WO2011019074A1

WO2011019074A1 - Nucleic acid that controls fibrosis of cells or organs

Info

Publication number: WO2011019074A1
Application number: PCT/JP2010/063728
Authority: WO
Inventors: 伸晃江藤; 将矩兼松
Original assignee: 協和発酵キリン株式会社
Priority date: 2009-08-12
Filing date: 2010-08-12
Publication date: 2011-02-17

Abstract

Provided are a nucleic acid and fibrosis control agent that are useful in controlling fibrosis of cells or organs. Also provided is a method for using said nucleic acid and fibrosis control agent. The following are provided: a nucleic acid containing a base sequence represented by one of sequence numbers 1 to 1281; a nucleic acid at least 90% identical to the aforementioned nucleic acid; a nucleic acid hybridized with a complementary strand of the aforementioned nucleic acid under stringent conditions; a nucleic acid containing the second through eighth base sequences of a base sequence represented by one of sequence numbers 1 to 594; a cell or organ fibrosis control agent using a nucleic acid or the like comprising a base sequence that is complementary to the base sequence of an abovementioned nucleic acid; a diagnostic or therapeutic agent for disorders resulting from cell or organ fibrosis; an expression control agent for target genes of nucleic acids such as microRNAs; a control method or the like for cells and organs; and a method for screening for cell or organ fibrosis control agents.

Description

Nucleic acids that control fibrosis of cells or organs

The present invention relates to a cell or organ fibrosis control agent using a nucleic acid, a diagnostic or therapeutic agent for a disease caused by a fibrotic disorder, a cell or organ fibrosis control method, and a screening method for a cell or organ fibrosis control agent About.

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 Documents). 1, 2). MicroRNAs are generated from genes that are transcribed into single or clustered microRNA precursors. In other words, 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 mRNA translation, 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 To do. Although the above complementarity may not be perfect, it has been shown that complementarity of the second to eighth bases from the 5′-end side of the microRNA is particularly important. Is 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 March 2009, the microRNA database miRBase (http://microrna.sanger.ac.uk/) contains 706 human, 547 mouse, and 286 rat microRNAs. . 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).

Fibrosis is a phenomenon in which the part called connective tissue that constitutes tissues such as internal organs grows abnormally and occurs in various tissues and organs such as lung, kidney, liver, heart, skin, blood vessels, and causes disease It is known. For example, when fibrosis occurs in the myocardium, abnormalities occur in the function of the heart, and symptoms such as dyspnea and increased heart rate (palpitations) appear. In addition, bone atrophy and degeneration in rheumatoid arthritis and the pathology of cirrhosis, which indicates fibrosis of the entire liver, are well known as examples of abnormal growth of the connective tissue, that is, fibrosis. In the kidney, the stromal hypertrophy occurs due to fibrosis, and eventually progresses to renal failure due to necrosis or decrease of tubules. Tissue fibrosis is mainly caused by abnormal production of the extracellular matrix of fibroblasts present in the tissue, but the epithelial cells constituting the tissue are transformed into mesenchymal cells together with the extracellular matrix. It has been clarified that starting to produce a substrate also contributes, and these are called epithelial-mesenchymal transition (EMT) (Non-patent Document 8). MicroRNA research so far has been progressing mainly in cancer research, and in many cancers, microRNA expression is different from normal tissues, and cancer can be classified by microRNA expression profile analysis. Have been reported (Non-Patent Document 9). It is also known that about half of the human microRNAs found so far exist in chromosomal abnormalities or fragile sites of chromosomes known in human cancer (Non-patent Document 10). On the other hand, general immunosuppressive drugs such as corticosteroids and other anti-inflammatory treatments are currently used for treatment of organ fibrosis. However, the mechanisms involved in controlling fibrosis appear to be a separate phenomenon from that of inflammation, and anti-inflammatory therapy is not always effective in reducing or preventing fibrosis. It cannot be said that the clinical needs are satisfied (Non-patent Document 11). Thus, there is still a need to develop new therapies that modulate fibrotic disorders, including reducing or preventing fibrosis.

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. In cancer, miR-15a / miR-16 cluster is included in chromosome 13q14, which is deleted in miR-29cB cell chronic lymphocytic leukemia (B-CLL), and this deletion is one of the causes of B-CLL. That are predicted to be connected (Non-patent Document 12). In B-CLL, miR-29 and miR-181 expression is further reduced, and one of the targets is Tcl1, which is known as a proto-oncogene. (Non-patent Document 13) is known. 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 14 and 15). Moreover, the expression of microRNAs such as miR-127 and miR-124a is reduced by hypermethylation modification of genes in cancer, and Bcl6 and Cdk6 known as proto-oncogenes are the targets (Non-patent Document 16, 17). 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 generation of the above is promoted (Non-patent Document 18). It has also been clarified that a gene called BIC, which has been previously overexpressed in Hodgkin's lymphoma and was a candidate oncogene that does not encode a protein, encodes miR-155 (Non-patent Document 19).

On the other hand, as an example of the relationship between fibrosis and microRNA reported so far, miR-29 expression is decreased in mice or human myocardial infarction that caused myocardial infarction due to left coronary artery occlusion in the heart, It has been shown that when miR-29 expression is suppressed in cultured cardiac fibroblasts, the expression of Collagen A1, A2, A3, which is one of the fibrosis-related molecules, is significantly increased (Non-patent Document 20). Similarly, with regard to cardiac fibrosis, in a β1-adrenergic receptor transgenic mouse with spontaneous heart failure, analysis of microRNA expression in the heart found that miR-21 increased with time. (Non-patent document 21). In this paper, we performed database analysis by bioinformatics using miR-21 seed sequence and found Sprouty-1 (SPRY1), a molecule that inhibits Ras / ERK signal as one of miR-21 targets. In the progress of fibrosis, miR-21 suppresses the expression of SPRY1, activates the ERK-MAPK signal, and regulates the survival of fibroblasts.

As described above, many relations between disease states and microRNAs have been reported in recent years, but many of them show abnormal expression in cancer cells, and reports showing the function of microRNAs include Let-7, miR-143. Report that the growth of cancer cell lines was inhibited by forcibly expressing miR-145 and miR-145 (Non-Patent Documents 22, 23, 24), cancer cells by forcibly expressing miR-16 and miR-34 There are reports that the cell cycle of the strain was stopped (Non-patent Documents 25 and 26), reports that cell transformation was caused by forced expression of miR-372 and miR-373 (Non-patent Document 27), and the like. .
On the other hand, administration of microRNA or a precursor thereof from outside the body increases the expression of the microRNA, or administration of the antisense oligonucleotide reduces the expression of the microRNA to reduce fibrosis of the organ. There has been almost no report of improvement, and in the above-mentioned report in the heart, an example in which the expression of Collagen A1, A2, A3 in the heart was significantly increased by administration of the precursor of miR-29 (Non-patent Document 20) Although there is an example (Non-patent Document 21) that cardiac fibrosis is enhanced when a cardiac disorder is applied by administering an antisense oligonucleotide of miR-21 or miR-21, there is no report analyzing the fibrosis of the kidney There is the present situation.

It is expected that new therapeutic agents and diagnostic agents will be developed by identifying microRNAs whose expression changes in fibrotic organs, analyzing their functions, and elucidating the relationship with diseases.
In particular, finding microRNAs that promote or suppress fibrosis of cells or organs not only helps understand the mechanism of fibrosis, but also develops diagnostic and therapeutic agents for human organ fibrosis, and further It is expected to lead to new diagnostic methods and treatments for the fibrosis used. Acute renal failure and glomerulonephritis characterized by fibrosis of tubulointerstitium, vasculitis, diabetic nephropathy, hypertensive nephropathy, HIV nephropathy, IgA nephropathy, lupus nephritis, interstitial nephritis, Obstructed kidney due to ureteral obstruction, as well as pulmonary fibrosis, cirrhosis, arteriosclerosis, scleroderma, coronary restenosis after percutaneous transluminal coronary vasodilation (PTCA), interstitial myocarditis, interstitial bladder Diagnostic and therapeutic agents for diseases caused by abnormalities in extracellular matrix production of cells, tissue hyperplasia, such as inflammation, skin scarring after burns, fibrosis associated with poisoning, etc., and diagnosis using them It is expected to contribute to the development of methods and treatments.

An object of the present invention is to provide a nucleic acid and a fibrosis control agent useful for controlling fibrosis of cells or organs, and methods for using them. In this case, the fibrosis regulator refers to a fibrosis inhibitor and a fibrosis promoter.

The present invention relates to the following [1] to [31].
[1] A cell or organ fibrosis control agent comprising any one 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: 1-27, 28-594 (b) Containing a nucleic acid comprising a base sequence represented by any of SEQ ID NOs: 1-27, 28-594 A nucleic acid consisting of a base sequence having a nucleotide sequence of 90% or more (d) a sequence number 1 to 1 to a nucleic acid of 17 to 28 bases (c) a sequence represented by any one of SEQ ID NOs: 1 to 27 and 28 to 594 A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleotide sequence represented by any one of 27 and 28 to 594 (e) a base represented by any one of SEQ ID NOs: 1 to 27 and 28 to 594 A nucleic acid comprising the second to eighth base sequences of the sequence (f) a nucleic acid comprising the base sequence represented by any one of SEQ ID NOs: 595 to 1281 (g) a base sequence represented by any one of the sequence numbers 595 to 1281; 90% or more identity (H) Nucleic acid that hybridizes under stringent conditions with the complementary strand of the nucleic acid consisting of the base sequence represented by any of SEQ ID NOs: 595 to 1281 [2] The nucleic acid is microRNA or microRNA precursor The cell or organ fibrosis regulator according to [1] above, which is a body.
[3] A cell or organ fibrosis control agent comprising, as an active ingredient, a nucleic acid having a base sequence complementary to the base sequence of the nucleic acid described in [1].
[4] A cell or organ fibrosis control agent comprising a vector that expresses the nucleic acid according to any one of [1] to [3] as an active ingredient.
[5] A cell or organ fibrosis control agent 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 or organ fibrosis control agent comprising as an active ingredient a substance that promotes expression of a gene having the target base sequence of the nucleic acid according to [1].
[7] The cell or organ fibrosis regulator according to [5] or [6] above, wherein the substance that suppresses or promotes expression is a nucleic acid.
[8] The cell or organ fibrosis regulator according to [7] above, wherein the nucleic acid is siRNA or antisense oligonucleotide.
[9] A cell or organ fibrosis control agent comprising the vector expressing the nucleic acid according to [7] as an active ingredient.
[10] The nucleic acid according to any one of [1] to [3] above, the vector according to [4] above, or the substance according to any of [5] to [8] above as an active ingredient A therapeutic or diagnostic agent for diseases caused by fibrosis of cells or organs.
[11] A disease caused by fibrosis of a cell or an organ containing as an active ingredient a reagent for detecting the expression level of the nucleic acid according to [1], a mutation of the nucleic acid, or a mutation of a genome encoding the nucleic acid Diagnostics.
[12] Diseases caused by fibrosis of cells or organs are acute renal failure, glomerulonephritis, vasculitis, diabetic nephropathy, hypertensive nephrosclerosis, HIV nephropathy, IgA nephropathy, lupus nephritis, stroma Nephritis, obstructed kidney due to ureteral obstruction, pulmonary fibrosis, cirrhosis, arteriosclerosis, scleroderma, coronary restenosis after percutaneous transluminal coronary vasodilation (PTCA), interstitial myocarditis, interstitial The therapeutic or diagnostic agent according to the above [10] or [11], which is cystitis, skin scarring after burn injury, or fibrosis associated with poisoning.
[13] Diseases caused by fibrosis of cells or organs are acute renal failure, glomerulonephritis, vasculitis, diabetic nephropathy, hypertensive nephrosclerosis, HIV nephropathy, IgA nephropathy, lupus nephritis, stroma The therapeutic agent or diagnostic agent according to [10] or [11] above, which is fibrosis associated with renal disorder selected from the group consisting of pneumonia and obstructive kidney due to ureteral obstruction.
[14] Administering an effective amount of the nucleic acid according to any one of [1] to [3] above, the vector according to [4] above, or the substance according to any of [5] to [8] above. A method for treating a disease caused by fibrosis of a cell or organ, characterized by comprising:
[15] Diagnosis of a disease caused by fibrosis of a cell or organ, wherein the expression level of the nucleic acid according to [1], a mutation of the nucleic acid, or a mutation of a genome encoding the nucleic acid is detected Method.
[16] Diseases caused by fibrosis of cells or organs are acute renal failure, glomerulonephritis, vasculitis, diabetic nephropathy, hypertensive nephrosclerosis, HIV nephropathy, IgA nephropathy, lupus nephritis, stroma Nephritis, obstructed kidney due to ureteral obstruction, pulmonary fibrosis, cirrhosis, arteriosclerosis, scleroderma, coronary restenosis after percutaneous transluminal coronary vasodilation (PTCA), interstitial myocarditis, interstitial The method of treatment or diagnosis according to [14] or [15] above, which is cystitis, skin scarring after burn injury, or fibrosis associated with poisoning.
[17] Diseases caused by fibrosis of cells or organs are acute renal failure, glomerulonephritis, vasculitis, diabetic nephropathy, hypertensive nephrosclerosis, HIV nephropathy, IgA nephropathy, lupus nephritis, stroma The method of treatment or diagnosis according to [14] or [15] above, wherein the disease is fibrosis associated with renal disorder selected from the group consisting of nephritis and obstructed kidney due to ureteral obstruction.
[18] The nucleic acid according to any one of [1] to [3] above, the vector according to [4] above, or the above [for manufacturing a therapeutic agent for a disease caused by fibrosis of a cell or organ Use of the substance according to any one of [5] to [8].
[19] Diseases caused by fibrosis of cells or organs are acute renal failure, glomerulonephritis, vasculitis, diabetic nephropathy, hypertensive nephrosclerosis, HIV nephropathy, IgA nephropathy, lupus nephritis, stroma Nephritis, obstructed kidney due to ureteral obstruction, pulmonary fibrosis, cirrhosis, arteriosclerosis, scleroderma, coronary restenosis after percutaneous transluminal coronary vasodilation (PTCA), interstitial myocarditis, interstitial The use according to [18] above, which is fibrosis associated with cystitis, skin scarring after burns, or poisoning.
[20] Diseases caused by fibrosis of cells or organs are acute renal failure, glomerulonephritis, vasculitis, diabetic nephropathy, hypertensive nephrosclerosis, HIV nephropathy, IgA nephropathy, lupus nephritis, stroma. The use according to [18] above, which is fibrosis associated with renal disorder selected from the group consisting of pneumonia and obstructive kidney due to ureteral obstruction.
[21] An agent for controlling expression of a target gene of a nucleic acid, which is an active ingredient of a fibrosis regulator for cells or organs, containing the nucleic acid according to any one of [1] to [3] as an active ingredient.
[22] A nucleic acid target gene expression control agent, which is an active ingredient of a cell or organ fibrosis control agent, comprising the vector according to [4] as an active ingredient.
[23] A method for controlling fibrosis of a cell or organ, comprising using the nucleic acid according to any one of [1] to [3] above.
[24] A method for controlling fibrosis of a cell or organ, characterized by using the vector according to [4] above.
[25] A method for controlling fibrosis of a cell or organ, comprising using a substance that suppresses or promotes the expression of the target gene of the nucleic acid according to [1].
[26] The method for controlling fibrosis of a cell or organ according to the above [25], wherein the substance that suppresses or promotes expression is a nucleic acid.
[27] The method for controlling fibrosis of a cell or organ according to the above [26], wherein the nucleic acid is siRNA or antisense oligonucleotide.
[28] A method for controlling fibrosis of a cell or organ using the vector expressing the nucleic acid according to [26].
[29] A method for controlling expression of a target gene of a nucleic acid that is an active ingredient of a fibrosis control agent for cells or organs, wherein the nucleic acid according to any one of [1] to [3] above is used.
[30] A method for controlling expression of a target gene of a nucleic acid, which is an active ingredient of a fibrosis control agent for cells or organs, using the vector according to [4] above.
[31] A screening method for a cell or organ fibrosis regulator using as an index the promotion or suppression of the expression or function of the nucleic acid according to [1].

According to the present invention, a fibrosis inhibitor or fibrosis promoter for cells or organs, a diagnostic or therapeutic agent for diseases caused by abnormal extracellular matrix production of cells or EMT, and an expression inhibitor for target genes of nucleic acids such as microRNA Alternatively, an expression promoter, a method for suppressing cell fibrosis, or a method for promoting fibrosis can be provided.

FIG. 1 shows the results of analysis of collagen-I gene expression by real-time RT-PCR. The expression of Collagen I in the liver after administration of control RNA, miR-30b, miR-486, let-7i is shown as a relative ratio to the control. FIG. 2 shows the results of analysis of TGF-β gene expression by real-time RT-PCR. The expression of TGF-β in the liver after administration of control microRNA, miR-30b, miR-486, let-7i is shown as a relative ratio to the control.

The nucleic acid used in the present invention may be any molecule as long as nucleotides and molecules having functions equivalent to the nucleotides are polymerized, such as RNA that is a ribonucleotide polymer, DNA that is a deoxyribonucleotide polymer. , A polymer in which ribonucleotides and deoxyribonucleotides are mixed, and a nucleotide polymer containing a nucleotide analog, and further 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 nuclease resistance, to stabilize, 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 molecule obtained by modifying ribonucleotides, deoxyribonucleotides, RNA, or DNA. Examples thereof include sugar moiety-modified nucleotide analogs and phosphodiester bond-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)].

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 a nucleic acid, a different chemical is used. Any molecule can be used as long as it is a molecule to which a substance is added, 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, 6-FAM Examples include addition derivatives, biotin addition derivatives, and the like.

Examples of the nucleic acid 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 594 (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 594 (C) a nucleic acid comprising a base sequence represented by any one of SEQ ID NOs: 1 to 594 and having a nucleotide sequence having 90% or more identity (d) comprising a base sequence represented by any of SEQ ID NOs: 1 to 594 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 594 (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 of SEQ ID NOs: 595 to 1281 (j) represented by any of SEQ ID NOs: 595 to 1281 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: 595 to 1281 .

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 microRNA include human microRNA having a base sequence represented by any of SEQ ID NOs: 1 to 27. Furthermore, as a microRNA having the same function as the human microRNA consisting of the base sequence represented by any of SEQ ID NOs: 1-27, it is represented by any of SEQ ID NOs: 28-486, which is an ortholog of the human microRNA. A nucleic acid having a base sequence can be mentioned. As a specific example, the ortholog of the human microRNA of SEQ ID NO: 1 includes those consisting of the base sequences represented by SEQ ID NOs: 28 to 33 and 37 to 39. Table 1 shows a correspondence table between microRNAs having the base sequences represented by SEQ ID NOs: 1 to 27 and their orthologs.

The species shown in the top row of Table 1 and Table 2 to be described later are as follows. hsa, Homo sapiens, humans; mmu, Mus musculus, mice; rno, Rattus norvegicus, rats; cgr, Cricetulus griseus, Chinese hamsters; age, Ateles geoffroyi, red spider monkeys; 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 trogopy; Ssy, Symphalangus syndactylus, octopus gibbon; lca, Lemur catta, ring-tailed lemur; oan, Ornithorhynchus anatinus, platypus; cfa, Canis familiaris, dog; mdo, Monodelphis Xla, Xenopus laevis, Xenopus laevis; xtr, Xenopus tropicalis, Nishimegaru; bta, Bos taurus, cattle; oar, Ovis aries, sheep; ssc, Sus scrofa, pig; dre, Danio rerio, zebrafish , Trufffish; tni, Tetraodon nigroviridis, greenfish human microRNA and its ortholog are considered to have similar functions because of their high sequence identity. 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 recognized as a 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 base represented by any one of SEQ ID NOs: 487 to 594 as a microRNA having the same base sequence at the 5 'end side from the microRNA having the base sequence represented by any one of SEQ ID NOs: 1 to 27 A nucleic acid consisting of a sequence can be mentioned. As a specific example of the microRNA, for the microRNA consisting of the base sequence represented by SEQ ID NO: 1, microRNA consisting of the base sequence represented by SEQ ID NOs: 487 and 488 can be mentioned. Artificial microRNA is also included in microRNA. Table 2 shows a correspondence table between the microRNAs having the base sequences represented by SEQ ID NOs: 1 to 27 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 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 above-mentioned nucleic acid, and can form a hairpin structure. MicroRNA is produced from a microRNA precursor through processing by a protein called Dicer.

Examples of the microRNA precursor include a nucleic acid having a base sequence represented by SEQ ID NO: 595 for human microRNA of SEQ ID NO: 1. In addition, for a microRNA having a base sequence represented by any of SEQ ID NOs: 2 to 594, a nucleic acid having a base sequence represented by any of SEQ ID NOs: 596 to 1281 can be exemplified. Table 3 shows the correspondence 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 nucleotide sequence represented by any of SEQ ID NOs: 1-1811 is BLAST [J. Mol. Biol., 215 , 403 (1990)] or FASTA [ Methods in Enzymology, 183 , 63 (1990)], etc., and at least 90% or more, preferably 93% of the nucleic acid consisting of the base sequence represented by any of SEQ ID NOs: 1-1811 It means that the nucleic acid has the identity of 95% or more, more preferably 96% or more, particularly preferably 97% or more, and most preferably 98% or more.

The stringent conditions in the above are, for example, 7.5 mL, 1 M Na ₂ HPO ₄ (pH 7.2) 0.6 mL, 10% SDS 21 mL, 50x Denhardt's solution 0.6 mL, 10 Add the other strand labeled with ³² P-ATP to the Hybridization buffer consisting of 0.3 mL of mg / mL sonicated salmon sperm DNA, react at 50 ° C overnight, then react at 50 ° C for 10 minutes, 5x SSC / 5% SDS solution And then washed with 1 × SSC / 1% SDS solution at 50 ° C. for 10 minutes, and then the membrane is taken out and exposed to an X-ray film to detect the signal. Those skilled in the art can appropriately select the hybridization conditions that give the same stringency.

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

As a method for detecting a mutation in a nucleic acid such as microRNA using a nucleic acid, any method can 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. 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, A method for detecting the presence or absence can be given.

The vector expressing nucleic acid may be any vector as long as it is designed to biosynthesize nucleic acid such as microRNA by being introduced into a cell and transcribed. Specific examples of vectors capable of expressing nucleic acids such as microRNA in cells include pCDNA6.2-GW / miR (Invitrogen), pSilencer4.1-CMV (Ambion), pSINsi-hH1 DNA (Takara Bio), pSINsi-hU6UDNA (Takara Bio), pENTR / U6 (Invitrogen), microRNA Archive (Takara Bio), pMIRRNA1 (System Bioscience), etc. it can.

As a method for suppressing the expression of a gene having a target base sequence of a nucleic acid such as microRNA (hereinafter referred to as a target gene), the activity of suppressing the expression of mRNA having a target base sequence of a nucleic acid such as microRNA is utilized. Any method may be used as long as it suppresses the expression of a gene having the target base sequence. In addition, suppression of expression here includes a case where the translation of 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.

The microRNA is a single-stranded RNA having a length of 17 to 28 bases containing a sequence complementary to a continuous 7 base sequence existing in the 3 ′ untranslated region of the mRNA of the target gene as the second to eighth base sequences. Some artificial microRNAs are also included. 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 nucleic acid such as microRNA is a base sequence consisting of several bases recognized by 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. The base sequence complementary to the 2-8th base sequence on the 5 ′ end side of the microRNA is that translation of mRNA having the base sequence is suppressed by the microRNA [Current Biology, 15 , R458-R460]. (2005)], a base sequence complementary to the second to eighth base sequences on the 5 ′ end side of nucleic acids such as microRNA in the present invention can be mentioned as the target base sequence. For example, an mRNA containing a sequence that perfectly matches the 3 'UTR base sequence group of human mRNAs by preparing a target sequence complementary to the 2-8th base sequence on the 5' end side of the microRNA. Can be determined by selecting by a method such as a character string search. The 3'UTR base sequence group of human mRNA should be 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)". Can do. As a specific example of a gene having a target base sequence of microRNA consisting of the base sequence represented by any one of SEQ ID NOs: 1 to 27, EntreGene of National Center for Biotechnology Information (NCBI) Examples of the genes listed in Table 4 are the names (Official Symbol and Gene ID) used in the database (http://www.ncbi.nlm.nih.gov/Entrez/).

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 the microRNA 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 can be designed in the same manner as Pre-miRTMmimiRNA Precursor Molecules (Ambion) or miRIDIAN microRNA Mimics (GE Healthcare) to express nucleic acids such as microRNA in cells. it can. 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 (manufactured by B-Bridge), miCENTURY OX siMature (manufactured by B-Bridge), and miCENTURY OX miNatural (manufactured by B-Bridge).

The method for synthesizing the nucleic acid used in the present invention is not particularly limited, and it can be produced by a method using a known chemical synthesis, an enzymatic transcription method or the like. 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.

Examples of a method for screening for a substance that promotes or suppresses the expression or function of the nucleic acid using the nucleic acid used in the present invention include, for example, introducing a vector that expresses the nucleic acid into a cell, and expressing an mRNA having the target base sequence. Alternatively, a method of screening for a substance that promotes or suppresses the function can be mentioned.

The pharmaceutical comprising the nucleic acid used in the present invention as an active ingredient can be used for diagnosis or treatment of diseases caused by fibrosis of cells or organs, tissue hyperplasia, or the like. The nucleic acid can also be used as a fibrosis control agent for cells or organs. Cell or organ fibrosis as used herein refers to a state in which extracellular matrix production is abnormally increased in the living body or a state in which epithelial-mesenchymal transition (EMT) occurs. An agent refers to an inhibitor or an accelerator. In the present invention, “fibrosis of cells or organs” is not limited to fibrosis of only one of cells or organs, but includes cases where both cells and organs are fibrotic.

Examples of diseases caused by cell or organ fibrosis or tissue hyperplasia include, for example, fibrosis associated with damage to fibrous tissue. Here, the fibrous tissue is not particularly limited as long as it has a connective tissue, for example, kidney, lung, liver, heart, pancreas, spleen, stomach, spinal cord, pituitary gland, gonad, thyroid, gallbladder, Bone marrow, adrenal gland, skin, digestive tract (eg, large intestine, small intestine), brain, brain parts (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), bladder , Testis, ovary, placenta, uterus, bone, joint, skin, blood vessel, adipose tissue, skeletal muscle and the like. Specific examples of the disease include, but are not limited to, acute renal failure, glomerulonephritis, vasculitis, diabetic nephropathy, hypertensive nephrosclerosis, HIV nephropathy, IgA nephropathy, lupus nephritis, interstitial Nephritis, fibrosis associated with renal disorders such as obstructed kidney due to ureteral obstruction, pulmonary fibrosis, cirrhosis, arteriosclerosis, scleroderma, coronary restenosis after percutaneous transluminal coronary vasodilation (PTCA), between Examples include interstitial myocarditis, interstitial cystitis, skin scarring after burn injury, and fibrosis associated with poisoning.

Hereinafter, the present invention will be described in detail.
1. Method for detecting expression of nucleic acid such as microRNA and microRNA precursor expressed in fibrotic cells (1-1) Identification of microRNA Whether a small RNA sequence is microRNA is determined by RNA (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 in 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 weight RNA Extracting total RNA from a cell line or a specimen derived from a patient in which organ fibrosis is observed, and using the RNA, microRNA expressed in fibrotic cells or fibrotic tissue Small molecule RNA containing can be obtained as follows.

As a method for obtaining low molecular weight RNA, specifically, low molecular weight RNA can be obtained by 15% polyacrylamide gel electrophoresis in accordance with the method described in Jeans & Development (Genes & Development), 15 , 188-200 (2000). The method of separating is mentioned. 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, for example, according to the method described in Science, 294 , 858-862 (2001), 5′-adenylation, 3′-adapter ligation, 5′-adapter ligation, reverse transcription, PCR amplification, concatamerization In addition, after sequentially ligating to a vector, 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.

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, a probe labeled appropriately based on the base sequence of the nucleic acid is prepared, and hybridization and washing are performed. Thus, this is a method for detecting a band specifically bound to a nucleic acid. Specifically, for example, it can be performed according to the method described in Science, 294 , 853-858 (2001).

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

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 tissue and nucleic acid of a nucleic acid by microscopic observation. It is a method for examining distribution and localization in 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 nucleic acid and a base sequence corresponding to the surrounding genomic sequence.

For example, after synthesizing a specimen-derived cDNA, using this as a template, a template-specific design designed from a nucleotide sequence corresponding to a nucleic acid such as microRNA or a microRNA precursor and its surrounding genomic sequence, or a nucleotide sequence corresponding to a reverse transcription primer PCR is performed using a typical primer, a cDNA fragment containing the nucleic acid is amplified, and the amount of the nucleic acid contained in the sample-derived RNA is detected from the number of cycles until a certain amount is reached. As a template-specific primer, an appropriate region corresponding to the nucleic acid and the surrounding genomic sequence is selected, and consists of a sequence of 15 to 40 residues, preferably 20 to 30 residues at the 5 ′ end of the base sequence of the region. A set of DNA or LNA consisting of DNA or LNA and a sequence complementary to 15 to 40 residues, preferably 20 to 30 residues at the 3 ′ end 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), QuantiMir RT Kit (System Biosciences), PrimeScript 1st strand cDNA Synthesis (Takara Bio). Furthermore, by subjecting the sample-derived RNA or cDNA to hybridization to a filter or slide glass or silicon substrate or the like to which DNA or LNA corresponding to a base sequence containing at least one nucleic acid is immobilized, and washing. The variation in the amount of nucleic acid 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 nucleic acid between the control sample and the target sample can be accurately detected by immobilizing an internal control such as a base sequence corresponding to U6 RNA on a filter or substrate. 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. Accurate nucleic acid quantification can be performed by simultaneously hybridizing labeled cDNA. Furthermore, the nucleic acid can be quantified by directly labeling and hybridizing RNA derived from the control sample and / or the 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 and SP6 promoter is bound to the 3 ′ end of the nucleotide sequence corresponding to the nucleic acid or its surrounding genomic sequence, and labeled NTP (mixture of ATP, GTP, CTP, UTP) and Labeled antisense RNA is synthesized by an in vitro transcription system using 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 digest is subjected to gel electrophoresis, and RNA fragments protected from digestion by forming RNA-RNA hybrids are detected or quantified as nucleic acids. Specifically, it can be detected or quantified using mirVana miRNA Detection Kit (Ambion).

2. Synthesis of Nucleic Acid As described in 1 above, microRNAs or microRNA precursors that are expressed in cells or fibrotic tissues with increased fibrosis, or whose expression is increased or decreased compared to normal tissues, etc. After the nucleic acid is 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 is not particularly limited, and the nucleic acid can be produced by a method using 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 single-stranded into the cell. By measuring the expression of a reporter gene when RNA is expressed, it can be detected whether or not it has a function of 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), pMIR-REPORT® System (Ambion). 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 specimen-derived DNA or a specimen-derived cDNA as a template, a primer designed based on the genomic base sequence including the nucleic acid base sequence 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), using primers derived from the base sequence of the genome, including the base sequence of the nucleic acid, 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 electrophoresed 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), DNA fragments amplified with primers designed based on the base sequence of the genome including the base sequence of the nucleic acid using the sample-derived DNA or the sample-derived cDNA as a template are used as isotopes or fluorescence in the nucleic acid. By hybridizing with a labeled nucleic acid incorporating a label and treating with osmium tetroxide, one strand of DNA at a position where a mismatch exists can be cleaved to detect a mutation. CCM 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 genomic base sequences, including nucleic acid base sequences, are used chemically, using specimen-derived DNA or specimen-derived cDNA as a template. Electrophoresis is performed using a gel having a denaturant concentration gradient and a 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.

Also, nucleic acid mutations can be detected by directly determining and analyzing the base sequence of the sample-derived DNA or the sample-derived cDNA.

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

The nucleic acid can be expressed by using a vector that is biosynthesized by introduction into a cell and transcription. Specifically, based on the base sequence of the nucleic acid or the genomic base sequence containing the base sequence, a DNA fragment containing the hairpin portion is prepared and inserted downstream of the promoter of the expression vector to construct an expression plasmid. Next, the nucleic acid can be expressed by introducing the expression plasmid into a host cell suitable for the expression vector.

As an 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 a nucleic acid base sequence can be transcribed is used. Any promoter can be used as long as it can be expressed in the host cell. For example, RNA polymerase II (pol II) type promoter or U RNA or H1 RNA transcription system RNA polymerase III (pol III) system. A promoter etc. can be mention | raise | lifted. Examples of the Pol II promoter include a promoter of cytomegalovirus (human CMV) IE (immediate early) gene, an early promoter of SV40, 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).

A gene containing a nucleic acid base sequence is inserted downstream of a promoter in a viral vector to construct a recombinant viral vector, the vector is introduced into a packaging cell to produce a recombinant virus, and the nucleic acid base sequence is determined. Genes containing can also be expressed.

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 the proteins required 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 retrovirus vectors, and HIV virus-derived gag, pol, env, vpr, vpu for lentiviral vectors. , Vif, tat, rev, nef, etc., in the case of adenovirus vectors, proteins such as E1A and E1B derived from adenovirus, and in the case of adeno-associated virus vectors, Rep (p5, p19, p40), Vp (Cap), etc. Can be used.

In addition to using an expression vector, the nucleic acid used in the present invention can 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 microRNAs and microRNA precursors can be expressed in the same manner as Pre-miRTM miRNA Precursor Molecules (Ambion) or miRIDIAN microRNA Mimics (GE Healthcare). 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. Products that use these methods include miCENTURY OX Precursor (B-Bridge), miCENTURY OX siMature (B-Bridge), and miCENTURY OX miNatural (B-Bridge).

6). Methods for suppressing the activity of nucleic acids such as microRNAs and microRNA precursors Nucleic acids such as microRNAs and microRNA precursors are antisense technologies [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 DNA method [Nippon Rinsho-Japanese Journal of Clinical Medicine, 56 , 563 (1998), Circulation Research, 82 , 1023 (1998), Experimental Nephrology, 5 , 429 (1997), Nippon R insho-Japanese Journal of Clinical Medicine, 54 , 2583 (1996)], or siRNA (short interfering RNA) can be used to suppress the activity.

Antisense refers to a nucleic acid that can specifically hybridize a nucleic acid having a base sequence complementary to the base sequence of a certain target nucleic acid to suppress the expression of the target nucleic acid. As the nucleic acid used for antisense, in addition to DNA, RNA or nucleotide analogs, 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. Further, in the same manner as Anti-miRTM miRNA Inhibitors (manufactured by Ambion) and miRIDIAN microRNA Hairpin Inhibitors (manufactured by Dharmacon), the expression of nucleic acids such as microRNAs and microRNA precursors can be suppressed.

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). sequence of the siRNA, the literature of the nucleotide sequence [Genes Dev., 13, 3191 (1999)] targeting can be appropriately designed based on the conditions. 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. The siRNA that suppresses a nucleic acid such as microRNA used in the present invention may be any siRNA that can suppress the activity of the nucleic acid, but each of the nucleotide sequences represented by any of SEQ ID NOs: 1 to 594 SiRNA designed from continuous sequence information after the 9th base is preferred. 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.

MicroRNA expressed in fibrotic cells using antisense or siRNA specific to nucleic acids such as microRNA and microRNA precursors expressed in fibrotic cells or tissues (hereinafter also referred to as “fibrotic cells”) And the expression of microRNA precursors can be suppressed. For example, by administering antisense DNA or siRNA specific to the microRNA or the microRNA precursor, the activity of the microRNA is suppressed and the action of the microRNA or microRNA precursor in fibrotic cells is controlled. can do.

In the case of a patient with abnormal expression of microRNA or its precursor expressed in fibrotic cells, fibrotic cells can be obtained by administering to the patient an antisense oligonucleotide or siRNA specific to the microRNA or its precursor. The disease which develops by the said expression abnormality can be treated. That is, an antisense oligonucleotide or siRNA specific to the microRNA or a precursor thereof is useful as a therapeutic agent for diseases caused by abnormal production of extracellular matrix, that is, fibrosis.

When an antisense oligonucleotide or siRNA specific to a nucleic acid 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 retroviral vector, After being inserted into an appropriate vector such as an adenovirus vector or an adeno-associated virus vector, it can be administered as a pharmaceutical preparation according to the conventional method described in 9 below.

7). Method of suppressing gene function using nucleic acid such as microRNA or microRNA precursor As a method of suppressing function or expression of target gene of nucleic acid, nucleic acid such as microRNA is used for expression of mRNA having target base sequence. Any method may be used as long as it uses the inhibitory activity. For example, by expressing a nucleic acid such as microRNA and increasing the amount of nucleic acid such as microRNA in the cell, it is possible to suppress the translation of mRNA having the target sequence and to suppress the expression of the gene. . The nucleic acid 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 594 can include the gene groups shown in Table 4 above, for example.

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

8). Method for screening a substance that promotes or suppresses expression or function of nucleic acid using nucleic acid such as microRNA or microRNA precursor Using nucleic acid to promote expression or function of nucleic acid such as microRNA or its precursor Substances to be suppressed can be screened. For example, by selecting a base sequence to be screened from the base sequence of a nucleic acid and utilizing a cell that expresses a nucleic acid having the base sequence, the expression or function of the selected microRNA or its precursor is promoted or Substances to be suppressed can be screened.

As a cell expressing microRNA and a microRNA precursor used for screening, in addition to a cell with enhanced fibrosis, as described in 5 above, a vector expressing a nucleic acid having the base sequence may be an animal cell, A transformed cell obtained by introduction into a host cell such as yeast, a cell into 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 a nucleic acid such as a microRNA or a precursor thereof as a target for screening The test substance is contacted with a cell expressing the nucleic acid, and the expression level of the selected nucleic acid is determined. Using the change 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.
(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 the target sequence of a nucleic acid such as microRNA 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 a test substance is contacted with the cell, Using a change in the expression level of the reporter gene as an indicator, a substance that promotes or suppresses the expression or function of nucleic acids such as microRNA and its precursor is obtained.

Selection of the target sequence can be performed by the method described in 7 above. Examples of mRNA having a nucleic acid target sequence such as a microRNA comprising a base sequence represented by any of SEQ ID NOs: 1 to 594 include: Examples of the gene groups shown in Table 4 above can be given.

9. Cell fibrosis inhibitor or fibrosis promoter using nucleic acid such as microRNA or microRNA precursor Nucleic acid such as microRNA or microRNA precursor, and nucleic acid having a base sequence complementary to the base sequence are: By controlling the expression of a gene having a target sequence, it can be used as a cell fibrosis inhibitor or fibrosis promoter.

Examples of the organ or cell fibrosis inhibitor include the following nucleic acids mentioned in (a) to (h).
(A) a nucleic acid comprising a base sequence represented by any one of SEQ ID NOs: 1 to 23, 28 to 411, and 487 to 589 (b) represented by any one of SEQ ID NOs: 1 to 23, 28 to 411, and 487 to 589 A nucleic acid having a nucleotide sequence of 17 to 28 nucleotides (c) having 90% or more identity with the nucleotide sequence represented by any of SEQ ID NOs: 1 to 23, 28 to 411, and 487 to 589 Nucleic acid consisting of a base sequence (d) Nucleic acid (e) that hybridizes under stringent conditions with a complementary strand of a nucleic acid consisting of the base sequence represented by any of SEQ ID NOs: 24-27, 412-486, 590-594 Nucleic acids comprising the second to eighth base sequences of the base sequences represented by any of Nos. 1 to 23, 28 to 411, and 487 to 589 (f) SEQ ID Nos. 595 to 618, 624 to 1073, and 1156 to 1277 Nucleic acid comprising a base sequence represented by any of the above (g) A nucleic acid comprising a base sequence represented by any one of SEQ ID NOs: 595 to 618, 624 to 1073, and 1156 to 1277 and having a nucleotide sequence of 90% or more (H) a nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the base sequence represented by any of SEQ ID NOs: 619 to 623, 1074 to 1155, and 1278 to 1281.

Examples of organ or cell fibrosis promoters include nucleic acids listed in (i) to (p) below.
(I) a nucleic acid comprising a base sequence represented by any of SEQ ID NOs: 24-27, 412-486, 590-594 (j) represented by any of SEQ ID NOs: 24-27, 412-486, 590-594 A nucleic acid having a nucleotide sequence of 17 to 28 nucleotides (k) having 90% or more identity with the nucleotide sequence represented by any of SEQ ID NOs: 24 to 27, 412 to 486, and 590 to 594 Nucleic acid consisting of a base sequence (l) Nucleic acid (m) sequence that hybridizes under stringent conditions with a complementary strand of a nucleic acid consisting of a base sequence represented by any one of SEQ ID NOs: 1 to 23, 28 to 411, and 487 to 589 Nucleic acids containing the second to eighth base sequences of the base sequences represented by any of Nos. 24 to 27, 412 to 486, and 590 to 594 (n) SEQ ID Nos. 619 to 623, 1074 to 1155, 12 Nucleic acid consisting of a base sequence represented by any one of 8 to 1281 (o) A base having 90% or more identity with the base sequence represented by any one of SEQ ID NOs: 619 to 623, 1074 to 1155, 1278 to 1281 Nucleic acid consisting of sequence (p) A nucleic acid that hybridizes under stringent conditions with a complementary strand of nucleic acid consisting of a base sequence represented by any of SEQ ID NOs: 595 to 618, 624 to 1073, and 1156 to 1277.
As the nucleic acid, microRNA or a microRNA precursor is preferably used.

Examples of the fibrosis promoter for cells or organs include nucleic acids having a base sequence complementary to the base sequences of the nucleic acids described in (a) to (h) above. Examples thereof include nucleic acids having a base sequence complementary to the base sequences of the nucleic acids described in (i) to (p) above. In addition, the above-mentioned nucleic acids and vectors expressing nucleic acids complementary thereto can also be used as cell growth inhibitors or growth promoters.

On the other hand, a substance that suppresses the expression of a target gene of a nucleic acid such as the above microRNA, or a substance that promotes the expression of the target gene can also be used as a fibrosis inhibitor or a proliferation promoter of cells or organs. As this substance, a nucleic acid or a vector expressing it can also be used. Examples of substances that suppress the expression of the target gene include siRNA against the mRNA of the target gene and antisense to the target gene. Substances that promote the expression of the target gene include those against the target gene-specific microRNA. Examples include siRNA and antisense to the target gene.

The preparation form and administration method of the cell or organ fibrosis inhibitor or growth promoter are the same as those for diagnostic agents and therapeutic agents containing nucleic acids such as microRNA and microRNA precursor described later in 10.

10. Diagnostic and therapeutic agents that contain nucleic acids such as microRNA and microRNA precursors Nucleic acids such as microRNA and microRNA precursors control the expression of genes that have target sequences or control the expression of nucleic acids such as microRNAs By controlling, it can be used as a therapeutic agent for diseases caused by fibrosis of cells or organs, that is, abnormal production of extracellular matrix and EMT. Furthermore, siRNA against the target gene of the nucleic acid controls fibrosis of cells or organs, that is, as a disease caused by abnormal production of extracellular matrix and EMT, etc. Examples include fibrosis associated with tissue damage.

Further, by quantifying nucleic acids or detecting mutations, it is possible to diagnose a disease caused by fibrosis of cells or organs, that is, abnormal production of extracellular matrix and EMT.
A diagnostic agent containing a nucleic acid is a reagent necessary for quantifying a nucleic acid or detecting a mutation, such as a buffer, a salt, a reaction enzyme, a labeled protein that binds to the nucleic acid, depending on the target diagnostic method, And a coloring agent for detection.

A therapeutic agent containing a nucleic acid as an active ingredient can be administered alone, but usually mixed with one or more pharmacologically acceptable carriers and well known in the pharmaceutical arts. It is desirable to administer it as a pharmaceutical preparation produced by any method.
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, as well as the carrier used, a formulation such as an aerosol or dry powder is 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.

A therapeutic agent containing a nucleic acid as an active ingredient can also be produced by preparing a nucleic acid-expressing vector and a base used for the nucleic acid therapeutic agent [Nature Genet., 8, 42 (1994)].
The base used for the therapeutic agent may be any base as long as it is usually used for injections, such as distilled water, sodium chloride or a salt solution such as a mixture of sodium chloride and an inorganic salt, mannitol, lactose, dextran. And a solution such as glucose, 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 can be used for treatment as it is in the case of a liquid just before the treatment, or in the case of a solid, dissolved in the above sterilized base if necessary.

Examples of the nucleic acid-expressing vector include the recombinant virus vector prepared by the above method 5, and more specifically, a retrovirus vector and a lentivirus vector.
For example, a virus vector can be prepared by combining a nucleic acid with a polylysine-conjugated antibody specific for an adenovirus hexon protein to produce a complex, and binding the resulting complex to an adenovirus vector. The virus vector stably reaches a target cell, is taken up into the cell by an endosome, 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 also been developed (WO97 / 16538, WO97 / 16539), and using the Sendai virus, a Sendai virus incorporating a nucleic acid can be produced. Can do.
Nucleic acids 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)].

In the membrane fusion-mediated transfer method via liposomes, nucleic acid can be taken up and expressed locally in the tissue by administering the liposome preparation directly to the target tissue [Hum. Gene Ther., 3 , 399 (1992)]. Direct DNA uptake techniques are preferred for targeting DNA directly to a lesion.
For example, receptor-mediated DNA transfer can be performed by binding DNA (usually 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 Fibrosis of Organs and Cells The method for measuring fibrosis is not particularly limited as long as it is a method capable of measuring increased production of extracellular matrix. For example, staining of extracellular matrix can be used. A method for evaluating the extracellular matrix includes Masson's trichrome staining. In this method, the nucleus can be dyed in black with iron hematoxylin, the cytoplasm in red with acidic fuchsin, and the collagen fibers in blue with aniline blue. Generally, the blue region and the fibrosis region in the tissue are in a proportional relationship. It is recognized [J Clin Pathol. 59 (4) , 377-81 (2006)].

12 Method for measuring the degree of organ and cell fibrosis As a method for measuring the degree of organ and cell fibrosis, gene expression of the extracellular matrix can be analyzed by quantitative PCR. Specifically, the expression of Collagen and Fibronectin, which are extracellular substrates, is performed by PCR using appropriate reagents such as Syber-Green (QIAGEN) and Taqman-probe (Applied Biosystems), and the fluorescence intensity is measured. Methods and the like. There are also methods for quantifying extracellular matrix by Western blot, ELISA (Enzyme-Linked Immunosorbent Assay) method, colorimetric method and the like. More specifically, Sircol Soluble Collagen Assay (manufactured by Biocolor) and the like can be mentioned.

In addition, fibrosis can be measured only in EMT, which is said to be a particularly important phenomenon in the process of organ and cell fibrosis.
Methods for evaluating EMT include reduction of cell markers that are specifically expressed in epithelial cells such as β-catenin and ZO-1, E-cadherin, and mesenchymal cells such as FSP-1, αSMA, and Vimentin. In addition to measuring the increase in cell markers expressed in each antibody by immunostaining with each antibody, quantitative analysis is also possible by analyzing their gene expression by quantitative PCR [J Clin Invest., 117 , 482-91 (2007)].

Hereinafter, the present invention will be described specifically by way of examples. However, the present invention is not limited to these examples.

Expression of microRNA during renal fibrosis The difference in microRNA expression in fibrotic kidney was examined using a unilateral ureteral obstruction (UUO) model, which is known as a general animal model of renal fibrosis.
The left renal ureter of 7-week-old BALB / c mice (purchased from Charles River Japan) was ligated under anesthesia, 4 days later, the left kidney was removed, Trizol reagent (Invitrogen) was added, and the product was added to the product. Total RNA was extracted according to the method described. Using mirVana (registered trademark) miRNA Bioarrays V9.2 (manufactured by Ambion), microRNA microarray analysis was performed on each of two control mice and ureter-ligated mice. In addition, it was confirmed that the samples used for these analyzes showed increased expression of fibrosis-related genes such as TGF-β, collagen, fibronectin and the like.
The expression of microRNA in the control mouse was set to 1.00, and compared with the expression level of microRNA in the ureter-ligated mouse, the expression was observed in both ureter-ligated mice. Table 5 shows the average value of two urinary ligation mice relative to the control. As shown in Table 5, 31 candidate microRNAs were obtained.
In Table 5, mmu_miR_345 is SEQ ID NO: 28, hsa_miR_320 is SEQ ID NO: 2, hsa_miR_422b corresponds to hsa_miR_378, SEQ ID NO: 3, rno_miR_422b corresponds to rno_miR_378, SEQ ID NO: 45, hsa_miR_213 is SEQ ID NO: 4, mmu_miR_99h is SEQ ID NO: 4, SEQ ID NO: 6, hsa_miR_422a is SEQ ID NO: 7, hsa_miR_30e_5p is SEQ ID NO: 9, hsa_miR_30d is SEQ ID NO: 10, hsa_miR_30b is SEQ ID NO: 11, hsa_miR_30c is SEQ ID NO: 12, hsa_miR_30a_5p is SEQ ID NO: 13, hsa_miR_u is SEQ ID NO: 13 266, hsa_miR_185 is SEQ ID NO: 16, mmu_miR_203 is SEQ ID NO: 289, hsa_miR_451 is SEQ ID NO: 18, mmu_miR_451 is SEQ ID NO: 301, hsa_miR_29a is SEQ ID NO: 19, hsa_miR_29c is SEQ ID NO: 20, rno_miR_140_AS is SEQ ID NO: 399, hsa_miR mmu_miR_192 is SEQ ID NO: 72, hsa_miR_146b is SEQ ID NO: 25, hsa_miR_21 is SEQ ID NO: 26, and hsa_miR_223 is SEQ ID NO: It represents a micro RNA having the nucleotide sequence represented by 27.
In Table 5, the microRNAs indicated by ambi_miR_3998, ambi_miR_7029, ambi_miR_13268, and ambi_miR_13143 are hsa_miR_213 (SEQ ID NO: 4), hsa_miR_451 (SEQ ID NO: 18), hsa_miR_140-3p (SEQ ID NO: 21) registered in miRBase, respectively. This corresponds to hsa_miR_1974 (SEQ ID NO: 23).

Analysis of microRNA expression during cell fibrosis To further investigate whether microRNA fluctuations in ureter-ligated mice are involved in fibrosis, TGF-β, the central factor of fibrosis, was used to measure renal fibroblasts and renal urine. Changes in microRNA expression upon stimulation of tubule epithelial cells were examined using quantitative PCR.
A rat renal fibroblast cell line, NRK-49F cells (hereinafter referred to as NRK-49F), is a DMEM medium (GIBCO) containing 5% fetal calf serum (GIBCO) and 5% CO ₂ at 37 ° C. Cultured in a concentration incubator.
NRK-49F was seeded in a 6-well plate at 3 × 10 ⁴ per well and cultured overnight in the above medium. One day later, the medium was replaced with DMEM medium without serum. After another 16 hours, the medium was replaced with a recombinant human TGF-β1 (manufactured by R & D) diluted to a final concentration of 5 ng / ml using DMEM medium without serum, and the culture was continued. As a negative control, the medium was changed to serum-free DMEM not containing TGF-β1.
After 48 hours of stimulation with TGF-β1, the medium was removed, and total RNA was extracted using Trizol reagent (Invitrogen) according to the method described in the product. After measuring the RNA concentration, react with TaqMan MicroRNA assays (Applied Biosystems) according to the method described in the product, and use Real-time PCR with Applied Biosystems 7900HT Fast real-time PCR system (Applied Biosystems). Went.
The relative expression level of each control cell was calculated by setting the microRNA expression level of control cells to which TGFβ-1 was not added to 1.00. As a result, as shown in Table 6, it is clear that miR-378, 192, 30e-5p, 30b, 30c, 30d, 486, 185, 320, 451, 200a, 29c expression is reduced by TGF-β stimulation. It became. In particular, miR-378, 30b, 30c, 486, 451, 29c showed an expression decrease of 50% or more.
In Table 6, miR-378 is SEQ ID NO: 3, miR-192 is SEQ ID NO: 6, miR-30e-5p is SEQ ID NO: 9, miR-30d is SEQ ID NO: 10, miR-30c is SEQ ID NO: 12, and miR-30b is SEQ ID NO: 11, miR-486 is SEQ ID NO: 14, miR-185 is SEQ ID NO: 16, miR-320 is SEQ ID NO: 2, miR-451 is SEQ ID NO: 18, miR-200a is SEQ ID NO: 19, and miR-29c is SEQ ID NO: The microRNA which has a base sequence represented by 20 is represented. In addition, probes represented by human sequences were used for TaqMan MicroRNA assays.

As another experiment, expression analysis of the target microRNA was similarly performed using MDCK cells, which are canine renal epithelial cells.
MDCK was seeded in a 24-well plate at 1 × 10 ⁴ per well and cultured overnight in the above medium. One day later, the medium was replaced with recombinant human TGF-β1 (R & D) diluted with DMEM medium containing 10% fetal bovine serum to a final concentration of 10 ng / ml, and the culture was continued. .
72 hours after stimulation with TGF-β1, the medium was removed, and total RNA was extracted using Trizol reagent (Invitrogen) according to the method described in the product.After concentration measurement, TaqMan MicroRNA assays (Applied Biosystems) ), Followed by real-time PCR using Applied Biosystems 7900HT Fast real-time PCR system (Applied Biosystems), and expression of control cells that do not stimulate TGF-β and microRNA The amount was compared.
The relative expression level of each control cell was calculated by setting the microRNA expression level of the control cells to which TGF-β1 was not added to 1.00. As a result, as shown in Table 7, it was revealed that miR-30b, 30c, and 486 expression was reduced by TGF-β stimulation. This is consistent with the result in the UUO model as a result of NRK-49F. In addition, miR-146b, 21, 223 expression was increased by TGF-β stimulation.
In Table 7, miR-30b is SEQ ID NO: 11, miR-30c is SEQ ID NO: 12, miR-486 is SEQ ID NO: 14, miR-146b is SEQ ID NO: 25, miR-21 is SEQ ID NO: 26, and miR-223 is SEQ ID NO: The microRNA which has a base sequence represented by 27 is represented. In addition, probes represented by human sequences were used for TaqMan MicroRNA assays.

Influence on extracellular matrix production of renal tubular epithelial cells in which microRNA was forcibly expressed A precursor of microRNA was introduced into renal tubular epithelial cells, and the influence of microRNA produced from the molecules on collagen production was examined. Renal fibrosis is known to involve tubule cells, and the cell line NRK-52E is used as a model. Collagen I and the like are used as an index of tubule cell fibrosis [JASN., 16 , 2702-13 (2005)], [Journal of Cellular Biochemistry, 103 , 1999-2009 (2008)].
NRK-52E cells (hereinafter referred to as NRK-52E), a rat renal tubular epithelial cell line, are 5% CO at 37 ° C in DMEM medium (GIBCO) containing 5% fetal calf serum (GIBCO). The cells were cultured in a ₂ concentration incubator.
NRK-52E was seeded in a 24-well plate at 1 × 10 ⁴ per well and cultured overnight in the above medium. One day later, the final concentration of pre-miR miRNA precursor molecules (Ambion) producing miR-21, 223, 30b, 30c, 486, 194, let-7i was determined by lipofection using lipofectamine 2000 (Invitrogen). Was introduced into NRK-52E to be 5 nM. MiRNA precursor molecules-Negative control (Ambion) was also introduced as a negative control. Lipofection followed the method described in the instructions attached to the product.
24 hours after the introduction of the molecule by lipofection method, the medium was diluted with DMEM medium containing 5% fetal bovine serum to a final concentration of 2 ng / ml of recombinant human TGF-β1 (R & D). The culture was continued.
Two days after changing to a medium containing TGF-β1, the medium was removed, and using Trizol reagent (Invitrogen), total RNA was extracted according to the method described in the product. Using PrimeScript 1st strand cDNA Synthesis Kit (manufactured by Takara Bio Inc.), cDNA was synthesized according to the method described in the product.
Using the prepared cDNA, the expression of fibrosis-related genes was analyzed by real-time RT-PCR. Samples were prepared using the QuantiTect SYBR Green PCR Kit (QIAGEN) according to the method described in the product, and the expression level of collagen I was quantified using an Applied Biosystems 7900HT Fast real-time PCR system (Applied Biosystems). As a result of calculating the expression level of collagen I in cells into which each microRNA was introduced, assuming that the expression level of collagen I in cells into which miRNA precursor molecules-Negative control used as negative control was introduced was 1.00, let -7i (SEQ ID NO: 8), miR-30b (SEQ ID NO: 11), miR-30c (SEQ ID NO: 12), and miR-486 (SEQ ID NO: 14) are introduced to reduce the expression of collagen I It became clear to do. It was also clarified that the expression of collagen I was increased by introducing molecules that generate miR-194 (SEQ ID NO: 24), miR-21 (SEQ ID NO: 26), and miR-223 (SEQ ID NO: 27). .

Effects of fibroblasts forced to express microRNAs on extracellular matrix production Pre-miR miRNA precursor molecules (Ambion) were introduced into fibroblasts, and the effects of microRNAs generated from these molecules on collagen production were examined. It was. Renal fibrosis is known to involve fibroblasts in kidney tissue, and the model cell line NRK-49F is used as the model. Collagen I or the like is used as an index of fibrosis [JASN., 16 , 2702-13 (2005)], [Kidney International, 66 , 112-120 (2004)].
NRK-49F was seeded in a 24-well plate at 2 × 10 ⁴ per well and cultured overnight in the above medium. One day later, the final concentration of pre-miR miRNA precursor molecules that produce miR-21, 30b, 30c, 192, 486, let-7i is 5 nM by lipofection using lipofectamine 2000 (Invitrogen). Introduced into NRK-49F. MiRNA precursor molecules-Negative control (Ambion) was also introduced as a negative control. Lipofection followed the method described in the instructions attached to the product.
Eight hours after the introduction of the molecule by the lipofection method, the medium was replaced with serum-free DMEM medium. After another 16 hours, the medium was replaced with recombinant human TGF-β1 (R & D) diluted to a final concentration of 1 ng / ml using DMEM medium without serum, and the culture was continued.
Two days after replacement with a medium containing TGF-β1, the medium was removed, and using Trizol reagent (Invitrogen), total RNA was extracted according to the method described in the product. After measuring the concentration, PrimeScript 1st strand cDNA Synthesis Using Kit (manufactured by Takara Bio Inc.), cDNA was synthesized according to the method described in the product.
Using the prepared cDNA, the expression of fibrosis-related genes was analyzed by real-time RT-PCR. Samples were prepared using the QuantiTect SYBR Green PCR Kit (QIAGEN) according to the method described in the product, and the expression level of collagen I was quantified using an Applied Biosystems 7900HT Fast real-time PCR system (Applied Biosystems). As a result, as shown in Table 9, let-7i (SEQ ID NO: 8), miR-30b (SEQ ID NO: 11), miR-30c (SEQ ID NO: 12), miR-192 (SEQ ID NO: 6), miR-486 ( It was revealed that the expression of collagen I is reduced by introducing a molecule that generates SEQ ID NO: 14). It was also revealed that collagen I expression was increased by introducing a molecule that generates miR-21 (SEQ ID NO: 26).

Effect on renal epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells in which microRNA is forcibly expressed Cells in which renal tubular epithelial cells are transformed into mesenchymal cells (EMT), resulting in excessive production of extracellular matrix proteins It has become clear that this is part of the cause of promoting fibrosis. Therefore, pre-miR miRNA precursor molecules (manufactured by Ambion) were introduced into renal tubular epithelial cells, and the influence of microRNA produced from the molecules on EMT was examined. For the analysis of epithelial-mesenchymal transition (EMT), increased expression of α smooth muscle actin (αSMA) in MDCK cells, a canine tubular epithelial cell line, is used [Am J Physiol Renal Physiol, 291 , F1332- 42 (2006), JASN., 18 , 1497-1507, (2007)].
MDCK was seeded in a 24-well plate at 1 × 10 ⁴ per well and cultured overnight in the above medium. One day later, pre-miR miRNA precursor molecules that generate miR-21, 146b, 223, 194, let-7i were made MDCK to a final concentration of 5 nM by lipofection using lipofectamine 2000 (Invitrogen). Introduced. MiRNA precursor molecules-Negative control (Ambion) was also introduced as a negative control. Lipofection followed the method described in the instructions attached to the product.
24 hours after introduction of the molecule by the lipofection method, the medium was diluted with DMEM medium containing 10% fetal calf serum to a final concentration of 2 ng / ml of recombinant human TGF-β1 (R & D). The culture was continued.
Two days after replacement with a medium containing TGF-β1, the medium was removed, and using Trizol reagent (Invitrogen), total RNA was extracted according to the method described in the product. After measuring the concentration, PrimeScript 1st strand cDNA Synthesis Using Kit (manufactured by Takara Bio Inc.), cDNA was synthesized according to the method described in the product.
Using the prepared cDNA, the expression of fibrosis-related genes was analyzed by real-time RT-PCR. Prepare samples using the QuantiTect SYBR Green PCR Kit (QIAGEN) according to the method described in the product, and use the Applied Biosystems 7900HT Fast real-time PCR system (Applied Biosystems) to express αSMA, which is an EMT indicator Was quantified. As a result, as shown in Table 10, it was clarified that the expression of αSMA is reduced by introducing a molecule that generates let-7i (SEQ ID NO: 8). In addition, by introducing molecules that generate miR-194 (SEQ ID NO: 24), miR-146b (SEQ ID NO: 25), miR-21 (SEQ ID NO: 26), miR-223 (SEQ ID NO: 27), expression of αSMA Became clear.

Effect on extracellular matrix production of human hepatic stellate cells in which microRNA was forcibly expressed A microRNA precursor was introduced into human hepatic stellate cells, and the influence of microRNA produced from the molecule on collagen production was examined. Hepatic stellate cells are said to play a central role in liver fibrosis and collagen production, and collagen I and the like are used as an index of fibrosis [HEPATOLOGY, 34 , 89-100 (2001) ].
Li-90 cells (hereinafter referred to as Li-90), which are human hepatic stellate cells, are DMEM medium (GIBCO) containing 20% fetal calf serum (GIBCO) and a 5% CO ₂ concentration at 37 ° C. Cultured in an incubator.
Li-90 was seeded in a 24-well plate at 1.5 × 10 ⁴ per well and cultured overnight in the above medium. One day later, pre-miR miRNA precursor molecules (manufactured by Ambion) that produce miR-30b, 486, let-7i were Lipofected using lipofectamine 2000 (manufactured by Invitrogen) to a final concentration of 10 nM. -90 introduced. MiRNA precursor molecules-Negative control (Ambion) was also introduced as a negative control. Lipofection followed the method described in the instructions attached to the product.
Eight hours after the introduction of the molecule by the lipofection method, the medium was replaced with serum-free DMEM medium. After another 16 hours, the medium was replaced with recombinant human TGF-β1 (manufactured by R & D) diluted to a final concentration of 1 ng / ml using DMEM medium without serum, and the culture was continued.
Two days after changing to a medium containing TGF-β1, the medium was removed, and using Trizol reagent (Invitrogen), total RNA was extracted according to the method described in the product. cDNA was synthesized using superscript VILO (Invitrogen) according to the method described in the product.
Using the prepared cDNA, the expression of fibrosis-related genes was analyzed by real-time RT-PCR. Samples were prepared using the QuantiTect SYBR Green PCR Kit (QIAGEN) according to the method described in the product, and the amount of collagen I expression was quantified using an Applied Biosystems 7900HT Fast real-time PCR system (Applied Biosystems). As a result of calculating the expression level of collagen I in the cells into which each microRNA was introduced, assuming that the expression level of collagen I in the cells into which miRNA precursor molecules-Negative control used as negative control was introduced was 1.00, let It was revealed that the expression of collagen I is reduced by introducing molecules that generate -7i (SEQ ID NO: 8) and miR-486 (SEQ ID NO: 14).

Effects of microRNA on forced extracellular matrix production of pulmonary fibroblasts We examined the action of microRNA in the lung, an organ in which fibrosis is a cause of disease progression. Pre-miR miRNA precursor molecules (manufactured by Ambion) were introduced into human lung fibroblasts, and the influence of microRNA generated from the molecules on collagen production was examined. It is known that fibroblasts in lung tissue are involved in lung fibrosis, and collagen I and the like are used as an index of the fibrosis [Respiration, 77 , 311-319 (2009) ].
Human lung fibroblast IMR-90 cells (hereinafter referred to as IMR-90) are prepared in MEM medium containing 10% fetal calf serum (GIBCO), 2 mM L-glutamine, 0.1 mM NEAA, 1 mM pyruvate ( (Manufactured by GIBCO) at 37 ° C. in an incubator with 5% CO ₂ concentration.
IMR-90 was seeded in a 24-well plate at 1.5 × 10 ⁴ per well and cultured overnight in the above medium. One day later, pre-miR miRNA precursor molecules that generate miR-30b, 30c, 486, and let-7i were added to IMR-90 by lipofection using lipofectamine 2000 (Invitrogen) to a final concentration of 10 nM. Introduced. MiRNA precursor molecules-Negative control (Ambion) was also introduced as a negative control. Lipofection followed the method described in the instructions attached to the product.
Eight hours after the introduction of the molecule by the lipofection method, the medium was replaced with a serum-free MEM medium. After another 16 hours, the medium was replaced with a recombinant human TGF-β1 (manufactured by R & D) diluted to a final concentration of 0.5 ng / ml using DMEM medium without serum, and the culture was further continued.
Two days after changing to a medium containing TGF-β1, the medium was removed, and using Trizol reagent (Invitrogen), total RNA was extracted according to the method described in the product. cDNA was synthesized using superscript VILO (Invitrogen) according to the method described in the product.
Using the prepared cDNA, the expression of fibrosis-related genes was analyzed by real-time RT-PCR. Samples were prepared using the QuantiTect SYBR Green PCR Kit (QIAGEN) according to the method described in the product, and the expression level of collagen I was quantified using an Applied Biosystems 7900HT Fast real-time PCR system (Applied Biosystems). As a result, as shown in Table 12, molecules that generate let-7i (SEQ ID NO: 8), miR-30b (SEQ ID NO: 11), miR30c (SEQ ID NO: 12), miR-486 (SEQ ID NO: 14) are introduced. This revealed that the expression of collagen I was reduced.

Effect on extracellular matrix production in renal tubular epithelial cells in which microRNA expression was suppressed The effect of reduced microRNA expression on extracellular matrix production by introducing antisense oligonucleotides complementary to microRNA into cells was investigated. Anti-miR miRNA inhibitors (manufactured by Ambion) were used as antisense oligonucleotides complementary to microRNA, and the influence of microRNA suppressed by the molecule on collagen production was examined.
NRK-52E was seeded in a 24-well plate at 1 × 10 ⁴ per well and cultured overnight in the above medium. One day later, anti-miR miRNA inhibitors that inhibit miR-30c, 486, 320a, 378, and 451 were introduced into NRK-52E by lipofection using lipofectamine 2000 (Invitrogen) to a final concentration of 30 nM. did. Anti-miR miRNA Inhibitors-Negative Control # 1 (Ambion) was also introduced as a negative control. Lipofection followed the method described in the instructions attached to the product.
24 hours after introduction of the molecule by the lipofection method, the medium was diluted with DMEM medium containing 5% fetal calf serum to a final concentration of 2 ng / ml of recombinant human TGF-β1 (R & D) The culture was continued.
Two days after changing to a medium containing TGF-β1, remove the medium, extract the total RNA according to the method described in the product using Trizol reagent (Invitrogen), measure the concentration, then PrimeScript 1st strand cDNA Synthesis Using Kit (manufactured by Takara Bio Inc.), cDNA was synthesized according to the method described in the product.
Using the prepared cDNA, the expression of fibrosis-related genes was analyzed by real-time RT-PCR. Samples were prepared using the QuantiTect SYBR Green PCR Kit (QIAGEN) according to the method described in the product, and the expression level of collagen I was quantified using an Applied Biosystems 7900HT Fast real-time PCR system (Applied Biosystems). As a result, as shown in Table 13, miR-320a (SEQ ID NO: 2), miR-378 (SEQ ID NO: 3), miR-30c (SEQ ID NO: 12), miR-486 (SEQ ID NO: 14), miR-451 ( It was revealed that the expression of collagen I was increased by suppressing the expression of SEQ ID NO: 18).

Effect on extracellular matrix production in renal fibroblasts with suppressed expression of microRNA We investigated the effect of reduced expression of microRNA on extracellular matrix production by introducing antisense oligonucleotides complementary to microRNA into cells. Anti-miR miRNA inhibitors (manufactured by Ambion) were used as antisense oligonucleotides complementary to microRNA, and the influence of microRNA suppressed by the molecule on collagen production was examined.
NRK-49F was seeded in a 24-well plate at 2 × 10 ⁴ per well and cultured overnight in the above medium. One day later, anti-miR miRNA inhibitors that inhibit miR-30c, 192, 320a, 378, and 451 were introduced into NRK-49F by lipofection using lipofectamine 2000 (Invitrogen) to a final concentration of 30 nM. did. Anti-miR miRNA Inhibitors-Negative Control # 1 (Ambion) was also introduced as a negative control. Lipofection followed the method described in the instructions attached to the product. For let-7i and miR-486, miRIDIAN microRNA Hairpin Inhibitor (manufactured by Dharmacon) was introduced into NRK-49F by a lipofection method using lipofectamine 2000 to a final concentration of 10 nM. MiRIDIAN microRNA Hairpin Inhibitor Negative Control # 2 (Dharmacon) was also introduced as a negative control.
Eight hours after the introduction of the molecule by the lipofection method, the medium was replaced with serum-free DMEM medium. After another 16 hours, the medium was replaced with a recombinant human TGF-β1 (manufactured by R & D) diluted to a final concentration of 1 ng / ml using DMEM medium without serum, and the culture was further continued.
Two days after replacement with a medium containing TGF-β1, the medium was removed, and using Trizol reagent (Invitrogen), total RNA was extracted according to the method described in the product. After measuring the concentration, PrimeScript 1st strand cDNA Synthesis Using Kit (manufactured by Takara Bio Inc.), cDNA was synthesized according to the method described in the product.
Using the prepared cDNA, the expression of fibrosis-related genes was analyzed by real-time RT-PCR. Samples were prepared using the QuantiTect SYBR Green PCR Kit (QIAGEN) according to the method described in the product, and the expression level of collagen I was quantified using an Applied Biosystems 7900HT Fast real-time PCR system (Applied Biosystems). As a result, as shown in Table 14, miR-320a (SEQ ID NO: 2), miR-378 (SEQ ID NO: 3), miR-192 (SEQ ID NO: 6), miR-30c (SEQ ID NO: 12), miR-486 ( It was revealed that the expression of collagen I was increased by suppressing the expression of SEQ ID NO: 14), miR-451 (SEQ ID NO: 18), and let-7i (SEQ ID NO: 8).

Effects on renal epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells with suppressed microRNA expression The effect of microRNA expression reduction on EMT by introduction of antisense oligonucleotide complementary to microRNA was examined.
MDCK was seeded in a 24-well plate at 1 × 10 ⁴ per well and cultured overnight in the above medium. One day later, anti-miR miRNA inhibitors that inhibit miR-320a, 378, 192, 30c, 486, and 451 were introduced into MDCK to a final concentration of 30 nM by lipofection using lipofectamine 2000 (Invitrogen). did. Anti-miR miRNA Inhibitors-Negative Control # 1 (Ambion) was also introduced as a negative control. Lipofection followed the method described in the instructions attached to the product.
24 hours after introduction of the molecule by the lipofection method, the medium was diluted with DMEM medium containing 10% fetal bovine serum to a final concentration of 2 ng / ml of recombinant human TGF-β1 (R & D). The culture was continued.
Two days after replacement with a medium containing TGF-β1, the medium was removed, and using Trizol reagent (Invitrogen), total RNA was extracted according to the method described in the product. After measuring the concentration, PrimeScript 1st strand cDNA Synthesis Using Kit (manufactured by Takara Bio Inc.), cDNA was synthesized according to the method described in the product.
Using the prepared cDNA, the expression of fibrosis-related genes was analyzed by real-time RT-PCR. Prepare samples using the QuantiTect SYBR Green PCR Kit (QIAGEN) according to the method described in the product, and use the Applied Biosystems 7900HT Fast real-time PCR system (Applied Biosystems) to express αSMA, which is an EMT indicator Was quantified. As a result, as shown in Table 15, miR-320a (SEQ ID NO: 2), miR-378 (SEQ ID NO: 3), miR-192 (SEQ ID NO: 6), miR-30c (SEQ ID NO: 12), miR-486 ( It was revealed that the expression of αSMA was increased by suppressing the expression of SEQ ID NO: 14) and miR-451 (SEQ ID NO: 18).

Effects of microRNA administration on organ fibrosis In order to examine whether microRNA administration affects organ fibrosis, we evaluated each of the three microRNAs administered to the liver fibrosis model. The microRNAs evaluated were miR-30b, miR-486, and let-7i. As a microRNA, miR-30b uses a nucleic acid (hsa-miR-30b) consisting of the base sequence represented by SEQ ID NO: 11 as an antisense strand, and is complementary to the first base to the 19th base on the 5 ′ side of the antisense strand. A nucleic acid consisting of the base sequence represented by SEQ ID NO: 1282, in which a TT overhang sequence was added to a typical sequence, was synthesized as a sense strand and annealed. miR-486 is obtained by annealing an antisense strand consisting of the base sequence represented by SEQ ID NO: 14 and a sense strand consisting of the base sequence represented by SEQ ID NO: 1283, and let-7i is SEQ ID NO: 8 An antisense strand consisting of the base sequence represented by the above and an annealed sense strand consisting of the base sequence represented by SEQ ID NO: 1284 were used. As a negative control, 21mer luciferase siRNA was used. Each RNA was synthesized by Hokkaido System Science and described in Stable nucleic acid lipid particle (SNALP), a lipid particle with high delivery efficiency to the liver, described in Mol Ther., 13 (3) , 494-505 (2006). After conjugation according to the established method, it was used for evaluation.
Hepatology, 50 , 185-197 (2009), Am J Physiol Gastrointest Liver Physiol, 296 , G582-G592 using the Bile Duct Ligation (BDL) model known as a general animal model for liver fibrosis (2009)].
Bile ducts from 9-week-old male C57BL / 6J mice (purchased from Charles River Japan) were ligated under anesthesia and 7 SNALP conjugate microRNAs were administered at a dose of 150 μg / head immediately after surgery and 24 hours later, respectively. Administered. Thereafter, the liver was removed 48 hours later and quickly frozen in liquid nitrogen. Frozen liver is crushed using Trizol reagent (Invitrogen), added with chloroform (Wako Pure Chemicals) and centrifuged, and then the supernatant is described in the product using RNeasy kit (QIAGEN) Total RNA was extracted according to the method described. After measuring the concentration of total RNA, cDNA was synthesized using Super Script VILO ^™ cDNA Synthesis Kit (manufactured by Invitrogen) according to the method described in the product. Using the prepared cDNA, the expression of fibrosis-related genes was analyzed by real-time RT-PCR. Prepare samples using the QuantiTect SYBR Green PCR Kit (manufactured by QIAGEN) according to the method described in the product, and use the Applied Biosystems 7900HT Fast real-time PCR system (manufactured by Applied Biosystems) to identify collagen I and fibrosis indicators. The expression level of TGF-β1 was quantified. As a result, by administering miR-30b (SEQ ID NO: 11), miR-486 (SEQ ID NO: 14), let-7i (SEQ ID NO: 8), collagen I (FIG. 1) and TGF-β1 (FIG. 2) It was revealed that the expression was significantly suppressed.

According to the present invention, an agent for controlling fibrosis of cells or organs, a diagnostic or therapeutic agent for diseases caused by fibrosis of cells or organs, an agent for controlling expression of target genes of nucleic acids such as microRNA, a method for controlling cells or organs, In addition, screening methods for cell or organ fibrosis control agents and the like are provided, which are useful in the prevention, diagnosis or treatment of diseases caused by cell or organ fibrosis. In this case, control refers to suppression and promotion.

This application is based on Japanese Patent Application No. 2009-187563 filed in Japan (filing date: August 12, 2009), the contents of which are incorporated in full herein.

Claims

An agent for controlling fibrosis of cells or organs, which contains 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: 1-27, 28-594 (b) Containing a nucleic acid comprising a base sequence represented by any of SEQ ID NOs: 1-27, 28-594 A nucleic acid consisting of a base sequence having a nucleotide sequence of 90% or more (d) a sequence number 1 to 1 to a nucleic acid of 17 to 28 bases (c) a sequence represented by any one of SEQ ID NOs: 1 to 27 and 28 to 594 A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleotide sequence represented by any one of 27 and 28 to 594 (e) a base represented by any one of SEQ ID NOs: 1 to 27 and 28 to 594 A nucleic acid comprising the second to eighth base sequences of the sequence (f) a nucleic acid comprising the base sequence represented by any one of SEQ ID NOs: 595 to 1281 (g) a base sequence represented by any one of the sequence numbers 595 to 1281; 90% or more identity (H) a nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid consisting of the base sequence represented by any of SEQ ID NOs: 595 to 1281
The cell or organ fibrosis regulator according to claim 1, wherein the nucleic acid is microRNA or a microRNA precursor.
A cell or organ fibrosis control agent 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 or organ fibrosis control agent comprising, as an active ingredient, a vector that expresses a nucleic acid that is an active ingredient of the cell or organ fibrosis control agent according to any one of claims 1 to 3.
A cell or organ fibrosis control agent comprising a substance that suppresses the expression of a gene having the target base sequence of the nucleic acid according to claim 1 as an active ingredient.
A cell or organ fibrosis control agent comprising a substance that promotes the expression of a gene having the target base sequence of the nucleic acid according to claim 1 as an active ingredient.
The cell or organ fibrosis regulator according to claim 5 or 6, wherein the substance that suppresses or promotes expression is a nucleic acid.
The cell or organ fibrosis regulator of claim 7, wherein the nucleic acid is siRNA or antisense oligonucleotide.
A cell or organ fibrosis control agent comprising the vector expressing the nucleic acid according to claim 7 as an active ingredient.
A cell or organ fiber containing 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 for diseases caused by oxidization.
A diagnostic agent for a disease caused by fibrosis of a cell or organ, comprising as an active ingredient a reagent for detecting the expression level of the nucleic acid, the mutation of the nucleic acid, or the mutation of the genome encoding the nucleic acid.
Diseases resulting from cell or organ fibrosis are acute renal failure, glomerulonephritis, vasculitis, diabetic nephropathy, hypertensive nephrosclerosis, HIV nephropathy, IgA nephropathy, lupus nephritis, interstitial nephritis, Obstructed kidney due to ureteral obstruction, pulmonary fibrosis, cirrhosis, arteriosclerosis, scleroderma, coronary restenosis after percutaneous transluminal coronary vasodilation (PTCA), interstitial myocarditis, interstitial cystitis, The therapeutic or diagnostic agent according to claim 10 or 11, which is scarring after burn or fibrosis associated with poisoning.
Diseases resulting from cell or organ fibrosis are acute renal failure, glomerulonephritis, vasculitis, diabetic nephropathy, hypertensive nephrosclerosis, HIV nephropathy, IgA nephropathy, lupus nephritis, interstitial nephritis, The therapeutic or diagnostic agent according to claim 10 or 11, which is a fibrosis associated with a renal disorder selected from the group consisting of obstructive kidneys due to ureteral obstruction.
A cell 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. Alternatively, a method for treating a disease caused by organ fibrosis.
A method for diagnosing a disease caused by fibrosis of a cell or organ, comprising detecting the expression level of the nucleic acid according to claim 1, a mutation of the nucleic acid, or a mutation of a genome encoding the nucleic acid.
Diseases resulting from cell or organ fibrosis are acute renal failure, glomerulonephritis, vasculitis, diabetic nephropathy, hypertensive nephrosclerosis, HIV nephropathy, IgA nephropathy, lupus nephritis, interstitial nephritis, Obstructed kidney due to ureteral obstruction, pulmonary fibrosis, cirrhosis, arteriosclerosis, scleroderma, coronary restenosis after percutaneous transluminal coronary vasodilation (PTCA), interstitial myocarditis, interstitial cystitis, The method of treatment or diagnosis according to claim 14 or 15, wherein the skin scarring after burns or fibrosis associated with poisoning.
Diseases resulting from cell or organ fibrosis are acute renal failure, glomerulonephritis, vasculitis, diabetic nephropathy, hypertensive nephrosclerosis, HIV nephropathy, IgA nephropathy, lupus nephritis, interstitial nephritis, The treatment or diagnosis method according to claim 16, which is a fibrosis associated with renal disorder selected from the group consisting of obstructive kidneys due to ureteral obstruction.
The nucleic acid according to any one of claims 1 to 3, the vector according to claim 4, or the vector according to any one of claims 5 to 8 for the manufacture of a therapeutic agent for a disease caused by fibrosis of cells or organs. Use of the substance according to claim 1.
Diseases resulting from cell or organ fibrosis are acute renal failure, glomerulonephritis, vasculitis, diabetic nephropathy, hypertensive nephrosclerosis, HIV nephropathy, IgA nephropathy, lupus nephritis, interstitial nephritis, Obstructed kidney due to ureteral obstruction, pulmonary fibrosis, cirrhosis, arteriosclerosis, scleroderma, coronary restenosis after percutaneous transluminal coronary vasodilation (PTCA), interstitial myocarditis, interstitial cystitis, 19. Use according to claim 18, wherein the skin scarring after burns or fibrosis associated with poisoning.
Diseases resulting from cell or organ fibrosis are acute renal failure, glomerulonephritis, vasculitis, diabetic nephropathy, hypertensive nephrosclerosis, HIV nephropathy, IgA nephropathy, lupus nephritis, interstitial nephritis, The use according to claim 18, which is a fibrosis associated with renal injury selected from the group consisting of obstructed kidneys due to ureteral obstruction.
An expression control agent for a target gene of a nucleic acid, which is an active ingredient of a cell or organ fibrosis control agent, comprising the nucleic acid according to any one of claims 1 to 3 as an active ingredient.
An agent for controlling expression of a target gene of a nucleic acid, which is an active ingredient of a cell or organ fibrosis regulator, comprising the vector according to claim 4 as an active ingredient.
A method for controlling fibrosis of a cell or organ, characterized by using the nucleic acid according to any one of claims 1 to 3.
A method for controlling fibrosis of a cell or organ, comprising using the vector according to claim 4.
A method for controlling fibrosis of a cell or organ, comprising using a substance that suppresses or promotes the expression of a target gene of the nucleic acid according to claim 1.
26. The method for controlling fibrosis of a cell or organ according to claim 25, wherein the substance that suppresses or promotes expression is a nucleic acid.
27. The method for controlling fibrosis of a cell or organ according to claim 26, wherein the nucleic acid is siRNA or antisense oligonucleotide.
A method for controlling fibrosis of a cell or organ using the vector expressing the nucleic acid according to claim 26.
A method for controlling the expression of a target gene of a nucleic acid that is an active ingredient of a fibrosis control agent for cells or organs, wherein the nucleic acid according to any one of claims 1 to 3 is used.
A method for controlling the expression of a target gene of a nucleic acid, which is an active ingredient of a cell or organ fibrosis regulator, characterized by using the vector according to claim 4.
A method for screening a fibrosis control agent for cells or organs, wherein the index is to promote or suppress the expression or function of the nucleic acid according to claim 1.