic acid consisting of a base sequence represented by any one of SEQ ID NOs: 408 to 920 (j) represented by any one of SEQ ID NOs: 408 to 920 A nucleic acid comprising a nucleotide sequence having 90% or more identity to the nucleotide sequence (k) a nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleotide sequence represented by any of SEQ ID NOs: 408 to 920 .

As the nucleic acid, microRNA is preferably used. MicroRNA refers to single-stranded RNA having a length of 17 to 28 bases. The surrounding genomic sequence containing the microRNA sequence has a sequence that can form a hairpin structure, and the microRNA can be excised from either strand of the hairpin. MicroRNAs complementarily bind to their target mRNA and suppress mRNA translation, or promote post-transcriptional control of gene expression by promoting mRNA degradation.

Examples of the microRNA used in the present invention include human microRNA having a base sequence represented by any of SEQ ID NOs: 1 to 38. Furthermore, as a microRNA having the same function as the human microRNA consisting of the base sequence represented by any of SEQ ID NOs: 1 to 38, a base sequence represented by SEQ ID NOs: 39 to 258, which is an ortholog of the human microRNA The nucleic acid which consists of can be mention | raise | lifted. As a specific example, an ortholog of the human microRNA of SEQ ID NO: 1 is composed of the base sequence represented by SEQ ID NO: 39. Table 1 shows a correspondence table between the microRNAs of SEQ ID NOs: 1 to 38 and their orthologs. The biological species shown at the top of Table 1 are as follows. hsa, Homo sapiens, human; mmu, Mus musculus, mouse; rno, Rattus norvegicus, rat; cgr, Cricetulus griseus, Chinese hamster; age, Ateles geoffroyi, red spider monkey; Mnea catamarin; mml, Macaca mulatta, rhesus monkey; mne, Macaca nemestrina, pigtail monkey; pbi, Pygathrix bieti, black goldfish; ggo, Gorilla gorilla, gorilla; ppa, Pan paniscus, bonobo; ptr, Pan troglodytes, chimpanzee; ppy, Pongo , Orangutan; ssy, Symphalangus syndactylus, black-tailed gibbon; lca, Lemur catta, ring-tailed lemur; oan, Ornithorhynchus anatinus, platypus, cfa, Canis familiaris, dog; Xenopus laevis, Xenopus laevis; xtr, Xenopus tropicali b, Bos taurus, cattle; oar, Ovis aries, sheep; ssc, Sus scrofa, pig; dre, Danio rerio, zebrafish; fru, Fugu rubripes, trough;
Since human-derived microRNA and its ortholog have high sequence identity, they are considered to have similar functions. In addition, microRNAs that are subgrouped by the alphabet added to the end of the microRNA name also have similar functions because of their high sequence identity.

As a mechanism by which microRNA suppresses translation of mRNA of its target gene, mRNA having a base sequence complementary to the 2-8th base sequence on the 5 'end side of microRNA is used as microRNA target gene. [Current Biology, 15 , R458-R460 (2005)]. By this mechanism, the expression of the mRNA is suppressed by the microRNA. Accordingly, microRNAs having the same base sequence on the 2nd to 8th positions on the 5 ′ end side have the same function by suppressing the expression of the same mRNA. A microRNA having the base sequence represented by any one of SEQ ID NOs: 1 to 258 and a microRNA having the same base sequence at the second to eighth positions on the 5 ′ end are composed of base sequences represented by SEQ ID NOs: 259 to 407 Nucleic acids can be raised. Specific examples of the microRNA include a microRNA consisting of SEQ ID NO: 261 for the microRNA of SEQ ID NO: 9, and a microRNA consisting of SEQ ID NO: 271 for the microRNA of SEQ ID NO: 12. be able to. Artificial microRNA is also included in the microRNA of the present invention. Table 2 shows a correspondence table between the microRNAs of SEQ ID NOs: 1 to 258 and the microRNAs having the same base sequence at the second to eighth positions on the 5 ′ end. MicroRNAs having a common seed sequence are considered to have the same function because their target base sequences are considered identical.

As the above nucleic acid, a microRNA precursor is also preferably used. The microRNA precursor is a nucleic acid having a length of about 50 to about 200 bases, more preferably about 70 to about 100 bases including the nucleic acid of the present invention, and can form a hairpin structure. MicroRNA is produced from a microRNA precursor through processing by a protein called Dicer.

As the microRNA precursor used in the present invention, for example, for the human microRNA of SEQ ID NO: 1, a nucleic acid having the base sequence represented by SEQ ID NO: 408 can be mentioned. In addition, for the microRNAs represented by SEQ ID NOs: 2 to 407, nucleic acids having the base sequences represented by SEQ ID NOs: 409 to 920 can be exemplified. Table 3 shows a correspondence table between the microRNA and the microRNA precursor in the present invention. In the present invention, the microRNA precursor includes an artificial microRNA precursor.

In the present invention, a nucleic acid having 90% or more identity with the base sequence represented by any of SEQ ID NOs: 1 to 920 is BLAST [J. Mol. Biol., 215 , 403 (1990)] or FASTA [ Methods in Enzymology, 183 , 63 (1990)], etc., and preferably at least 90% or more with a nucleic acid comprising a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 920, as the identity. Means 93% or more, more preferably 95% or more, still more preferably 96% or more, particularly preferably 97% or more, and most preferably 98% or more.

The nucleic acid having 90% or more identity with the base sequence represented by any of SEQ ID NOs: 1 to 920 used in the present invention is preferably the base sequence represented by any of SEQ ID NOs: 1 to 920 It has “the same function” as a nucleic acid consisting of “Same function” means that the target genes are the same.

The stringent conditions in the above are 7.5 mL, 1M Na ₂ HPO ₄ (pH 7.2) 0.6 mL, 10% SDS 21 mL, 50x Denhardt's solution 0.6 mL, 10 mg / Add the other strand labeled with ³² P-ATP to the hybridization buffer consisting of 0.3 mL of mL sonicated salmon sperm DNA, react at 50 ° C overnight, and then wash with 5xSSC / 5% SDS solution at 50 ° C for 10 minutes. Further, the condition is such that the signal can be detected by washing with 1 × SSC / 1% SDS solution at 50 ° C. for 10 minutes, and then removing the membrane and exposing it to an X-ray film.

The nucleic acid that hybridizes under stringent conditions with the complementary sequence of the nucleic acid consisting of the base sequence represented by any of SEQ ID NOs: 1 to 920 used in the present invention is preferably any of SEQ ID NOs: 1 to 920 It has “the same function” as the nucleic acid comprising the base sequence represented. “Same function” means that the target genes are the same.

As a method for detecting the expression of nucleic acid such as microRNA using the nucleic acid of the present invention, any method may be used as long as it can detect the presence of nucleic acid such as microRNA or microRNA precursor in a sample. , (1) Northern hybridization, (2) dot blot hybridization, (3) in situ hybridization, (4) quantitative PCR, (5) differential hybridization, (6) microarray, (7) ribonuclease protection assay Etc.

As a method for detecting a mutation in a nucleic acid such as microRNA using the nucleic acid of the present invention, any method may be used as long as it can detect a mutation in the base sequence of a nucleic acid such as microRNA or a microRNA precursor in a sample. For example, a method for detecting a heteroduplex formed by hybridization of a nucleic acid having a non-mutated base sequence and a nucleic acid having a mutant base sequence, or by directly sequencing a base sequence derived from a specimen And a method for detecting the presence or absence of a mutation.

The vector for expressing the nucleic acid of the present invention may be any vector designed so that the nucleic acid of the present invention such as microRNA is biosynthesized by introduction into a cell and transcription. Good. As a vector capable of expressing the nucleic acid of the present invention such as microRNA in a cell, specifically, pCDNA6.2-GW / miR (Invitrogen), pSilencer4.1-CMV (Ambion), Examples include pSINsi-hH1 DNA (Takara Bio), pSINsi-hU6 DNA (Takara Bio), pENTR / U6 (Invitrogen).

As a method for suppressing the expression of a gene having the target base sequence of the nucleic acid of the present invention such as microRNA (hereinafter referred to as target gene), the target base of the nucleic acid of the present invention such as microRNA is used using the nucleic acid of the present invention. Any method may be used as long as it suppresses the expression of the gene having the target base sequence by utilizing the activity of suppressing the expression of mRNA having the sequence. In addition, suppression of expression here includes a case where translation from mRNA is suppressed, and a case where the amount of protein translated from mRNA is reduced by cleaving or decomposing mRNA. Specific examples of substances that suppress the expression of mRNA having the target base sequence include nucleic acids such as siRNA and antisense oligonucleotides. The siRNA can be prepared based on the continuous sequence information of the mRNA [Genes Dev., 13 , 3191 (1999)]. The number of residues of the base constituting one strand of siRNA is preferably 17 to 30 residues, more preferably 18 to 25 residues, still more preferably 19 to 23 residues.

In the present invention, the microRNA has a length of 17 to 28 bases containing a sequence complementary to a continuous 7 base sequence present in the 3 ′ untranslated region of the mRNA of the target gene as the second to eighth base sequences. Also included are artificial microRNAs that are single-stranded RNAs. When the sequence including the sequence and extending it back and forth forms a hairpin structure, and the microRNA sequence is RNA that can be excised from any one strand of the hairpin structure in the cell by the microRNA biosynthetic pathway, The extended sequence is called an artificial microRNA precursor. Artificial microRNAs and artificial microRNA precursors can be designed using the method as described above using a gene whose expression is to be suppressed as a target gene.

The target base sequence of a nucleic acid such as microRNA is a base sequence consisting of several bases recognized by the nucleic acid such as microRNA in the present invention, and the expression of mRNA having the base sequence is such as microRNA in the present invention. A base sequence that is suppressed by a nucleic acid. Translation from mRNA having a base sequence complementary to the 2-8th base sequence on the 5 ′ end side of the microRNA is suppressed by the microRNA [Current Biology, 15 , R458-R460 (2005)] In the present invention, a base sequence complementary to the 2-8th base sequence on the 5 ′ end side of a nucleic acid such as microRNA in the present invention can be mentioned as a target base sequence. For example, a target sequence complementary to the 2-8th base sequence on the 5 ′ end side of microRNA is prepared, and contains a sequence that completely matches the 3′-UTR base sequence group of human mRNA. mRNA can be determined by selecting by a method such as character string search. The 3'-UTR base sequence group of human mRNA is prepared using the genome sequence and gene position information that can be obtained from "UCSC Human Genome Browser Gateway (http://genome.ucsc.edu/cgi-bin/hgGateway)" be able to. Specific examples of genes having the target base sequences of microRNAs represented by SEQ ID NOs: 1 to 407 include the EntreGene database (http: // www) of the National Center for Biotechnology Information (NCBI) in the United States. .ncbi.nlm.nih.gov / Entrez /) and the genes listed in Table 4 represented by the names (Official Symbol and Gene ID) used.

In the present invention, a method for expressing a nucleic acid such as microRNA in a cell includes a method using a nucleic acid that expresses a gene encoding microRNA or the like when introduced into the cell. As the nucleic acid, in addition to DNA, RNA, or nucleotide analogues, these chimeric molecules or derivatives of the nucleic acids can also be used. Specifically, the nucleic acid is designed in the same manner as Pre-miR ^™ miRNA Precursor Molecules (Ambion) or miRIDIAN microRNA Mimics (GE Healthcare), and the nucleic acid such as the microRNA of the present invention is expressed in the cell. Can be made. When microRNA is expressed, any method may be used as long as microRNA can finally be produced in the cell. For example, (1) In addition to introducing single-stranded RNA as a microRNA precursor, (2) There is a method for introducing microRNA itself and RNA consisting of complementary strands of microRNA and 100% -matched double-stranded RNA, and (3) double-stranded RNA assuming the state after microRNA is cleaved into Dicer. can give. Examples of products using such a method include miCENTURY OX Precursor (B-Bridge), miCENTURY OX siMature (B-Bridge), and miCENTURY OX miNatural (B-Bridge).

The method for synthesizing the nucleic acid used in the present invention is not particularly limited, and the nucleic acid can be produced by a method using a known chemical synthesis or an enzymatic transcription method. Examples of methods using known chemical synthesis include phosphoramidite method, phosphorothioate method, phosphotriester method, etc. For example, synthesis with ABI3900 high-throughput nucleic acid synthesizer (Applied Biosystems) Can do. Examples of the enzymatic transcription method include transcription using a typical phage RNA polymerase, for example, T7, T3, or SP6 RNA polymerase, using a plasmid or DNA having the target base sequence as a template.

As a method of screening for a substance that promotes or suppresses the expression or function of a nucleic acid using the nucleic acid of the present invention, the nucleic acid of the present invention is used to promote or suppress the expression or function of a nucleic acid such as the microRNA of the present invention. Any method may be used as long as it is a method for screening a substance to be allowed to be screened. For example, a substance that enhances the effect of suppressing the expression of mRNA having the target base sequence by introducing the vector expressing the nucleic acid of the present invention into a cell, or a substance that promotes the expression of mRNA having the target base sequence A screening method is mentioned.

In the present specification, “control of cell growth” means suppression or promotion of cell growth.

As used herein, “cell cycle fluctuation” means increasing or decreasing the cell cycle rate at a specific stage (G1, S, M, G2, etc.). An increase in the cell cycle rate at a particular stage indicates the possibility that the cell cycle stops at that stage.

The nucleic acid of the present invention suppresses the expression of its target gene and changes the cell cycle. Therefore, the nucleic acid of the present invention, the vector that expresses the nucleic acid of the present invention, the substance that promotes the expression of the nucleic acid of the present invention, and the substance that suppresses the expression of the target gene of the nucleic acid of the present invention are those of mammalian cells such as humans. Since the cell cycle can be changed, it is useful as a drug for pharmaceuticals and research as a cell cycle changing agent. A nucleic acid of the present invention, a vector that expresses the nucleic acid of the present invention, a substance that promotes the expression of the nucleic acid of the present invention, or a substance that suppresses the expression of a target gene of the nucleic acid of the present invention is applied to cells of mammalian cells such as humans. When contacted, the cell cycle in the cell varies.

The type of cell is not limited as long as cell cycle fluctuation is observed, but is preferably a mammalian cell such as a human (eg, cancer cell, vascular endothelial cell, fibroblast, synovial cell, lymphocyte, etc.). .

The type of cancer is not particularly limited, non-solid cancer such as leukemia, malignant lymphoma, myeloma, or stomach cancer, esophageal cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, kidney cancer, bladder cancer, lung cancer, cervical cancer Solid cancers such as cancer, ovarian cancer, breast cancer, prostate cancer, skin cancer, brain tumor and the like are included. The cancer is preferably cervical cancer, stomach cancer, liver cancer or ovarian cancer.

The nucleic acid of the present invention is miR-153, miR-224, miR-320, miR-345, miR-363, miR-373, miR-375, miR-448, miR-519a, miR-519b, miR-519c, In the case of miR-526b or miR-92b, or an ortholog thereof, a microRNA having the same base sequence at the 2nd to 8th positions on the 5 ′ end side, or a microRNA precursor thereof, the nucleic acid of the present invention, the present invention The vector that expresses the nucleic acid of the present invention, the substance that promotes the expression of the nucleic acid of the present invention, and the substance that suppresses the expression of the target gene of the nucleic acid of the present invention increase the G1 phase.

The nucleic acid of the present invention is miR-129, miR-148a, miR-148b, miR-149, miR-152, miR-214, miR-217, miR-326, miR-363 *, miR-379, miR-449 , MiR-449b, miR-450, miR-500, miR-518f *, miR-544, miR-549, miR-555, miR-583, miR-585, miR-644, miR-766, miR-769- 3p or miR-96, or an ortholog thereof, 5′-end side 2-8th microRNA having the same base sequence, or a microRNA precursor thereof, the nucleic acid of the present invention, the nucleic acid of the present invention , A substance that promotes the expression of the nucleic acid of the present invention, and a substance that suppresses the expression of the target gene of the nucleic acid of the present invention increase the G2M phase.

When the nucleic acid of the present invention is miR-154, or its ortholog, 5′-end micro RNA having the same base sequence at the 2nd to 8th positions, or a microRNA precursor thereof, the nucleic acid of the present invention, the present The vector that expresses the nucleic acid of the invention, the substance that promotes the expression of the nucleic acid of the invention, and the substance that suppresses the expression of the target gene of the nucleic acid of the invention increase the S phase and the G2M phase.

The nucleic acid of the present invention, the vector that expresses the nucleic acid of the present invention, the substance that promotes the expression of the nucleic acid of the present invention, and the substance that suppresses the expression of the target gene of the nucleic acid of the present invention Is useful as a cell growth inhibitor. Accordingly, the pharmaceutical comprising the nucleic acid of the present invention, the vector expressing the nucleic acid of the present invention, the substance that promotes the expression of the nucleic acid of the present invention, or the substance that suppresses the expression of the target gene of the nucleic acid of the present invention as an active ingredient It can be used for diagnosis or treatment of diseases caused by abnormalities in the cycle (particularly abnormal progression of the cell cycle) (for example, diseases caused by abnormal cell proliferation (particularly diseases caused by abnormally increased cell proliferation or tissue hyperplasia)). . By administering to a mammal an effective amount of the nucleic acid of the present invention, a vector that expresses the nucleic acid of the present invention, a substance that promotes the expression of the nucleic acid of the present invention, or a substance that suppresses the expression of the target gene of the nucleic acid of the present invention. Diseases resulting from cell cycle abnormalities in the mammal can be treated. Specifically, diseases caused by abnormal progression of the cell cycle include cancer, arteriosclerosis, rheumatoid arthritis, benign prostatic hyperplasia, vascular restenosis after percutaneous transvascular coronary angioplasty, pulmonary fibrosis, thread Spherical nephritis, autoimmune diseases and the like, preferably cancer.

On the other hand, a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid of the present invention, a vector for expressing the nucleic acid, a substance that suppresses the expression of the nucleic acid of the present invention, and the expression of the target gene of the gene of the present invention Since the substance to be promoted can also change the cell cycle of mammalian cells such as humans, it is useful as a drug for drug or research as a cell cycle changing agent. Promotes the expression of a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid of the present invention, a vector for expressing the nucleic acid, a substance that suppresses the expression of the nucleic acid of the present invention, and the target gene of the gene of the present invention When a substance is brought into contact with cells of mammalian cells such as humans, the cell cycle in the cells changes.

The nucleic acid of the present invention is miR-153, miR-224, miR-320, miR-345, miR-363, miR-373, miR-375, miR-448, miR-519a, miR-519b, miR-519c, miR-526b or miR-92b, or an ortholog thereof, or a microRNA having the same base sequence at the 2nd to 8th positions on the 5 ′ end, or a base sequence of the nucleic acid of the present invention when these are microRNA precursors A nucleic acid comprising a base sequence complementary to the above, a vector that expresses the nucleic acid, a substance that suppresses the expression of the nucleic acid of the present invention, and a substance that promotes the expression of the target gene of the gene of the present invention reduces G1 phase. Let

The nucleic acid of the present invention is miR-129, miR-148a, miR-148b, miR-149, miR-152, miR-214, miR-217, miR-326, miR-363 *, miR-379, miR-449 , MiR-449b, miR-450, miR-500, miR-518f *, miR-544, miR-549, miR-555, miR-583, miR-585, miR-644, miR-766, miR-769- In the case of 3p or miR-96, or these orthologs, microRNAs having the same base sequence at the 2nd to 8th positions on the 5 ′ end, or these microRNA precursors, the base sequence of the nucleic acid of the present invention Thus, a nucleic acid having a complementary base sequence, a vector for expressing the nucleic acid, a substance that suppresses the expression of the nucleic acid of the present invention, and a substance that promotes the expression of the target gene of the gene of the present invention reduce G2M phase.

When the nucleic acid of the present invention is miR-154, or its ortholog, 5′-end micro RNA having the same base sequence at the second to eighth positions, or a microRNA precursor thereof, the base of the nucleic acid of the present invention A nucleic acid comprising a base sequence complementary to the sequence, a vector that expresses the nucleic acid, a substance that suppresses the expression of the nucleic acid of the present invention, and a substance that promotes the expression of the target gene of the gene of the present invention are the S phase and Reduce G2M phase.

Promotes the expression of a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid of the present invention, a vector for expressing the nucleic acid, a substance that suppresses the expression of the nucleic acid of the present invention, and the target gene of the gene of the present invention Since the substance can promote cell growth of mammalian cells such as humans, it is useful as a cell growth promoter. Accordingly, the expression of a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid of the present invention, a vector that expresses the nucleic acid, a substance that suppresses the expression of the nucleic acid of the present invention, or a target gene of the gene of the present invention. A medicament containing a promoting substance as an active ingredient can be used, for example, as a medicament for increasing the sensitivity of an anticancer agent or radiation therapy by temporarily and locally promoting the growth of cancer cells in cancer treatment.

Hereinafter, the present invention will be described in detail.

1. Methods for detecting the expression of nucleic acids such as microRNAs and microRNA precursors expressed in mammalian cells such as cancer cells

(1-1) Identification of microRNA Whether a small RNA sequence is microRNA can be determined by following the criteria described in RNA, 9 , 277-279 (2003). For example, in the case of a low molecular weight RNA that has been newly acquired and whose base sequence has been determined, it can be performed as follows.

A peripheral genomic sequence obtained by extending the DNA sequence corresponding to the obtained low molecular RNA base sequence by about 50 nt to the 5 'end side and 3' end side, respectively, is obtained, and RNA predicted to be transcribed from the genomic sequence Predict secondary structure. As a result, when it has a hairpin structure and the base sequence of the small RNA is located on one strand of the hairpin, it can be determined that the small RNA is a microRNA. Genomic sequences are publicly available and are available, for example, from UCSC Genome Bioinformatics (http://genome.ucsc.edu/). In addition, various programs for secondary structure prediction have been published, such as RNAfold [Nucleic Acids Research, 31 , 3429-3431 (2003)], Mfold [Nucleic Acids Research, 31 , 3406-3415 (2003)], etc. Can be used. Further, existing microRNA sequences are registered in a database called miRBase at Sanger Center in the UK, and it can be determined whether or not they are identical to existing microRNAs based on whether or not they are identical to the sequences described here.

(1-2) Acquisition of sequence information of low molecular RNA Extracting total RNA from a sample containing mammalian cells such as a human cancer cell line or a cancer patient-derived sample, and containing the microRNA expressed in the cell using the RNA The low molecular weight RNA can be obtained as follows.

As a method for obtaining low molecular weight RNA, specifically, a method for separating low molecular weight RNA by 15% polyacrylamide gel electrophoresis according to the method described in Genes & Development, 15 , 188-200 (2000). can give. From this, 5'-terminal dephosphorylation, 3'-adapter ligation, phosphorylation, 5'-adapter ligation, reverse transcription, PCR amplification, concatamerization, and ligation to vector are sequentially cloned, and the clone is cloned. Can be determined. Alternatively, according to the method described in, for example, Science, 294 , 858-862 (2001), 5′-adenylation, 3′-adapter ligation, 5′-adapter ligation, reverse transcription, PCR amplification, concatamerization, to a vector Through the ligation, the low molecular RNA can be cloned and the base sequence of the clone can be determined.

In addition, according to the method described in Nucleic Acids Research, 34 , 1765-1771 (2006), 5 ′ end dephosphorylation, 3′-adapter ligation, phosphorylation, 5′-adapter ligation, reverse transcription, PCR amplification, microbeads By sequentially ligating to a vector, low molecular RNA is cloned, and the base sequence information of the low molecular RNA can be obtained by determining the base sequence by reading the base sequence of the microbead.

As another method for obtaining low-molecular-weight RNA, there is a method using a small RNA Cloning kit (manufactured by Takara Bio Inc.).

(1-3) Method for detecting expression level of nucleic acid such as microRNA Examples of methods for detecting the expression level of nucleic acid such as microRNA and its precursor include (1) Northern hybridization and (2) dot blot high. Hybridization, (3) in situ hybridization, (4) quantitative PCR, (5) differential hybridization, (6) microarray, (7) ribonuclease protection assay and the like.

Northern blotting is a method in which sample-derived RNA is separated by gel electrophoresis, then transferred to a support such as a nylon filter, and a probe appropriately labeled based on the base sequence of the nucleic acid of the present invention is prepared. This is a method for detecting a band specifically bound to the nucleic acid of the present invention by washing, and specifically, for example, according to the method described in Science, 294 , 853-858 (2001). it can.

The labeled probe may be a radioisotope, biotin, digoxigenin, a fluorescent group, a chemiluminescent group, etc., and a base sequence of the nucleic acid of the present invention by a method such as nick translation, random priming or phosphorylation at the 5 ′ end. It can be prepared by incorporating DNA or RNA having a complementary sequence, or LNA. Since the binding amount of the labeled probe reflects the expression level of the nucleic acid of the present invention, the expression level of the nucleic acid of the present invention can be quantified by quantifying the amount of bound labeled probe. Electrophoresis, membrane transfer, probe preparation, hybridization, and nucleic acid detection can be performed by the methods described in Molecular Cloning 3rd Edition [Cold Spring Harbor Press, (2001) Cold Spring Harbor, NY] .

In dot blot hybridization, RNA extracted from tissues and cells is spot-fixed on a membrane in a dotted manner, and then hybridized with a labeled polynucleotide that serves as a probe to detect RNA that specifically hybridizes with the probe. It is a method to do. As the probe, the same probe as in Northern hybridization can be used. Preparation of RNA, RNA spot, hybridization, and detection of RNA can be performed by the methods described in Molecular Cloning 3rd edition.

In situ hybridization uses a paraffin or cryostat section of tissue obtained from a living body or immobilized cells as a specimen, performs hybridization and washing steps with a labeled probe, and observes the nucleic acid of the present invention by microscopic observation. This is a method for examining the distribution and localization in tissues and cells [Methods in Enzymology, 254 , 419 (1995)]. As the probe, the same probe as in Northern hybridization can be used. Specifically, microRNA can be detected according to the method described in Nature Method, 3 , 27 (2006).

In quantitative PCR, cDNA synthesized from a sample-derived RNA using a reverse transcription primer and a reverse transcriptase (hereinafter, the cDNA is referred to as a sample-derived cDNA) is used for measurement. As a reverse transcription primer used for cDNA synthesis, a random primer or a specific RT primer can be used. The specific RT primer refers to a primer having a sequence complementary to a nucleotide sequence corresponding to the nucleic acid of the present invention and its surrounding genomic sequence.

For example, after synthesizing a specimen-derived cDNA, using this as a template, design from the base sequence corresponding to the nucleic acid of the present invention such as microRNA or microRNA precursor and the surrounding genomic sequence, or the base sequence corresponding to the primer for reverse transcription. PCR is performed using the template-specific primer, the cDNA fragment containing the nucleic acid of the present invention is amplified, and the amount of the nucleic acid of the present invention contained in the sample-derived RNA is detected from the number of cycles until a certain amount is reached. To do. As a template-specific primer, an appropriate region corresponding to the nucleic acid of the present invention and its surrounding genomic sequence is selected, and the 5 ′ end 15 to 40 residues, preferably 20 to 30 residues, of the base sequence of the region is selected. A DNA or LNA set consisting of a sequence and a DNA or LNA set consisting of a sequence complementary to the 3 ′ end 15 to 40 residues, preferably 20 to 30 residues can be used. Specifically, it can be performed according to the method described in Nucleic Acids Research, 32 , e43 (2004).

A specific RT primer having a stem-loop structure can also be used as a reverse transcription primer for cDNA synthesis. Specifically, the method described in Nucleic Acid Research, 33, e179 ( 2005) or can be performed using the TaqMan MicroRNA Assays (Applied Biosystems).
As yet another sample-derived cDNA synthesis method, a reverse transcription reaction can also be performed by adding a polyA sequence to a sample-derived RNA with polyA polymerase and using a base sequence containing an oligo dT sequence as a primer for reverse transcription. . Specifically, it can be performed using miScript System (Qiagen) or QuantiMir RT Kit (System Biosciences). Furthermore, the sample-derived RNA or cDNA is hybridized with a filter or slide glass or silicon or other substrate on which DNA or LNA corresponding to the base sequence containing at least one nucleic acid of the present invention is immobilized, and washing is performed. By doing so, a change in the amount of the nucleic acid of the present invention can be detected. Examples of the method based on such hybridization include a method using differential hybridization [Trends Genet., 7 , 314 (1991)] and a microarray [Genome Res., 6 , 639 (1996)]. In either method, the difference in the amount of the nucleic acid of the present invention between the control sample and the target sample can be accurately detected by immobilizing an internal control such as a nucleotide sequence corresponding to U6 RNA on a filter or substrate. Can do. In addition, labeled cDNA synthesis using differently labeled dNTPs (mixtures of dATP, dGTP, dCTP, and dTTP) based on RNA derived from the control sample and the target sample, and two filters on one filter or one substrate. By simultaneously hybridizing the labeled cDNA, the nucleic acid of the present invention can be accurately quantified. Furthermore, the nucleic acid of the present invention can be quantified by directly labeling and hybridizing RNA derived from a control sample and / or target sample. For example, 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), etc. MicroRNA can be detected. Specifically, it can be detected or quantified in the same manner as mirVana miRNA Bioarray (Ambion) and miRNA microarray kit (Agilent Technology).

In the ribonuclease protection assay, first, a promoter sequence such as T7 promoter or SP6 promoter is bound to the 3 ′ end of the nucleotide sequence corresponding to the nucleic acid of the present invention or its surrounding genomic sequence, and labeled NTP (ATP, GTP, CTP, UTP). The labeled antisense RNA is synthesized by an in vitro transcription system using a mixture) and RNA polymerase. The labeled antisense RNA is bound to the sample-derived RNA to form an RNA-RNA hybrid, and then digested with ribonuclease A that degrades only single-stranded RNA. The digested product is subjected to gel electrophoresis, and an RNA fragment protected from digestion by forming an RNA-RNA hybrid is detected or quantified as the nucleic acid of the present invention. Specifically, it can be detected or quantified using mirVana miRNA Detection Kit (Ambion).

2. Synthesis of Nucleic Acid As described in 1 above, it is expressed in mammalian cells or tissues in which cell proliferation is enhanced, such as cancer cells or cancer tissues, or the expression is increased or decreased compared to normal tissues After nucleic acids such as microRNA and microRNA precursor are identified, not only RNA that is a polymer of ribonucleotides but also DNA that is a polymer of deoxyribonucleotides can be synthesized based on the base sequence. For example, the base sequence of DNA can be determined based on the base sequence of microRNA identified in 1 above. The base sequence of DNA corresponding to the base sequence of RNA can be uniquely determined by replacing U (uracil) contained in the RNA sequence with T (thymine). In addition, a polymer in which ribonucleotides and deoxyribonucleotides are mixed, a polymer containing nucleotide analogues, and a nucleic acid derivative can be synthesized in the same manner.

The method for synthesizing the nucleic acid of the present invention is not particularly limited, and can be produced by a method using a known chemical synthesis or an enzymatic transcription method. Examples of methods using known chemical synthesis include phosphoramidite method, phosphorothioate method, phosphotriester method, etc. For example, synthesis with ABI3900 high-throughput nucleic acid synthesizer (Applied Biosystems) Can do. Examples of the enzymatic transcription method include a transcription method using a typical phage RNA polymerase such as T7, T3, or SP6 RNA polymerase using a plasmid or DNA having the target base sequence as a template.

3. Method for detecting the function of a nucleic acid such as microRNA or microRNA precursor As a method for detecting the function of a nucleic acid such as microRNA, there is a method for detecting whether or not the translation of mRNA having a target base sequence is suppressed. be able to.

It is known that microRNA suppresses translation of mRNA containing the target base sequence in the 3′-terminal untranslated region (3′-UTR) [Current Biology, 15 , R458-R460 (2005)]. Therefore, a DNA in which the target base sequence for the single-stranded RNA to be measured is inserted into the 3′-UTR of an appropriate reporter gene expression vector is prepared, introduced into a host cell suitable for the expression vector, and one DNA is inserted into the cell. By measuring the expression of the reporter gene when the strand RNA is expressed, it can be detected whether or not it has the function of a microRNA.

The reporter gene expression vector may be any vector as long as it has a promoter upstream of the reporter gene and can express the reporter gene in the host cell. Any reporter gene can be used as the reporter gene, for example, firefly luciferase gene, Renilla luciferase gene, chloramphenicol acetyltransferase gene, β-glucuronidase gene, β-galactosidase gene, β -Lactamase gene, aequorin gene, green fluorescent protein gene and DsRed fluorescent gene can be used. Reporter gene expression vectors having such properties include, for example, psiCHECK-1 (Promega), psiCHECK-2 (Promega), pGL3-Control (Promega), pGL4 (Promega), pRNAi-GL ( Takara Bio), pCMV-DsRed-Express (CLONTECH) and the like. Single-stranded RNA can be expressed by the method described in 6 below.

The function of single-stranded RNA as a microRNA can be specifically detected as follows. First, host cells are cultured in a multi-well plate or the like to express a reporter gene expression vector having a target sequence and single-stranded RNA. Then, the reporter activity is measured, and the function of the single-stranded RNA as a microRNA is detected by measuring the reporter activity when the single-stranded RNA is expressed compared to the case where the single-stranded RNA is not expressed. can do.

4). Method for detecting mutations in nucleic acids such as microRNA and microRNA precursors As a method for detecting mutations in nucleic acids such as microRNAs and microRNA precursors, heterogeneous nucleic acids formed by hybridization of normal and mutant nucleic acids A method for detecting this strand can be used.

Methods for detecting heteroduplex include: (1) heteroduplex detection 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 method of mismatch [Nature Genetics, 9 , 103-104 (1996)], (5) Denaturing gel electrophoresis [Mutat. Res., 288 , 103-112 (1993)], etc. There are methods.

The heteroduplex detection method by polyacrylamide gel electrophoresis is performed as follows, for example. First, using a sample-derived DNA or a sample-derived cDNA as a template, a primer designed based on the genomic base sequence including the base sequence of the nucleic acid of the present invention is amplified as a fragment smaller than 200 μbp. When heteroduplexes are formed, the mobility is slower than homoduplexes without mutations, and they can be detected as extra bands. If the fragment is smaller than 200 bp, most insertions, deletions and substitutions of 1 base or more can be detected. Heteroduplex analysis is preferably performed on a single gel combined with single-strand conformation analysis described below.

In single-strand conformation polymorphism analysis (SSCP analysis; single-strand-conformation-polymorphism-analysis), primers designed based on the base sequence of the genome containing the base sequence of the nucleic acid of the present invention using the sample-derived DNA or the sample-derived cDNA as a template The DNA amplified as a fragment smaller than 200 bp is denatured and then migrated in a native polyacrylamide gel. When DNA amplification is performed, the amplified DNA can be detected as a band by labeling the primer with an isotope or a fluorescent dye, or silver-staining the unlabeled amplification product. In order to clarify the difference from the wild type pattern, when a control sample is also run at the same time, a fragment having a mutation can be detected from the difference in mobility.

In the mismatch chemical cleavage method (CCM method), a DNA fragment amplified with a primer designed based on the base sequence of the genome including the base sequence of the nucleic acid of the present invention using the sample-derived DNA or the sample-derived cDNA as a template, By hybridizing with a labeled nucleic acid in which an isotope or a fluorescent label is incorporated into the nucleic acid and treating with osmium tetroxide, one strand of DNA at a mismatched site can be cleaved to detect a mutation. The CCM method is one of the most sensitive detection methods and can be applied to specimens of kilobase length.

In place of the above osmium tetroxide, the mismatch can be cleaved enzymatically by combining RNase A with an enzyme involved in mismatch repair in cells such as T4 phage resol base and endonuclease VII.

In denaturing gel electrophoresis (DGGE method), DNA fragments amplified with primers designed based on the base sequence of the genome including the base sequence of the nucleic acid of the present invention using the sample-derived DNA or the sample-derived cDNA as a template. Is electrophoresed using a gel having a chemical denaturant concentration gradient or temperature gradient. The amplified DNA fragment moves in the gel to a position where it is denatured into a single strand and does not move after denaturation. Since there is a difference in the movement of the amplified DNA in the gel with and without the mutation, the presence of the mutation can be detected. In order to increase the detection sensitivity, a poly (G: C) terminal is preferably attached to each primer.

In addition, the nucleic acid mutation of the present invention can be detected by directly determining and analyzing the base sequence of the specimen-derived DNA or the specimen-derived cDNA.

5. Methods for expressing nucleic acids such as microRNAs and microRNA precursors of the present invention Known microRNA sequences and the sequences of the precursors are registered in a database called miRBase at Sanger Center in the UK. Nucleic acids such as the inventive microRNA and microRNA precursors can be made. It can also be prepared using the microRNA sequence obtained by the method described in 1.

The nucleic acid of the present invention can be expressed by using a vector in which the nucleic acid of the present invention is biosynthesized by introduction into a cell and transcription. Specifically, based on the nucleotide sequence of the nucleic acid of the present invention or the genomic nucleotide sequence containing the nucleotide sequence, a DNA fragment containing a hairpin portion is prepared and inserted downstream of the promoter of the expression vector to express the expression plasmid. The nucleic acid of the present invention can be expressed by constructing and then introducing the expression plasmid into a host cell suitable for the expression vector.

As the expression vector, a vector that can replicate autonomously in a host cell or can be integrated into a chromosome and contains a promoter at a position where a gene containing the nucleotide sequence of the nucleic acid of the present invention can be transcribed is used. As the promoter, any promoter can be used so long as it can express the nucleic acid of the present invention in a host cell. For example, RNA polymerase II (pol II) type promoter or RNA polymerase III which is a transcription system of U6 RNA or H1 RNA. Examples include (pol III) promoters. Examples of the pol II promoter include cytomegalovirus (human CMV) IE (immediate early) gene promoter, SV40 early promoter, and the like. Examples of expression vectors using them include pCDNA6.2-GW / miR (Invitrogen), pSilencer® 4.1-CMV (Ambion), and the like. Examples of pol III promoters include U6 RNA, H1 RNA, and tRNA gene promoters. Examples of expression vectors using them include pSINsi-hH1 DNA (Takara Bio), pSINsi-hU6 DNA (Takara Bio), and pENTR / U6 (Invitrogen).

Alternatively, a gene containing the nucleotide sequence of the nucleic acid of the present invention is inserted downstream of the promoter in the viral vector to construct a recombinant viral vector, the vector is introduced into a packaging cell to produce a recombinant virus, A gene containing the nucleotide sequence of the nucleic acid of the present invention can also be expressed by infecting a desired host cell with a recombinant virus.
The packaging cell may be any cell as long as it can replenish the deficient protein of the recombinant viral vector deficient in any of the genes encoding proteins necessary for virus packaging, for example, from human kidney HEK293 cells, mouse fibroblast NIH3T3-derived cells, and the like can be used. Proteins supplemented by packaging cells include mouse retrovirus-derived gag, pol, env, etc. for retroviral vectors, and HIV virus-derived gag, pol, env, vpr, vpu for lentiviral vectors. , Vif, tat, rev, nef and other proteins, adenovirus vectors such as E1A and E1B derived from adenovirus, and adeno-associated virus vectors such as Rep (p5, p19, p40), Vp (Cap), etc. Can be used.

In addition to using an expression vector, the nucleic acid of the present invention can also be directly introduced into a cell without using a vector. As a nucleic acid used in this method, in addition to DNA, RNA, or nucleotide analogues, these chimeric molecules or derivatives of the nucleic acids can be used. Specifically, nucleic acids such as the microRNA and microRNA precursor of the present invention can be expressed in the same manner as Pre-miR ^™ miRNA Precursor Molecules (Ambion) and miRIDIAN microRNA Mimics (GE Healthcare). it can. When microRNA is expressed, any method can be used as long as microRNA can finally be produced in the cell. For example, (1) In addition to introducing single-stranded RNA as a microRNA precursor, (2) There is a method of introducing microRNA itself and RNA consisting of a complementary strand of microRNA and 100% -matched double-stranded RNA, and (3) double-stranded RNA assuming a state after microRNA is cleaved into Dicer. can give. Examples of products using such a method include miCENTURY OX Precursor (B-Bridge), miCENTURY OX siMature (B-Bridge), miCENTURY OX miNatural (B-Bridge), and the like.

6). Method for suppressing the activity of the nucleic acid of the present invention such as microRNA or microRNA precursor The nucleic acid of the present invention such as microRNA or microRNA precursor is an antisense technology [Bioscience and Industry, 50 , 322 (1992), Chemistry, 46, 681 (1991), Biotechnology, 9 , 358 (1992), Trends in Biotechnology, 10 , 87 (1992), Trends in Biotechnology, 10 , 152 (1992), Cell engineering, 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 nucleic acid 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), Nucleic Acids Res. 37 , e43, (2009)], or siRNA (short interfering RNA) to suppress its activity be able to.

An antisense nucleic acid refers to a nucleic acid capable of suppressing the expression of a target nucleic acid by hybridizing a nucleic acid having a base sequence complementary to the base sequence of a certain target nucleic acid in a specific base sequence. As the nucleic acid used for the antisense nucleic acid, in addition to DNA, RNA or nucleotide analogues, these chimeric molecules or derivatives of the nucleic acids can also be used. Specifically, antisense can be produced and expression can be suppressed by following the method described in Nature, 432 , 226 (2004) and the like. In addition, the expression of the nucleic acid of the present invention such as microRNA or microRNA precursor can be suppressed in the same manner as Anti-miR ^™ miRNA Inhibitors (Ambion) or miRIDIAN microRNA Inhibitors (GE Healthcare).

The siRNA is a short double-stranded RNA containing a base sequence of a certain target nucleic acid and can suppress the expression of the target nucleic acid by RNA interference (RNAi). The sequence of siRNA can be appropriately designed based on the conditions of the literature [Genes Dev, 13 , 3191 (1999)] from the target nucleotide sequence. For example, siRNA can be prepared by synthesizing and annealing two RNAs having a sequence of 19 bases selected and a sequence obtained by adding TT to the 3 ′ end of each complementary sequence and annealing. Further, by inserting a DNA corresponding to the selected 19-base sequence into a siRNA expression vector such as pSilencer 1.0-U6 (Ambion) or pSUPER (OligoEngine), the expression of the gene can be suppressed. A vector expressing siRNA can be prepared. As the siRNA that suppresses the nucleic acid of the present invention such as microRNA, any siRNA that can suppress the activity of the nucleic acid may be used. The number of residues of the base constituting one strand of siRNA is preferably 17 to 30 residues, more preferably 18 to 25 residues, still more preferably 19 to 23 residues.

The decoy nucleic acid method is a method of reducing the activity of a molecule by introducing a large amount of nucleic acid molecule into a cell and binding a target molecule. A decoy nucleic acid for miRNA can be designed using a single-stranded nucleic acid sequence similar to the target sequence of the miRNA. It is also possible to express such decoy nucleic acid using an expression vector.

MicroRNA expressed in the cell using antisense or siRNA or decoy nucleic acid specific to the nucleic acid of the present invention such as microRNA and microRNA precursor expressed in cells whose cell cycle is suppressed and cell growth is reduced And the expression of microRNA precursors can be suppressed. For example, by administering an antisense DNA or siRNA or decoy nucleic acid specific for the microRNA or the microRNA precursor, the activity of the microRNA is suppressed, and the action of the microRNA or microRNA precursor in the cell Can be controlled.

In addition, in the case of a patient due to abnormally increased expression of the nucleic acid of the present invention such as microRNA expressed in cells or a precursor thereof, an antisense oligonucleotide, siRNA or decoy nucleic acid specific for the microRNA or a precursor thereof is used in the patient. Can be used to control the function of the cells and treat diseases caused by the abnormal expression. That is, the antisense oligonucleotide or siRNA or decoy nucleic acid specific for the microRNA or its precursor is useful as a therapeutic agent for a disease caused by abnormally increased expression of the nucleic acid of the present invention.

When an antisense oligonucleotide or siRNA specific to the nucleic acid of the present invention such as microRNA or a precursor thereof is used as the above therapeutic agent, the antisense oligonucleotide or siRNA alone or the nucleic acid encoding them is a plasmid. After inserting into an appropriate expression vector such as a vector, retrovirus vector, adenovirus vector, adeno-associated virus vector, etc., it can be administered as a pharmaceutical preparation according to the conventional method described in the following 9.

7). Method of suppressing gene function using nucleic acid of the present invention such as microRNA or microRNA precursor As a method of suppressing gene function using nucleic acid of the present invention, the expression of mRNA having a target base sequence is microscopic. Any method may be used as long as the method uses the activity suppressed by nucleic acids such as RNA. For example, by expressing the nucleic acid of the present invention and increasing the amount of nucleic acid such as microRNA in the cell, there can be mentioned a method of suppressing the expression of the gene by suppressing the translation of mRNA having the target sequence. The nucleic acid of the present invention can be expressed by the method described in 5 above. Examples of the mRNA having the target base sequence of the nucleic acid consisting of the base sequence represented by any of SEQ ID NOs: 1 to 407 include the gene groups shown in Table 4 above, respectively.

Moreover, the function of the target gene can be suppressed using siRNA for the target gene shown in Table 4.

8). A method for screening a substance that promotes or suppresses the expression or function of a nucleic acid of the present invention such as microRNA or a microRNA precursor of the present invention Using a nucleic acid of the present invention, a nucleic acid such as microRNA or a precursor thereof Substances that promote or suppress expression or function can be screened. For example, expression or function of a selected microRNA or a precursor thereof using a cell expressing a nucleic acid having the base sequence by selecting a base sequence to be screened from the base sequence of the nucleic acid of the present invention. It is possible to screen for a substance that promotes or suppresses.

As a cell expressing microRNA and a microRNA precursor used for screening, in addition to cancer cells, a vector expressing a nucleic acid having the base sequence is used as a host such as an animal cell or yeast as described in 5 above. A transformed cell obtained by introduction into a cell, a cell in which a nucleic acid having the base sequence is directly introduced without using a vector, and the like can also be used.

Specific screening methods include methods that use changes in the expression level of nucleic acids such as microRNAs or their precursors to be screened as indicators, as well as mRNAs that have nucleic acid target sequences such as microRNAs, and encoded by them. A method using the change in the expression level of the gene product as an index can be mentioned.

(A) Screening method using as an index a change in the expression level of the nucleic acid of the present invention, such as a microRNA to be screened or a precursor thereof, and a cell expressing the nucleic acid. Using a change in expression level as an indicator, a substance that promotes or suppresses nucleic acids such as expression of microRNA and its precursor is obtained. The expression level of the nucleic acid can be detected by the method described in 3 above.

More specifically, it is as follows.
(ai) A test substance is brought into contact with a cell expressing the nucleic acid of the present invention and cultured.
(a-ii) The cells expressing the nucleic acid of the present invention are cultured without contacting the test substance.
(a-iii) The expression level of the nucleic acid of the present invention in the cells cultured in (ai) and (a-ii) is measured.
(a-iv) The expression level of the nucleic acid of the present invention in the cell of (ai) is compared with that in the cell of (a-ii), and the expression of the nucleic acid of the present invention is statistically significant in the cell of (ai). When the expression has been promoted, the test substance is selected as a substance that can change the cell cycle (suppress cell cycle progression) or a candidate substance that can suppress cell growth. Alternatively, in the cell (ai), when the expression of the nucleic acid of the present invention is statistically significantly suppressed, the test substance is a substance capable of changing the cell cycle (promoting the progression of the cell cycle). Or it selects as a candidate substance of the substance which can enhance cell growth.

The culture conditions in (a-i) and (a-ii) may be culture conditions that are usually used for culturing the cells, and those skilled in the art can appropriately set the culture conditions according to the type of the cells. Preferably, the culture conditions in (a-i) and (a-ii) are the same except for the presence or absence of the test substance.

(B) Screening method using as an index the change in the expression level of mRNA having a nucleic acid target sequence such as microRNA to be screened and the gene product encoded by it Contact test cells with cells expressing the mRNA Thus, a substance that promotes or suppresses the expression or function of nucleic acids such as microRNA and its precursor is obtained using changes in the expression level of mRNA having the target sequence of the selected nucleic acid and the gene product encoded thereby as an index. Alternatively, a DNA in which a target sequence of a nucleic acid such as the microRNA of the present invention 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 test substance is introduced into the cell. To obtain a substance that promotes or suppresses the expression or function of nucleic acids such as microRNA and its precursor, using the change in the expression level of the reporter gene as an indicator.

Selection of the target sequence can be performed by the method described in 8 above. Examples of mRNA having a nucleic acid target sequence such as a microRNA comprising a base sequence represented by any one of SEQ ID NOs: 1 to 407 include: Examples of the gene groups shown in Table 4 above can be given.

More specifically, it is as follows.
(bi) A test substance is brought into contact with a cell expressing the target gene of the nucleic acid of the present invention and cultured.
(b-ii) The cell expressing the target gene of the nucleic acid of the present invention is cultured without contacting the test substance.
(b-iii) The expression level of the target gene of the nucleic acid of the present invention in the cells cultured in (bi) and (b-ii) is measured.
(b-iv) The expression level of the target gene of the nucleic acid of the present invention in the cell of (bi) is compared with that in the cell of (b-ii), and the present invention is statistically significant in the cell of (bi). If the expression of the target gene of the nucleic acid is suppressed, the test substance is selected as a substance that can change the cell cycle (suppress cell cycle progression) or a candidate substance that can suppress cell growth . Alternatively, if the expression of the nucleic acid of the present invention is statistically significantly promoted in the cell (bi), the test substance is a substance capable of changing the cell cycle (promoting the progression of the cell cycle). Or it selects as a candidate substance of the substance which can enhance cell growth.

The culture conditions in (b-i) and (b-ii) may be any culture conditions usually used for culturing the cells, and those skilled in the art can appropriately set the culture conditions according to the type of the cells. Preferably, the culture conditions in (b-i) and (b-ii) are the same except for the difference in the presence or absence of the test substance.

The candidate substance selected in (a-iv) or (b-iv) above actually changes the cell cycle (suppresses or promotes cell cycle progression) or suppresses (or promotes) cell proliferation. You may check.

More specifically, it is as follows.
(ci) A cell is brought into contact with a candidate substance and cultured.
(c-ii) The cells are cultured without contacting the candidate substance.
(c-iii) The cell cycle or cell proliferation in the cells cultured in (ci) and (c-ii) is measured.
(c-iv) The cell cycle or cell proliferation in the cell of (ci) was compared with that of the cell of (c-ii), and the cell cycle was statistically significantly changed in the cell of (ci) When cell cycle progression is suppressed or when cell growth is suppressed, the candidate substance is used as a substance that changes the cell cycle (suppresses cell cycle progression) or cell proliferation. Get as a substance. Alternatively, in the cell of (ci), if the cell cycle is statistically significantly changed (progression of cell cycle is promoted) or cell proliferation is promoted, the candidate substance is Or a substance that fluctuates the cell cycle (promotes the progression of the cell cycle) or a substance that promotes cell proliferation.

The cells used in this confirmation step are preferably mammalian cells that proliferate by culture. Examples of the cells include cancer cells. The culture conditions in (c-i) and (c-ii) may be any culture conditions that are usually used for culturing the cells, and those skilled in the art can appropriately set them according to the type of the cells. Preferably, the culture conditions in (c-i) and (c-ii) are the same except for the presence or absence of candidate substances. Measurement of the cell cycle is as follows. In addition, the measurement of cell proliferation is the following 12. It can be performed by the method described in 1.

In one embodiment, the nucleic acids of the present invention include miR-106a, miR-106b, miR-122a, miR-124a, miR-147, miR-153, miR-17-5p, miR-224, miR-302d, miR- 320, miR-337, miR-345, miR-363, miR-373, miR-375, miR-431, miR-448, miR-491, miR-493-3p, miR-493-5p, miR-506, miR-507, miR-509, miR-511, miR-512-5p, miR-519a, miR-519b, miR-519c, miR-526b, miR-630 or miR-92b, or their orthologs, 5 'end When microRNAs having the same base sequence on the second to eighth sides or these microRNA precursors are selected, the increase in G1 phase is preferably used as an index. That is, when the candidate substance increases the G1 phase, the substance can be obtained as a substance that increases the G1 phase of the cell cycle. Such a substance has a high possibility of suppressing cell growth like the above-described nucleic acid of the present invention. On the other hand, when the candidate substance decreases the G1 phase, the substance can be obtained as a substance that decreases the G1 phase of the cell cycle. In contrast to the nucleic acid of the present invention described above, such a substance is likely to promote cell proliferation.

In one embodiment, when miR-196b, or an ortholog thereof, or a microRNA having the same base sequence at the 2nd to 8th positions on the 2 ′ end, or a microRNA precursor thereof is selected as the nucleic acid of the present invention, It is preferable to use the increase in S phase as an index. That is, when a candidate substance increases the S phase, the substance can be obtained as a substance that increases the S phase of the cell cycle. Such a substance has a high possibility of suppressing cell growth like the above-described nucleic acid of the present invention. On the other hand, when the candidate substance decreases the S phase, the substance can be obtained as a substance that decreases the S phase of the cell cycle. In contrast to the nucleic acid of the present invention described above, such a substance is likely to promote cell proliferation.

In one embodiment, the nucleic acids of the present invention include let-7b, let-7d, let-7e, let-7f, let-7g, miR-129, miR-134, miR-142-3p, miR-148a, miR- 148b, miR-149, miR-152, miR-193a, miR-193b, miR-197, miR-202, miR-214, miR-217, miR-221, miR-326, miR-329, miR-34a, miR-34b, miR-34c, miR-363STAR, miR-379, miR-449, miR-449b, miR-450, miR-500, miR-504, miR-512-3p, miR-517a, miR-517b, miR-518fSTAR, miR-542-3p, miR-544, miR-549, miR-552, miR-555, miR-561, miR-583, miR-585, miR-604, miR-637, miR-644, miR-661, miR-766, miR-769-3p or miR-96, or an ortholog thereof, 5′-end micro RNA having the same base sequence at the second to eighth positions, or a microRNA precursor thereof was selected. In some cases, it is preferable to use an increase in the G2M phase as an index. That is, when a candidate substance increases the G2M phase, the substance can be obtained as a substance that increases the G2M phase of the cell cycle. Such a substance has a high possibility of suppressing cell growth like the above-described nucleic acid of the present invention. On the other hand, when the candidate substance decreases the G2M phase, the substance can be obtained as a substance that decreases the G2M phase of the cell cycle. In contrast to the nucleic acid of the present invention described above, such a substance is likely to promote cell proliferation.

In one embodiment, miR-452 or miR-494, or an ortholog thereof, microRNA having the same base sequence at the 2nd to 8th positions on the 5 ′ end, or a precursor of these microRNAs was selected as the nucleic acid of the present invention. In some cases, it is preferable to use the increase in the G1 and G2M phases as an index. That is, when a candidate substance increases the G1 and G2M phases, the substance can be obtained as a substance that increases the G1 and G2M phases of the cell cycle. Such a substance has a high possibility of suppressing cell growth like the above-described nucleic acid of the present invention. On the other hand, when the candidate substance decreases the G1 and G2M phases, the substance can be obtained as a substance that decreases the G1 and G2M phases of the cell cycle. In contrast to the nucleic acid of the present invention described above, such a substance is likely to promote cell proliferation.

In one embodiment, the nucleic acid of the present invention includes let-7c, miR-154, miR-196a, miR-222, or miR-98, or an ortholog thereof, 5′-terminal microarrays having the same base sequence at the 2nd to 8th positions. When RNA or a microRNA precursor thereof is selected, it is preferable to use an increase in S phase and G2M phase as an index. That is, when a candidate substance increases S phase and G2M phase, the substance can be obtained as a substance that increases S phase and G2M phase of the cell cycle. Such a substance has a high possibility of suppressing cell growth like the above-described nucleic acid of the present invention. On the other hand, when the candidate substance decreases the S phase and the G2M phase, the substance can be obtained as a substance that decreases the S phase and the G2M phase of the cell cycle. In contrast to the nucleic acid of the present invention described above, such a substance is likely to promote cell proliferation.

In the confirmation step, it is sufficient to confirm either the effect on the cell cycle or the effect on cell proliferation, but the effect on both may be confirmed. For example, the effect on the cell cycle may be confirmed first, and the effect on cell proliferation may be confirmed for candidate substances for which the effect has been confirmed.

9. Cell growth inhibitor containing the nucleic acid of the present invention such as microRNA or microRNA precursor Among the nucleic acids of the present invention, those that suppress cell growth by controlling the expression of a gene having a target sequence are cell growth inhibitors Can be used as

Examples of the active ingredient of the cell growth inhibitor of the present invention include the following nucleic acids (a) to (h).
(A) Nucleic acid comprising a base sequence represented by any of SEQ ID NOs: 1 to 407 (b) Nucleic acid comprising 17 to 28 bases comprising a nucleic acid comprising a base sequence represented by any of SEQ ID NOs: 1 to 407 (C) a nucleic acid comprising a base sequence represented by any one of SEQ ID NOs: 1 to 407 and having a nucleotide sequence having 90% or more identity (d) comprising a base sequence represented by any of SEQ ID NOs: 1 to 407 Nucleic acid that hybridizes with a complementary strand of nucleic acid under stringent conditions (e) A nucleic acid comprising the second to eighth base sequences of the base sequence represented by any one of SEQ ID NOs: 1 to 407 (f) SEQ ID NOs: 408 to 920 A nucleic acid consisting of a base sequence represented by any of (g) a nucleic acid comprising a base sequence represented by any one of SEQ ID NOs: 408 to 920 and a base sequence having 90% or more identity (h) SEQ ID NOs: 408 to Any of 920 A nucleic acid that hybridizes to a complementary strand under stringent conditions nucleic acid consisting of the nucleotide sequence.

The nucleic acids described in (a) to (h) above may be microRNA or a microRNA precursor.

Also, a vector that expresses the above nucleic acid can be used as a cell growth inhibitor.

On the other hand, a substance that suppresses the expression of a target gene of a nucleic acid such as the above microRNA can also be used as a cell growth inhibitor. As this substance, a nucleic acid or a vector expressing it can also be used. Examples of the substance that suppresses the expression of the target gene include siRNA for the mRNA of the target gene and antisense oligonucleotide for the target gene.

For the formulation form and administration method of the cell growth inhibitor of the present invention, refer to 10. These are the same as diagnostic agents and therapeutic agents containing the nucleic acid of the present invention such as microRNA and microRNA precursor described later.

10. Diagnostic and therapeutic agents containing nucleic acids such as microRNAs and microRNA precursors of the present invention The nucleic acids of the present invention control the expression of genes having a target sequence or the expression of the nucleic acids of the present invention such as microRNAs. By controlling, it can be used as a therapeutic agent for diseases caused by abnormal growth of cells such as cancer. Furthermore, siRNA against the target gene of the nucleic acid of the present invention such as microRNA can be used as a therapeutic agent for diseases caused by abnormal growth or differentiation of cells by controlling the expression of the gene. Diseases caused by abnormal cell proliferation include cancer, arteriosclerosis, rheumatoid arthritis, benign prostatic hyperplasia, vascular restenosis after percutaneous transvascular coronary angioplasty, pulmonary fibrosis, glomerulonephritis and autoimmune disease Etc.

Further, by quantifying the nucleic acid of the present invention or detecting a mutation, it is possible to diagnose a disease caused by abnormal growth or differentiation of cells such as cancer.
The diagnostic agent containing the nucleic acid of the present invention is a reagent necessary for quantifying the nucleic acid of the present invention or detecting a mutation, for example, a buffer, a salt, a reaction enzyme, a It may contain a labeled protein that binds to the nucleic acid, a color former for detection, and the like.

The therapeutic agent containing the nucleic acid of the present invention as an active ingredient can be administered alone, but usually mixed with one or more pharmacologically acceptable carriers, and the technical field of pharmaceutical sciences It is desirable to administer it as a pharmaceutical formulation produced by any method well known in the art.
It is desirable to use the most effective route for treatment, and oral administration or parenteral administration such as buccal, respiratory tract, rectal, subcutaneous, intramuscular and intravenous is desirable. Can be given intravenously.

Examples of the dosage form include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.
Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
Liquid preparations such as emulsions and syrups include sugars such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, p-hydroxybenzoic acid Preservatives such as esters, and flavors such as strawberry flavor and peppermint can be used as additives.

For capsules, tablets, powders, granules, etc., excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, hydroxy A binder such as propylcellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin can be used as additives.

Formulations suitable for parenteral administration include injections, suppositories, sprays and the like.
The injection is prepared using a carrier made of a salt solution, a glucose solution, or a mixture of both. Suppositories are prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid. The spray is prepared using a carrier that does not irritate the recipient's oral cavity and airway mucosa, and that facilitates absorption by dispersing the active ingredient as fine particles.

Specific examples of the carrier include lactose and glycerin. Depending on the nature of the nucleic acid or microRNA precursor of the present invention and the carrier used, preparations such as aerosols and dry powders are possible. In these parenteral preparations, the components exemplified as additives for oral preparations can also be added.
The dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, weight, etc., but is usually 10 μg / kg to 20 mg / kg per day for an adult.

The therapeutic agent containing the nucleic acid of the present invention as an active ingredient can also be produced by preparing a vector that expresses the nucleic acid of the present invention and a base used for the nucleic acid therapeutic agent [Nature Genet., 8 , 42 (1994)].
The base used in the therapeutic agent of the present invention may be any base as long as it is usually used in injections, salt water such as distilled water, sodium chloride or a mixture of sodium chloride and an inorganic salt, mannitol, Examples thereof include a solution of lactose, dextran, glucose and the like, an amino acid solution such as glycine and arginine, an organic acid solution or a mixed solution of a salt solution and a glucose solution, and the like. In addition, according to a conventional method, these bases are mixed with an osmotic pressure adjusting agent, a pH adjusting agent, a vegetable oil such as sesame oil and soybean oil, or an auxiliary such as a surfactant such as lecithin or a nonionic surfactant. An injection may be prepared as a suspension or dispersion. These injections can be prepared as preparations for dissolution at the time of use by operations such as pulverization and freeze-drying. The therapeutic agent of the present invention can be used for treatment as it is in the case of a liquid just before the treatment, or in the case of an individual, dissolved in the above sterilized base as necessary.

Examples of the vector for expressing the nucleic acid of the present invention include the recombinant virus vector prepared in 6 above, and more specifically, a retrovirus vector and a lentivirus vector.
For example, a viral vector can be prepared by preparing a complex by combining the nucleic acid of the present invention with a polylysine-conjugated antibody specific for an adenovirus hexon protein and binding the resulting complex to an adenovirus vector. . The virus vector stably reaches the target cell, is taken up into the cell by endosomes, is degraded in the cell, and the nucleic acid can be efficiently expressed.

In addition, viral vectors based on Sendai virus (-) strand RNA virus have been developed (WO97 / 16538, WO97 / 16539), and Sendai virus incorporating the nucleic acid of the present invention using the Sendai virus has been developed. Can be produced.
The nucleic acids of the present invention can also be transferred by non-viral nucleic acid transfer methods. For example, calcium phosphate coprecipitation method [Virology, 52 , 456-467 (1973); Science, 209 , 1414-1422 (1980)], microinjection method [Proc. Natl. Acad. Sci. USA, 77 , 5399-5403 ( Proc. Natl. Acad. Sci. USA, 77 , 7380-7384 (1980); Cell, 27 , 223-231 (1981); Nature, 294 , 92-94 (1981)], liposome-mediated membrane Fusion-mediated transfer [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 [Science, 247 , 1465-1468 (1990); J. Biol. Chem., 266 , 14338-14342 (1991); Proc. Natl. Acad. Sci. USA, 87 , 3655-3659 (1991); 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)].

The liposome-mediated membrane fusion-mediated transfer method involves direct administration of a liposome preparation to a target tissue, thereby incorporating the nucleic acid of the present invention and siRNA for a target gene of a nucleic acid such as microRNA into the tissue. And can be expressed [Hum. Gene Ther., 3 , 399 (1992)]. Direct DNA uptake techniques are preferred for targeting DNA directly to a lesion.

Receptor-mediated DNA transfer can be performed, for example, by binding DNA (typically in the form of a covalently closed supercoiled plasmid) to a protein ligand via polylysine. The ligand is selected based on the presence of the corresponding ligand receptor on the cell surface of the target cell or tissue. The ligand-DNA conjugate can be injected directly into the blood vessel, if desired, and can be directed to a target tissue where receptor binding and internalization of the DNA-protein complex occurs. In order to prevent intracellular destruction of DNA, adenovirus can be co-infected to disrupt endosomal function.

11. Method for measuring cell cycle fluctuations in cancer cells and other cells Cell cycle measurement is performed by staining the DNA in the nucleus with a fluorescent dye such as Hoechst dye or Propium Iodide, and then measuring the content per cell using a flow cytometer. A method of measuring can be used (Krishan A., et al: Cancer Res. 1978, 38 : 3656-3662.). In addition, M phase cells that are difficult to distinguish from G2 phase by DNA content can be distinguished by staining their chromosomes and observing their characteristic aggregation state (Angulo, R .. et al: Cytometry, 1998, 34 , 143.). In addition to DNA staining with Hoechst dye and the like, immunostaining with phosphorylated histone H3 antibody that is specifically expressed in M phase is also used for chromosome staining. In addition to the fluorescence microscope, a high content screening system (for example, In Cell Analyzer 1000 (GE Healthcare)) that can simultaneously observe multiple specimens can be used for observation.

12 Method for Measuring Cancer Cell Proliferation and Cell Degree Degree The cell proliferation measurement method is not particularly limited as long as it is a method capable of measuring an index reflecting the number of cells and the cell growth rate. Viable cell count measurement, DNA synthesis rate measurement, total protein mass measurement, and the like can be used. An example of a method for evaluating the number of living cells is a method of measuring the amount of ATP in the cells. It is known that the amount of ATP in cells is proportional to the number of cells in culture (J. Immunol. Meth., 160 , 81-88 (1993)). More specific methods for measuring the amount of ATP in cells include the MTT method and the XTT method (J. Immunol. Meth., 65 , 55-63 (1983)). Another example is a method of measuring ATP by luminescence of a luciferin substrate by an ATP-dependent enzyme luciferase. As a kit for measuring the amount of ATP in cells, for example, CellTite-Glo ^R Luminescent Cell Viability Assay (manufactured by Promega) may be used. As a method for measuring the degree of cell death, a method of staining dead cells with a dye such as Propium Iodide, a method of measuring the activity of an enzyme leaked to the outside due to cell death, or the like can be used. For the latter, for example, a method for measuring the enzyme activity of adenylate kinase leaked out of the cell can be used. More specifically, ToxiLight Non-Destructive Cytotoxicity BioAssay Kit (manufactured by Lonza) may be used.

The present invention will be specifically described by the following examples. However, the present invention is not limited to these examples.

Cell cycle variability in cervical cancer-derived cell lines in which microRNA was forcibly expressed MicroRNA precursors were introduced into cervical cancer-derived cell lines, and the influence of microRNA precursors on cell cycle variability was examined.
HeLa human cervical cancer-derived cell line (hereinafter referred to as HeLa) was obtained from the American Type Culture Collection (hereinafter referred to as ATCC), and MEM medium (Invitrogen) containing 10% fetal calf serum (FBS, manufactured by JRH Biosciences). In the incubator with 5% CO ₂ concentration at 37 ° C.

HeLa cells were seeded in a 96-well plate at 3,000 cells per well and cultured overnight in MEM medium containing 10% FBS. One day later, the microRNA precursor was introduced into HeLa cells to a final concentration of 50 nM by a lipofection method, specifically, a method using oligofectamine (manufactured by Invitrogen). MicroRNA precursors include let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, miR-106a, miR-106b, miR-122a, miR-124a, miR- 129, miR-134, miR-142-3p, miR-147, miR-148a, miR-148b, miR-149, miR-152, miR-153, miR-154, miR-17-5p, miR-193a, miR-193b, miR-196a, miR-196b, miR-197, miR-202, miR-214, miR-217, miR-221, miR-222, miR-224, miR-302d, miR-320, miR- 326, miR-329, miR-337, miR-345, miR-34a, miR-34b, miR-34c, miR-363, miR-363 *, miR-373, miR-375, miR-379, miR-431 , MiR-448, miR-449, miR-449b, miR-450, miR-452, miR-491, miR-493-3p, miR-493-5p, miR-494, miR-500, miR-504, miR -506, miR-507, miR-509, miR-511, miR-512-3p, miR-512-5p, miR-517a, miR-517b, miR-518f *, miR-519a, miR-519b, miR- 519c, miR-526b, miR-542-3p, miR-544, miR-549, miR-552, miR-555, miR-561, miR-583, miR-585, miR-604, miR-630, miR- 637, miR-644, miR- 661, miR-766, miR-769-3p, miR-92b, miR-96 and miR-98 in the Pre-miR ^TM miRNA Precur sor Molecules (Ambion) was used. Pre-miR ^™ miRNA Precursor Molecules-Negative Control # 1 (hereinafter referred to as miR-NC # 1) (Ambion) was also introduced into HeLa cells to serve as a negative control. Lipofection followed the method described in the instructions attached to the product.

30 or 48 hours after the introduction of the microRNA precursor by the lipofection method, the cells were fixed with 70% ethanol and subjected to nuclear staining with NIM-DAPI (Beckman® Colter), and the flow cytometer Quanta SC MPL (Beckman Colter) The DNA content of each cell was measured. Set a gate for each cell cycle stage (G1 phase, S phase, G2M phase) in the histogram of DNA content after measurement, calculate the number of cells included in each cell cycle stage, and calculate the number of stages in the total number of living cells The percentage was calculated. For each microRNA precursor-introduced cell, the relative cell cycle rate was calculated with the ratio of each cell cycle stage of the HeLa cells in each control group (miR-NC # 1 introduced group) being 1.0. Tables 5-1 to 5-4 show the results of microRNAs that caused cell cycle fluctuations. Tables 6-1 and 6-2 show the correspondence between the microRNAs used in the examples and the sequence listing. The sequence is shown according to the microRNA database, miRBase 9.2, and the sequence of mature microRNA.

Each experiment was performed twice independently, and a microRNA showing a relative value exceeding 1.0 was regarded as a hit in either of 30 hours or 48 hours later. miR-106a, miR-106b, miR-122a, miR-124a, miR-147, miR-153, miR-17-5p, miR-224, miR-302d, miR-320, miR-337, miR-345, miR-363, miR-373, miR-375, miR-431, miR-448, miR-491, miR-493-3p, miR-493-5p, miR-506, miR-507, miR-509, miR- 511, miR-512-5p, miR-519a, miR-519b, miR-519c, miR-526b, miR-630 and miR-92b miRNA is introduced to increase G1 phase and G1 phase in the cell cycle The proportion of increased. The introduction of miR-196b increased S phase and increased the proportion of S phase in the cell cycle. let-7b, let-7d, let-7e, let-7f, let-7g, miR-129, miR-134, miR-142-3p, miR-148a, miR-148b, miR-149, miR-152, miR-193a, miR-193b, miR-197, miR-202, miR-214, miR-217, miR-221, miR-326, miR-329, miR-34a, miR-34b, miR-34c, miR- 363 *, miR-379, miR-449, miR-449b, miR-450, miR-500, miR-504, miR-512-3p, miR-517a, miR-517b, miR-518f *, miR-542- 3p, miR-544, miR-549, miR-552, miR-555, miR-561, miR-583, miR-585, miR-604, miR-637, miR-644, miR-661, miR-766, The introduction of miR-769-3p and miR-96 miRNAs increased G2M phase and increased the proportion of G2M phase in the cell cycle. By introducing miR-452 and miR-494 miRNAs, the G1 and G2M phases increased and the cell cycle rate increased. The introduction of let-7c, miR-154, miR-196a, miR-222, and miR-98 miRNAs increased S and G2M phases, and increased the proportion of S and G2M phases in the cell cycle.

Viable cell rate in cervical cancer-derived cell line in which microRNA was forcibly expressed A microRNA precursor was introduced into a cervical cancer-derived cell line, and the influence of the microRNA precursor on the viable cell rate was examined.

HeLa cells were seeded in a 384-well plate at 100 cells per well and cultured overnight in MEM medium containing 10% FBS. One day later, the microRNA precursor was introduced into HeLa cells to a final concentration of 50 nM by a lipofection method, specifically, a method using oligofectamine (manufactured by Invitrogen). MicroRNA precursors include let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, miR-106a, miR-106b, miR-122a, miR-124a, miR- 129, miR-134, miR-142-3p, miR-147, miR-148a, miR-148b, miR-149, miR-152, miR-153, miR-154, miR-17-5p, miR-193a, miR-193b, miR-196a, miR-196b, miR-197, miR-202, miR-214, miR-217, miR-221, miR-222, miR-224, miR-302d, miR-320, miR- 326, miR-329, miR-337, miR-345, miR-34a, miR-34b, miR-34c, miR-363, miR-363 *, miR-373, miR-375, miR-379, miR-431 , MiR-448, miR-449, miR-449b, miR-450, miR-452, miR-491, miR-493-3p, miR-493-5p, miR-494, miR-500, miR-504, miR -506, miR-507, miR-509, miR-511, miR-512-3p, miR-512-5p, miR-517a, miR-517b, miR-518f *, miR-519a, miR-519b, miR- 519c, miR-526b, miR-542-3p, miR-544, miR-549, miR-552, miR-555, miR-561, miR-583, miR-585, miR-604, miR-630, miR- 637, miR-644, miR- 661, miR-766, miR-769-3p, miR-92b, miR-96 and miR-98 in the Pre-miR ^TM miRNA Precur sor Molecules (Ambion) was used. Pre-miR ^™ miRNA Precursor Molecules-Negative Control # 1 (hereinafter referred to as miR-NC # 1) (Ambion) was also introduced into HeLa cells to serve as a negative control. Lipofection followed the method described in the instructions attached to the product.

Five days after the introduction of the microRNA precursor by the lipofection method, the cell viability was measured using the CellTiter-Glo ^™ Luminescent Cell Viability Assay (Promega) according to the method described in the instructions attached to the product. . The relative viable cell ratio of each control group (miR-NC # 1 introduction group) was calculated by setting the viable cell ratio of HeLa cells to 100. As a result, as shown in Table 7-1 and Table 7-2, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, miR-106a, miR-106b, miR-122a, miR-124a, miR-129, miR-134, miR-142-3p, miR-147, miR-148a, miR-148b, miR-149, miR-152, miR-153, miR-154, miR-17-5p, miR-193a, miR-193b, miR-196a, miR-196b, miR-197, miR-202, miR-214, miR-217, miR-221, miR-222, miR-224, miR-302d, miR-320, miR-326, miR-329, miR-337, miR-345, miR-34a, miR-34b, miR-34c, miR-363, miR-363 *, miR-373, miR -375, miR-379, miR-431, miR-448, miR-449, miR-449b, miR-450, miR-452, miR-491, miR-493-3p, miR-493-5p, miR-494 , MiR-500, miR-504, miR-506, miR-507, miR-509, miR-511, miR-512-3p, miR-512-5p, miR-517a, miR-517b, miR-518f *, miR-519a, miR-519b, miR-519c, miR-526b, miR-542-3p, miR-544, miR-549, miR-552, miR-555, miR-561, miR-583, miR-585, miR-604, miR-630, miR-637, miR-644, miR-661, miR-766, miR-769-3p, miR-92b, miR-96 and miR-98 Reduction in cell viability of 40% or more by the introduction of miRNA was observed.

Among microRNAs that affect the cell cycle in cervical cancer-derived cell lines and gastric cancer-derived cell lines, microRNAs that affect DNA methyltransferase 1 (Dnmt1)
MicroRNA precursors were introduced into cervical cancer-derived cell lines and gastric cancer-derived cell lines, and the influence of microRNA precursors on Dnmt1 was examined by qRT-PCR.
An AGS human gastric cancer-derived cell line (hereinafter referred to as AGS) was obtained from the American Type Culture Collection (hereinafter referred to as ATCC), and F12K medium (Invitrogen) containing 10% fetal calf serum (FBS, manufactured by JRH Biosciences). ) In an incubator at 37 ° C. with 5% CO ₂ concentration.
HeLa cells and AGS cells were seeded in 6-well plates at 10,000 cells per well, and cultured overnight in MEM medium or F12K medium containing 10% FBS. One day later, the microRNA precursor was introduced into HeLa cells and AGS cells at a final concentration of 50 nM by a lipofection method, specifically, a method using oligofectamine or Lipofectamine 2000 (manufactured by Invitrogen). As precursors of microRNA, miR-148a, miR-148b and miR-152 Pre-miR ^™ miRNA Precursor Molecules (manufactured by Ambion) were used. Pre-miR ^™ miRNA Precursor Molecules-Negative Control # 2 (hereinafter referred to as miR-NC # 2) (Ambion) was also introduced into HeLa cells and AGS cells to serve as negative controls. Furthermore, Dnmt1 siRNA (Qiagen, SI00300062) was used as a positive control. Lipofection followed the method described in the instructions attached to the product.
24 and 48 hours after the introduction of the microRNA precursor by lipofection, RNA was extracted using miRVana RNA isolation kit (Applied Biosystem) according to the method described in the instructions attached to the product. . Furthermore, reverse transcription reaction was performed using SuperScriptIII RT-reaction kit (manufactured by Invitrogen), and suppression of mRNA level of Dnmt1 was confirmed by qRT-PCR. The following primers were used. Forward: catcctcagggaccacatct (SEQ ID NO: 921), Reverse: gtgacggttgtgctgaagaa (SEQ ID NO: 922). As a result, as shown in FIG. 1, a decrease in Dnmt1 expression of about 20-40% was observed in both cells due to the introduction of miR-148a, miR-148b and miR-152 miRNAs.

Among microRNAs that affect the cell cycle in cervical cancer-derived cell lines and gastric cancer-derived cell lines, microRNAs that affect DNA methyltransferase 1 (Dnmt1)
MicroRNA precursors were introduced into cervical cancer-derived cell lines and gastric cancer-derived cell lines, and the influence of the microRNA precursors on the expression level of Dnmt1 protein was examined by immunoblotting.
HeLa cells and AGS cells were seeded in 6-well plates at 10,000 cells per well, and cultured overnight in MEM medium or F12K medium containing 10% FBS. One day later, the microRNA precursor was introduced into HeLa cells and AGS cells to a final concentration of 50 nM by a lipofection method, specifically, a method using oligofectamine or Lipofectamine 2000 (manufactured by Invitrogen). As precursors of microRNA, miR-148a, miR-148b and miR-152 Pre-miR ^™ miRNA Precursor Molecules (manufactured by Ambion) were used. Pre-miR ^™ miRNA Precursor Molecules-Negative Control # 2 (hereinafter referred to as miR-NC # 2) (Ambion) was also introduced into HeLa cells and AGS cells to serve as negative controls. Furthermore, Dnmt1 siRNA was used as a positive control. Lipofection followed the method described in the instructions attached to the product.
72 hours after introduction of the microRNA precursor by the lipofection method, proteins were extracted using RIPA buffer (Invitrogen) according to the method described in the instructions attached to the product. Furthermore, the Dnmt1 protein level was measured by immunoblotting using an anti-Dnmt1 antibody (NEB (M0231S)). As a result, as shown in FIG. 2, a decrease in the expression level of Dnmt1 protein was observed by introduction of miR-148a, miR-148b and miR-152 miRNAs in both cells.

M-phase accumulation rate in cervical cancer-derived cell line in which microRNA was forcibly expressed A microRNA precursor was introduced into a cervical cancer-derived cell line, and the influence of the microRNA precursor on the M-phase accumulation rate was examined.

HeLa cells were seeded in a 96-well plate at 3,000 cells per well and cultured overnight in MEM medium containing 10% FBS. One day later, the microRNA precursor was introduced into HeLa cells to a final concentration of 30 nM by a lipofection method, specifically, a method using oligofectamine (manufactured by Invitrogen). Pre-miR ^™ miRNA Precursor Molecules (made by Ambion) of miR-29b, miR-380-5p, miR-527, miR-193b, miR-485-5p and miR-409-3p as microRNA precursors Was used. In addition, Pre-miR ^™ miRNA Precursor Molecules-Negative Control # 2 (hereinafter referred to as miR-NC # 2) (Ambion) was introduced into HeLa cells as a negative control. Lipofection followed the method described in the instructions attached to the product.

Cells were fixed with 100% methanol 30 hours or 48 hours after introduction of the microRNA precursor by the lipofection method, and nuclear staining was performed with bisBenzimide H 33342 trihydrochoride (Hoechst, SIGMA), and IN cell analyzer 1000 (GE Healthcare ) Quantified the proportion of cells exhibiting a chromosome aggregation characteristic of M phase (M phase accumulation rate). The average value of the M-phase accumulation rate of HeLa cells in the control group (miR-NC # 2-introduced group) was 4.0%. Using this value as a reference value, the relative value of each M-phase accumulation rate was measured. Furthermore, the same methanol-fixed sample was stained with anti-phosphorylated histone H3 antibody (Cell Signaling, # 9701) showing specific expression in M phase, and phosphorylated histone H3 expression was similarly expressed with IN cell analyzer (GE Healthcare). The percentage of cells was measured. In the same manner as described above, the relative value of each M-phase accumulation rate was measured using the M-phase accumulation rate of the HeLa cells in the control group (miR-NC # 2 introduction group) as a reference value. As a result, as shown in Table 8, Hoechst staining and phosphorylation by introduction of miR-29b, miR-380-5p, miR-527, miR-193, miR-485-5p and miR-409-3p miRNAs. Increased M-phase accumulation rate due to oxidized histone H3 antibody was observed.

Five days after the introduction of the microRNA precursor by the lipofection method, the cell viability was measured using the CellTiter-Glo ^™ Luminescent Cell Viability Assay (Promega) according to the method described in the instructions attached to the product. . The relative viable cell ratio of each control group (miR-NC # 2-introduced group) HeLa cell viability was calculated as 100. As a result, as shown in Table 9, the introduction of miR-29b, miR-380-5p, miR-527, miR-193b, miR-485-5p and miR-409-3p miRNA increased to 40.0% or more. A decrease in the viable cell rate was observed.

Cell growth inhibition rate in liver cancer-derived cell lines in which microRNAs were forcibly expressed MicroRNA precursors were introduced into liver cancer-derived cell lines, and the influence of microRNA precursors on cell growth inhibition was examined.
HepG2 liver cancer cell line (hereinafter referred to as HepG2; ATCC HB-8065) is a MEM medium (Invitrogen) containing 10% fetal bovine serum (FBS, manufactured by JRH Biosciences) at 37 ° C and 5% CO ₂ concentration. In an incubator.
On a 384-well plate, the microRNA precursor was introduced into HepG2 cells by lipofection using oligofectamine (Invitrogen) to a final concentration of 25 nM. Specifically, microRNA and oligofectamine were mixed in advance and dispensed into a 384-well plate, and seeded at 1000 HepG2 cells per well. Details are attached to the product. The method described in the instructions was followed. As a precursor of microRNA, miRIDIAN miRNA Mimic Library (10.1) manufactured by Dharmacon was used. Pre-miR miRNA Precursor Molecules-Negative Control # 2 (hereinafter referred to as miR-NC # 2) (Ambion) was also introduced into HepG2 cells to serve as a negative control.
Three days after the introduction of the microRNA precursor by the lipofection method, the viable cell value was measured using CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the method described in the instructions attached to the product. The relative cell growth inhibition rate of each control group (miR-NC # 2-introduced group) HepG2 cell viable cell value was set to 0 and no cell was set to 100. As a result, the introduction of miR-363 *, miR-644 and miR-544 miRNAs showed inhibition rates of 77.2%, 65.7% and 62.9%, respectively.

Proliferation inhibition rate in ovarian cancer-derived cell lines in which microRNA was forcibly expressed MicroRNA precursors were introduced into ovarian cancer-derived cell lines, and the influence of microRNA precursors on cell growth inhibition was examined.
The RMG-I ovarian cancer-derived cell line (hereinafter referred to as RMG-I) was obtained from the Pharmaceutical Research Laboratories (JCRB), and F-12 medium containing 10% fetal bovine serum (FBS, manufactured by JRH Biosciences) ( Invitrogen) was cultured in an incubator at 37 ° C. with 5% CO ₂ concentration.
On a 384-well plate, a microRNA precursor was introduced into RMG-I cells by a lipofection method using HiPerfect (Qiagen) to a final concentration of 25 nM. Specifically, microRNA and HiPerfect were mixed in advance and dispensed into a 384-well plate, and RMG-I cells were seeded at 1000 cells per well. The method described in the attached instructions was followed. As a precursor of microRNA, miRIDIAN miRNA Mimic Library (10.1) manufactured by Dharmacon was used. A group in which only HiPerfect was introduced into RMG-I cells without a microRNA precursor was prepared and used as a negative control.
Three days after the introduction of the microRNA precursor by the lipofection method, the viable cell value was measured using CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the method described in the instructions attached to the product. The relative cell growth inhibition rate was calculated by setting the live cell value of RMG-I cells in the control group (Hiperfect only) to 0 and 100 to no cells. As a result, by introducing miR-544, miR-644 and miR-449 miRNAs, inhibition rates of 34.0%, 33.0% and 30.6% were shown, respectively.

Introducing an RNA precursor into another ovarian cancer cell line, the SK-OV-3 ovarian cancer cell line (hereinafter referred to as SK-OV-3), and the effect of the microRNA precursor on cell growth inhibition I investigated.
SK-OV-3 cells were obtained from ATCC and cultured in McCoy's 5A medium (Invitrogen) containing 10% fetal bovine serum (FBS, manufactured by JRH Biosciences) in an incubator at 37 ° C with 5% CO ₂ concentration. .
SK-OV-3 was seeded on a 384-well plate at 250 per well, and cultured overnight in McCoy's 5A medium containing 10% FBS. One day later, the microRNA precursor was introduced into SK-OV-3 cells to a final concentration of 25 nM by a lipofection method, specifically, a method using HiPerfect (Qiagen). As a precursor of microRNA, miRIDIAN miRNA Mimic Library (10.1) manufactured by Dharmacon was used. In addition, a group in which only HiPerfect was introduced into SK-OV-3 cells without a microRNA precursor was prepared and used as a negative control. Lipofection followed the method described in the instructions attached to the product.
Three days after the introduction of the microRNA precursor by the lipofection method, the viable cell value was measured using CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the method described in the instructions attached to the product. The relative cell growth inhibition rate was calculated by setting the SK-OV-3 cells in the control group (Hiperfect only) to 0 as the live cell value and 100 as the absence of cells. As a result, miR-644, miR-129, miR-96, miR-224 and miR-449b introduced miRNAs, and showed inhibition rates of 50.7%, 39.4%, 33.0%, 31.8% and 30.1%, respectively. .

According to the present invention, cell growth inhibitor, therapeutic agent and diagnostic agent for diseases caused by cell cycle fluctuation, expression inhibitor and promoter of target gene of nucleic acid such as microRNA, cell cycle fluctuation method, nucleic acid such as microRNA The method for suppressing the expression of the target gene, the method for promoting the expression, the screening method for the cell growth inhibitor, etc. are provided, and these are useful in the prevention, diagnosis and / or treatment of diseases caused by cell cycle fluctuations.

This application is based on Japanese Patent Application No. 2010-052417 filed in Japan (filing date: March 9, 2010), the contents of which are incorporated in full herein.

Claims

A cell growth regulator comprising any of the following nucleic acids (a) to (h) as an active ingredient:
(A) Nucleic acid comprising a base sequence represented by any of SEQ ID NOs: 1 to 407 (b) Nucleic acid comprising 17 to 28 bases comprising a nucleic acid comprising a base sequence represented by any of SEQ ID NOs: 1 to 407 (C) a nucleic acid comprising a base sequence represented by any one of SEQ ID NOs: 1 to 407 and having a nucleotide sequence having 90% or more identity (d) comprising a base sequence represented by any of SEQ ID NOs: 1 to 407 Nucleic acid that hybridizes with a complementary strand of nucleic acid under stringent conditions (e) A nucleic acid comprising the second to eighth base sequences of the base sequence represented by any one of SEQ ID NOs: 1 to 407 (f) SEQ ID NOs: 408 to 920 A nucleic acid consisting of a base sequence represented by any of (g) a nucleic acid comprising a base sequence represented by any one of SEQ ID NOs: 408 to 920 and a base sequence having 90% or more identity (h) SEQ ID NOs: 408 to Any of 920 A nucleic acid that hybridizes to a complementary strand under stringent conditions nucleic acid consisting of the nucleotide sequence.
The cell growth regulator according to claim 1, wherein the nucleic acid is microRNA or a microRNA precursor.
A cell growth regulator comprising, as an active ingredient, a nucleic acid having a base sequence complementary to the base sequence of the nucleic acid according to claim 1.
A cell growth regulator comprising as an active ingredient the vector that expresses the nucleic acid according to any one of claims 1 to 3.
A cell growth regulator comprising, as an active ingredient, a substance that suppresses the expression of a gene having the target base sequence of the nucleic acid according to claim 1.
A cell growth regulator comprising, as an active ingredient, a substance that promotes the expression of a gene having the target base sequence of the nucleic acid according to claim 1.
The cell growth regulator according to claim 5 or 6, wherein the substance that suppresses or promotes expression is a nucleic acid.
The cell growth regulator according to claim 7, wherein the nucleic acid is siRNA.
A cell growth regulator comprising the vector expressing the nucleic acid according to claim 7 as an active ingredient.
A disease caused by an abnormal cell cycle, comprising the nucleic acid according to any one of claims 1 to 3, the vector according to claim 4, or the substance according to any one of claims 5 to 8 as an active ingredient. Therapeutic or diagnostic agent.
A diagnostic agent for a disease caused by an abnormal cell cycle, comprising as an active ingredient a reagent for detecting the expression level of the nucleic acid according to claim 1, mutation of the nucleic acid, and mutation of a genome encoding the nucleic acid.
Groups of diseases caused by cell cycle abnormalities are cancer, arteriosclerosis, rheumatoid arthritis, benign prostatic hyperplasia, vascular restenosis after percutaneous transvascular coronary angioplasty, pulmonary fibrosis, glomerulonephritis and autoimmune disease The therapeutic or diagnostic agent according to claim 10 or 11, which is a disease selected from.
A cell cycle abnormality characterized by administering an effective amount of the nucleic acid according to any one of claims 1 to 3, the vector according to claim 4, or the substance according to any one of claims 5 to 8. To treat diseases caused by the disease.
A method for diagnosing a disease caused by an abnormal cell cycle, comprising detecting the expression level of the nucleic acid according to claim 1, a mutation of the nucleic acid, and a mutation of a genome encoding the nucleic acid.
Use of the nucleic acid according to any one of claims 1 to 3 and 7 for the manufacture of a therapeutic agent for a disease caused by an abnormal cell cycle.
An agent for controlling expression of a target gene of the nucleic acid, comprising the nucleic acid according to claim 1 as an active ingredient.
The expression regulator of the target gene of the nucleic acid according to claim 1, comprising the vector according to claim 4 as an active ingredient.
A cell cycle variation method using the nucleic acid according to claim 1 or 3.
A cell cycle variation method using the vector according to claim 4.
A cell cycle variation method comprising using a substance such as an antisense nucleic acid, siRNA, decoy nucleic acid or the like that suppresses or promotes expression of a target gene of the nucleic acid according to claim 1.
A cell growth control method using the nucleic acid according to claim 1 or 3.
A cell growth control method using the vector according to claim 4.
A method for controlling cell proliferation, comprising using a substance such as an antisense nucleic acid, siRNA, or decoy nucleic acid that suppresses or promotes expression of a target gene of the nucleic acid according to claim 1.
The method for suppressing expression or promoting expression of a target gene of nucleic acid according to claim 1, wherein the nucleic acid according to claim 1 or 3 is used.
The method for suppressing expression or promoting expression of a target gene of nucleic acid according to claim 1, wherein the vector according to claim 4 is used.
A method for screening a cell growth inhibitor, characterized by using the promotion of expression of the nucleic acid according to claim 1 as an index.
A method for screening a cell growth inhibitor, characterized by using suppression of expression of a target gene of the nucleic acid according to claim 1 as an index.
A method for screening a cell growth promoter, characterized by using suppression of nucleic acid expression according to claim 1 as an index.
A method for screening a cell growth promoter, characterized by using the promotion of expression of a target gene of the nucleic acid according to claim 1 as an index.