JP2017145222A - Preventive/therapeutic agent for liver fibrosis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a safe and effective agent for preventing/treating liver fibrosis, which suppresses or inhibits liver fibrosis.SOLUTION: According to the present invention, an agent for preventing/treating liver fibrosis comprises a microRNA (miRNA) as an active ingredient, the miRNA consisting of a sequence containing the nucleotide sequence represented by SEQ ID NO: 1. According to the present invention, an agent for preventing/treating liver fibrosis comprises a vector as an active ingredient, the vector being capable of expressing a microRNA (miRNA) consisting of a sequence containing the nucleotide sequence represented by SEQ ID NO: 1. According to the present invention, a method suppressing activation of hepatic stellate cells comprises the step of introducing miRNA consisting of a sequence containing the nucleotide sequence represented by SEQ ID NO: 1 into hepatic stellate cells in vitro. According to the present invention, a method for suppressing the production of an extracellular matrix comprises the step of introducing miRNA consisting of a sequence containing the nucleotide sequence represented by SEQ ID NO: 1 into cells in vitro.SELECTED DRAWING: None

Description

  The present invention relates to an agent for preventing / treating liver fibrosis.

  Although hepatic fibrosis develops due to persistent inflammation, there is currently no cure that focuses on liver fibrosis. For this reason, current treatment methods for liver fibrosis include sedation of inflammation by treating the primary disease (for example, in the case of chronic viral hepatitis, eliminating the causative virus) As a result, it can be expected to spontaneously heal fibrosis.

  On the other hand, in pulmonary fibrosis, treatment with pyrphenyldon is performed to suppress the activity of transforming growth factor β (TGF-β), which is a typical inflammatory cytokine for promoting fibrosis. ing.

SN Iyer., Et al., “Effects of Pirfenidone on Transforming Growth Factor-β Gene Expression at the Transcriptional Level in Bleomycin Hamster Model of Lung Fibrosis.”, J. PHARMACOL. EXP. THER., Vol.291, no.1 , 367-373, 1999.

Pyrphenyldon used in the treatment of pulmonary fibrosis is known to have liver toxicity and is difficult to adapt to patients with cirrhosis. At present, advanced liver fibrosis can only be handled by liver transplantation. Furthermore, liver transplantation in Japan is centered on living-donor liver transplantation where the family is a donor, and there are few cadaveric liver transplantations, so there is a problem that the number of patients that can be used for liver transplantation is very limited.
Therefore, establishment of a novel method for treating liver fibrosis focusing on liver fibrosis is desired.

  This invention is made | formed in view of the said situation, Comprising: It aims at providing the safe and effective liver fibrosis prevention and treatment agent which suppresses or inhibits hepatic fibrosis.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have a high safety and a nucleic acid to which RNA interference (RNAi) is applied because micro RNA (miRNA) is an endogenous gene. Focusing on the fact that it can be applied as a medicine, the present invention has been completed.

That is, the present invention includes the following aspects.
[1] A prophylactic / therapeutic agent for liver fibrosis comprising microRNA (miRNA) comprising a sequence containing the base sequence represented by SEQ ID NO: 1 as an active ingredient.
[2] A prophylactic / therapeutic agent for hepatic fibrosis comprising a vector capable of expressing a microRNA (miRNA) comprising a sequence comprising the base sequence represented by SEQ ID NO: 1 as an active ingredient.
[3] The prophylactic / therapeutic agent for liver fibrosis according to [1] or [2], wherein the miRNA is miR-29a or a precursor thereof.
[4] A method for inhibiting activation of hepatic stellate cells, comprising a step of introducing miRNA comprising the sequence represented by SEQ ID NO: 1 into hepatic stellate cells in vitro.
[5] A method for inhibiting the production of an extracellular matrix, comprising a step of introducing miRNA comprising a sequence containing the base sequence represented by SEQ ID NO: 1 into cells in vitro.

  ADVANTAGE OF THE INVENTION According to this invention, absorption of a liver fiber can be accelerated | stimulated, liver fibrosis can be suppressed or inhibited, and hepatic fibrosis can be treated safely and effectively. Further, since the degree of liver fibrosis is closely related to the liver carcinogenesis rate, it is possible to suppress carcinogenesis in the liver by suppressing or inhibiting liver fibrosis.

FIG. 3 is a diagram showing a production schedule and a miRNA administration schedule of chronic hepatitis model mice in Example 1. Hepatoxylin and eosin (HE) staining, picrosirius red (SR) and liver obtained from four groups of carbon tetrachloride treated mice treated for 1 week in Example 1 and It is an image which shows the result of the counterstaining using Fast Green (Fast Green). Hepatoxylin and eosin (HE) staining, picrosirius red (SR) and liver obtained from four groups of carbon tetrachloride treated mice treated for 2 weeks in Example 1 and It is an image which shows the result of the counterstaining using Fast Green (Fast Green). It is a graph which shows the result of having quantified the area of the positive area | region of SR in the liver obtained from the total of eight groups of carbon tetrachloride administration mice which were treated for 1 week or 2 weeks in Example 1. FIG. It is a graph which shows the result of having quantified hydroxypurine in the liver obtained from a total of eight groups of carbon tetrachloride administration mice which were treated for 1 week or 2 weeks in Example 1. It is a graph which shows the result of having quantified the collagen 1 (alpha) 1 (Col1a1) mRNA in the liver obtained from the carbon tetrachloride administration mouse | mouth of a total of eight groups which performed the treatment for 1 week or 2 weeks in Example 1. FIG. It is a graph which shows the result of having quantified mmu-miR-29a in the liver obtained from a total of eight groups of carbon tetrachloride administration mice treated for 1 week or 2 weeks in Example 1. Hepatoxylin and eosin (HE) staining and picrosirius red (SR) and fast of livers from four groups of thioacetamide-treated mice treated for 1 week in Example 1 It is an image which shows the result of the counterstaining using green (Fast Green). Hepatoxylin and eosin (HE) staining and picrosirius red (SR) and fast of liver obtained from four groups of thioacetamide treated mice treated for 2 weeks in Example 1 It is an image which shows the result of the counterstaining using green (Fast Green). It is a graph which shows the result of having quantified the area | region of the positive area | region of SR in the liver obtained from the thioacetamide administration mouse | mouth of a total of 8 groups which were treated for 1 week or 2 weeks in Example 1. FIG. It is a graph which shows the result of having quantified the hydroxypurine in the liver obtained from the thioacetamide administration mouse | mouth of a total of 8 groups which performed the treatment in Example 1 for 1 week or 2 weeks. The graph which shows the result of having quantified the relative expression level of collagen 1 (alpha) 1 (Col1a1) mRNA in the liver obtained from the thioacetamide administration mouse | mouth of a total of eight groups which were treated for 1 week or 2 weeks in Example 1. is there. It is a graph which shows the result of having quantified the relative expression level of mmu-miR-29a in the liver obtained from the thioacetamide administration mouse | mouth of a total of 8 groups which were treated for 1 week or 2 weeks in Example 1. FIG. The target gene of miR-29a was transferred to Targetscan, mirtarbase, and microRNA. It is a figure which shows the result predicted using the online search algorithm of org. It is a figure which shows the transforming growth factor- (beta) 1 (Transforming Growth Factor- (beta) 1: TGF (beta) 1) and miRNA administration schedule to the LX-2 cell in Example 2. FIG. It is a graph which shows the result of having quantified the relative expression level of mmu-miR-29a in the LX-2 cell which transfected miR-29a or NC miRNA after TGFβ1 treatment in Example 2. It is a graph which shows the result of having quantified the relative expression level of mRNA of Col1a1, PDGFC, and MMP2 in LX-2 cells transfected with miR-29a or NC miRNA after TGFβ1 treatment in Example 2. It is a figure which shows the result of having carried out immunostaining of Col1a1 protein in the LX-2 cell which transfected miR-29a or NC miRNA after TGF (beta) 1 treatment in Example 2. FIG. The graph which shows the result of having measured the cell viability using the cell proliferation kit II (XTT) (made by Roche) in the LX-2 cell which transfected miR-29a or NC miRNA after TGF (beta) 1 treatment in Example 2. is there. It is a graph which shows the result of having measured the activity of caspase-9 in the LX-2 cell which transfected miR-29a or NC miRNA after TGF (beta) 1 treatment in Example 2. FIG. It is a figure which shows the TGF (beta) 1 and miRNA administration schedule to the LX-2 cell in Example 3. FIG. It is a graph which shows the result of having quantified the relative expression level of mRNA of Col1a1, PDGFC, and MMP2 in LX-2 cells treated with TGFβ1 after transfection with miR-29a or NC miRNA in Example 3. It is a figure which shows the result of having carried out immunostaining of Col1a1 protein in the LX-2 cell which processed TGFβ1 after transfection with miR-29a or NC miRNA in Example 3. It is a figure which shows the suppression or inhibition mechanism of liver fibrosis by miR-29a with respect to a target gene. It is a figure which shows the suppression or inhibition mechanism of liver fibrosis by miR-29a with respect to genes other than a target gene.

<< Hepatic fibrosis prevention and treatment agent >>
As one embodiment, the present invention provides a prophylactic / therapeutic agent for hepatic fibrosis, which contains, as an active ingredient, microRNA (miRNA) comprising a sequence comprising the base sequence represented by SEQ ID NO: 1.

  The present inventors have paid attention to the fact that microRNA (miRNA) is an endogenous gene, and thus has high safety and can be applied as a nucleic acid medicine to which RNAi is applied. In addition, in the liver with advanced fibrosis, the expression of miR-132 and the miR-29 family (miR-29a, miR-29b and miR-29c) is remarkably decreased. It has been found that it is deeply involved in liver fibrosis, and has completed the present invention.

  According to the preventive / therapeutic agent for hepatic fibrosis according to the present embodiment, the absorption of liver fibrosis can be promoted, and the fibrosis can be suppressed or inhibited, and hepatic fibrosis can be treated safely and effectively. Moreover, carcinogenesis in the liver can be suppressed by suppressing or inhibiting liver fibrosis.

  In the present specification, “hepatic fibrosis” means that cells such as collagen increase in the liver and fiber changes such as collagen, and the liver changes due to alcohol intake, hepatitis caused by viruses, cirrhosis, etc. It is known that fibrosis proceeds. Moreover, it is known that the degree of liver fibrosis is closely related to the liver carcinogenesis rate.

<MiRNA>
The hepatic fibrosis prevention / treatment agent of this embodiment contains miRNA consisting of a sequence containing the base sequence represented by SEQ ID NO: 1 as an active ingredient. The miRNA used in the hepatic fibrosis preventive / therapeutic agent of the present embodiment targets genes involved in hepatic fibrosis, more specifically, the genes shown in Table 1 below, and the expression of proteins comprising these genes. It is miRNA which controls.

  Usually, microRNA (microRNA; miRNA) means what is called mature miRNA. A mature miRNA is an endogenous non-coding RNA of about 20 to 25 bases encoded on the genome. miRNA is first transcribed from a miRNA gene on genomic DNA as a primary transcript (Primary miRNA, hereinafter referred to as “Prim-miRNA”) having a length of several hundred to several thousand bases, and then subjected to processing. Pre-miRNA (precusor miRNA) having a hairpin structure of about 60 to 70 bases. After that, it moves from the nucleus into the cytoplasm and further undergoes processing to become a double-stranded mature miRNA of about 20-25 bases. It is known that double-stranded mature miRNAs function to inhibit the translation of target genes by forming a complex with a protein called RISC, with one strand forming a complex with a protein called RISC. .

In the present specification, “miRNA” includes not only endogenous miRNA but also synthetic nucleic acid having the same base sequence as endogenous miRNA.
Moreover, miRNA in this embodiment also includes the precursor.

  Usually, “a precursor of miRNA” means a nucleic acid capable of producing mature miRNA in a cell as a result of intracellular processing or cleavage of a double-stranded nucleic acid. Examples of miRNA precursors include pri-miRNA and pre-miRNA. The pri-miRNA is a primary transcription product (single-stranded RNA) of the miRNA gene, and usually has a length of about several hundred to several thousand bases. The pre-miRNA is a single-stranded RNA having a hairpin structure that is generated when the pri-miRNA undergoes intracellular processing, and usually has a length of 90 to 110 bases. The base sequence of the miRNA pri-miRNA or pre-miRNA is disclosed, for example, in the miRBBase database http://microrna.sanger.ac.uk/ produced by Sanger Laboratories.

  In order to improve the stability of the miRNA in this embodiment, for example, a nucleic acid derivative or a nucleic acid having a modified internucleoside bond may be used. As nucleic acid derivatives, known ones can be appropriately selected and used. Specifically, for example, phosphonate phosphorothioate, phosphorodithioate, phosphoramidate methoxyethyl phosphoramidate, formacetal, thioformacetal, diisopropylsilyl, acetamidate, carbamate, dimethylene-sulfide, dimethylene-sulfoxide, dimethylene -Nucleic acid containing an internucleoside bond of -sulfone, 2'-O-alkyl, and 2'-deoxy-2'-fluorophosphorothioate. Furthermore, derivatives such as Locked Nucleic Acid (LNA) are also included.

The miRNA in the present embodiment includes an RNA having the base sequence represented by SEQ ID NO: 1, and includes a nucleic acid that suppresses transcription of a gene involved in liver fibrosis by RNA interference (RNAi). Examples of the nucleic acid include RNA, RNA-DNA chimeric nucleic acid (hereinafter referred to as chimeric nucleic acid), hybrid nucleic acid, and the like.
In the present specification, “chimeric nucleic acid” means a nucleic acid containing RNA and DNA in a single nucleic acid in a single-stranded or double-stranded nucleic acid, and “hybrid nucleic acid” means in a double-stranded nucleic acid. Mean a nucleic acid in which one strand is RNA or a chimeric nucleic acid and the other strand is a DNA or a chimeric nucleic acid.

The miRNA in the present embodiment may be single-stranded or double-stranded. Examples of the double strand include double-stranded RNA, double-stranded chimeric nucleic acid, RNA / DNA hybrid, RNA / chimeric nucleic acid hybrid, chimeric nucleic acid / chimeric nucleic acid hybrid, and chimeric nucleic acid / DNA hybrid.
As miRNA in this embodiment, for example, single-stranded RNA, single-stranded chimeric nucleic acid, double-stranded RNA, double-stranded chimeric nucleic acid, RNA / DNA hybrid, RNA / chimeric nucleic acid hybrid, chimeric nucleic acid / chimeric nucleic acid hybrid or Examples include chimeric nucleic acid / DNA hybrids.

  The chain length of miRNA in this embodiment is preferably 15 bases or more and 40 bases or less, more preferably 15 bases or more and 30 bases or less, and further preferably 20 bases or more and 25 bases or less.

  The miRNA in this embodiment consists of a sequence having the base sequence (5'-UAGCACC-3 ') represented by SEQ ID NO: 1 as a seed sequence.

Usually, the “seed sequence” means a base sequence of 6 to 8 bases present in the 5 ′ terminal region of the mature miRNA, and it is known that this seed sequence determines the mRNA that is the target of the miRNA.
The miRNA in the present embodiment preferably has the base sequence represented by SEQ ID NO: 1 at the first to seventh bases at the 5 ′ end.

  In the miRNA of the present embodiment, a nucleic acid having a chain length of 8 to 33 bases is linked to the 3 ′ end of the RNA consisting of the base sequence represented by SEQ ID NO: 1 so as to have the above-mentioned chain length. Is preferred.

  More specifically, the miRNA in the present embodiment has 80% or more, for example, 85% or more, of the base sequence represented by SEQ ID NO: 2 at the 3 ′ end of the RNA consisting of the base sequence represented by SEQ ID NO: 1. For example, 90% or more, for example, 95% or more, for example, 99% or more, for example, 100% identity, nucleic acid consisting of a base sequence is linked, and further, from these genes targeting genes involved in liver fibrosis MiRNA that regulates the expression of the following protein. Among them, as the miRNA in the present embodiment, a nucleic acid comprising a sequence having 100% identity with the base sequence represented by SEQ ID NO: 2 is present at the 3 ′ end of the RNA comprising the base sequence represented by SEQ ID NO: 1. Further, miRNAs that are linked and further target the genes involved in liver fibrosis and control the expression of proteins composed of these genes are preferable. More specifically, the miRNA in the present embodiment is preferably miR-29a (SEQ ID NO: 5: 5'-UAGCACCAUCUGAAAUCCGGUUA-3 ').

<Expression vector>
The preventive / therapeutic agent for hepatic fibrosis according to the present embodiment can include an expression vector capable of expressing the above-described miRNA as an active ingredient. In the expression vector, the above-described miRNA or a nucleic acid (preferably DNA) encoding the same exhibits a promoter activity in a mammalian (preferably human) cell (for example, hepatic stellate cell) to be applied. Linked to the promoter obtained.

  The promoter used is not particularly limited as long as it can function in mammalian cells to be applied. As the promoter, for example, a pol I promoter, pol II promoter, pol III promoter and the like can be used. Specifically, SV40-derived early promoter, viral promoter such as cytomegalovirus LTR, mammalian constituent protein gene promoter such as β-actin gene promoter, and RNA promoter such as tRNA promoter are used.

  When miRNA expression is intended, a pol III promoter is generally used as the promoter. Examples of the polIII promoter include U6 promoter, H1 promoter, tRNA promoter and the like.

  The expression vector preferably contains a transcription termination signal, that is, a terminator region, downstream of the above-described miRNA or nucleic acid encoding the same. Furthermore, selection marker genes for selection of transformed cells (for example, genes that confer resistance to drugs such as tetracycline, ampicillin, kanamycin, genes that complement auxotrophic mutations, etc.) and fluorescent protein genes (for example, GFP ( (Green Fluorescent Protein), RFP (Red Fluorescent Protein), YFP (Yellow Fluorescent Protein), etc.).

<Dose conditions>
In the agent for preventing and treating hepatic fibrosis of the present embodiment, the above-described miRNA or the expression vector capable of expressing the above-described miRNA is an ordinary carrier that is preferably pharmaceutically or veterinarily acceptable. Or it can be administered to the administration subject together with a diluent in an effective amount.

  In addition, the hepatic fibrosis prevention / treatment agent of the present embodiment may be administered in combination with other drugs. For example, in the case of hepatic fibrosis due to hepatitis B virus, a nucleic acid analog preparation may be used. In the case of hepatic fibrosis caused by a virus, it is possible to treat hepatic fibrosis while suppressing replication of the causative virus by using an antiviral agent in combination.

  The above-described miRNA, which is an active ingredient of the hepatic fibrosis prevention / treatment agent of the present embodiment, can be produced by a known production method, for example, chemical synthesis, or inserted into an appropriate expression vector, and the gene It can also be engineered.

  When an expression vector capable of expressing the above-described miRNA is used as an active ingredient of the hepatic fibrosis prevention / treatment agent of the present embodiment, for example, a DNA encoding the above-described miRNA is used as an adenovirus vector, adeno-associated gene. The above-described miRNA can be expressed in the administration subject by administering to the administration subject an expression vector constructed by inserting it into a suitable gene therapy vector such as a virus (AAV) vector or a lentivirus vector.

  There are no particular limitations on the dosage form of the hepatic fibrosis prevention / treatment agent of the present embodiment. For example, powders, fine granules, granules, tablets, capsules, suspensions, emulsions, syrups, extracts Or oral preparations such as pills or pills, or parenteral preparations such as injections, externally applied liquids, ointments or creams for topical administration of suppositories.

  Oral preparations include, for example, gelatin, sodium alginate, starch, corn starch, sucrose, lactose, glucose, mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soy lecithin, sucrose, fatty acid ester, talc, magnesium stearate, Excipients such as polyethylene glycol, magnesium silicate, anhydrous silicic acid, or synthetic aluminum silicate, binders, disintegrants, surfactants, lubricants, fluidity promoters, diluents, preservatives, colorants, It can be produced according to a conventional method using a fragrance, a flavoring agent, a stabilizer, a moisturizing agent, an antiseptic, or an antioxidant.

Examples of parenteral administration methods include injection (subcutaneous, intravenous, etc.) and the like. Of these, the injection is most preferably used.
For example, in the preparation of an injection, in addition to the active ingredient, for example, a water-soluble solvent such as physiological saline or Ringer's solution, a water-insoluble solvent such as vegetable oil or fatty acid ester, an isotonic agent such as glucose or sodium chloride, Solubilizers, stabilizers, preservatives, suspending agents, or emulsifiers can be optionally used.
Further, the preventive / therapeutic agent for hepatic fibrosis according to the present embodiment may be administered by using a method of a sustained release preparation using a sustained release polymer or the like. For example, the preventive / therapeutic agent for hepatic fibrosis of the present embodiment can be incorporated into a pellet of an ethylene vinyl acetate polymer, and the pellet can be surgically transplanted into the liver tissue to be treated or prevented.

  The hepatic fibrosis prevention / treatment agent of the present embodiment is not limited to this, but contains an active ingredient in an amount of 0.01 to 99% by weight, preferably 0.1 to 80% by weight. Can do.

  The dose when using the hepatic fibrosis prevention / treatment agent of the present embodiment depends on, for example, the type of active ingredient to be used, the type of illness, the age, sex, weight, degree of symptoms, or administration method of the patient. And can be administered orally or parenterally. As a dosage of miRNA which is an active ingredient of the hepatic fibrosis prevention / treatment agent of the present embodiment, for example, when intravenously administered to one mouse, about 1 μg to about 1 mg is preferable, and about 1 μg to about 500 μg is preferable. More preferred is about 10 μg to about 100 μg. In addition, for example, when intravenously administered to one human (adult), about 10 μg / kg to about 100 mg / kg is preferable, about 10 μg / kg to about 50 mg / kg is preferable, and about 50 μg / kg to about 10 mg / kg. Is more preferable.

  As the administration frequency of the hepatic fibrosis prevention / treatment agent of the present embodiment, for example, administration once a day to once every several months (for example, once a week to once a month) Can do.

<< Usage >>
The miRNA that is an active ingredient of the preventive / therapeutic agent for liver fibrosis according to the present embodiment can be used for the following uses.

  As one embodiment, the present invention provides a method for inhibiting activation of hepatic stellate cells, comprising the step of introducing miRNA comprising a sequence comprising the base sequence represented by SEQ ID NO: 1 into hepatic stellate cells in vitro.

  In the present embodiment, by using the above-described miRNA, activation of hepatic stellate cells can be suppressed in vitro as shown in Examples described later.

  As one embodiment, the present invention provides an extracellular matrix production suppression method comprising a step of introducing miRNA comprising a sequence containing the base sequence represented by SEQ ID NO: 1 into cells in vitro.

  In the present embodiment, by using the above-described miRNA, the production of extracellular matrix can be suppressed in vitro as shown in Examples described later.

  Usually, “extracellular matrix (ECM)” means a supramolecular structure existing outside a cell. The components constituting the vertebrate ECM are mainly glycoproteins (partially cell adhesion molecules) such as collagen, proteoglycan, fibronectin, cadherin and laminin. Therefore, the production of extracellular matrix can be suppressed by suppressing the expression of the Col1a1 gene, which is one of the components of ECM.

Moreover, as one Embodiment, this invention provides the expression vector which can express the above-mentioned miRNA for the treatment of liver fibrosis, or the above-mentioned miRNA.
In one embodiment, the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the above-described miRNA or an expression vector capable of expressing the above-described miRNA, and a pharmaceutically acceptable carrier or diluent. .
Moreover, as one embodiment, the present invention provides an agent for preventing / treating liver fibrosis, which comprises the pharmaceutical composition.
Moreover, as one embodiment, the present invention provides the use of the above-described miRNA or an expression vector capable of expressing the above-described miRNA for producing an agent for preventing / treating liver fibrosis.
Moreover, as one embodiment, the present invention provides a method for treating liver fibrosis, comprising administering an effective amount of the above-described miRNA or an expression vector capable of expressing the above-described miRNA to a patient in need of treatment. To do.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, this invention is not limited to a following example.

[Example 1]
(1) Preparation of chronic hepatitis model mice (carbon tetrachloride-administered mice and thioacetamide-administered mice) C57BL / 6J male mice (6 weeks old) (manufactured by SCL Japan, Inc.) were raised for one week and acclimatized. The lighting conditions were controlled by a cycle of brightening for 12 hours and darkening for 12 hours. Carbon tetrachloride (CCl ₄ ) was mixed with corn oil and administered into the abdominal cavity at a dose of 0.5 μL / g body weight every 12 days for a total of 12 times over 5 weeks to produce carbon tetrachloride-administered mice. In addition, in thioacetamide-treated mice, thioacetamide (TAA) is dissolved in physiological saline, so that the first dose is 100 μg / g body weight, and the second to fifth dose is 200 μg / g body weight. Thus, it administered every 3 days so that it might become 300 microgram / g body weight in the 6th-9th administration, and it may become 400 microgram / g body weight in the 10th-12th administration. As a control mouse for each chronic hepatitis model mouse, a mouse given corn oil or physiological saline alone for 5 weeks was prepared. Five weeks later, liver fibrosis was observed in mice treated with carbon tetrachloride and mice treated with thioacetamide.

(2) Treatment evaluation of chronic hepatitis by administration of miR-29a For each mouse produced in (1), miR-29a was administered for 1 week or 2 weeks immediately after the 5-week hepatic fibrosis induction period. (See FIG. 1A). 50 μg of double-stranded mature mmu-miR-29a (manufactured by Bonac) or a negative control RNA (NC miRNA) (manufactured by Bonac), which is a 20-mer non-specific gene that does not recognize a human gene, is used as a control. It was mixed with atelocollagen (manufactured by Athelogin) and injected through the tail vein every 3 days. Mice treated for 1 week were administered miR-29a twice, and mice treated for 2 weeks were administered miR-29a four times. Therefore, the carbon tetrachloride-administered mouse and the thioacetamide-administered mouse are divided into the following four groups of mice, respectively. A group in which only observation was performed without doing anything (observation), a group in which only atelocollagen as a gene introduction reagent was administered (vehicle), a group in which a non-specific gene was administered (NC miRNA), and a group in which miR-29a was administered (See FIG. 1A). For each of the four groups, a group treated for 1 week and a group treated for 2 weeks were prepared, for a total of 8 groups.

(3) Histological analysis Each liver was surgically removed from the 8 groups of mice prepared in (2). The removed liver was fixed with a 10% formalin solution, and two 5 μm continuous paraffin sections were prepared. One section was used for haematoxylin and eosin (HE) staining (see images above FIGS. 1B, 1C, 2A and 2B). The other section was stained with Picrosirius red (SR) solution (Sigma Aldrich) and counterstained with Fast Green (FIGS. 1B, 1C, 2A and 2B). See image below). The positive region of SR was quantified using a microanalyzer (Nippon Pola Digital) (see FIG. 1D and FIG. 2C). Five images containing at least one portal vein were selected to verify the area of the SR positive region.

From the results of HE staining in FIGS. 1B, 1C, 2A and 2B, inflammatory cells are generally scattered in all chronic hepatitis model mice, but the liver of mice administered with miR-29a. Then, it was confirmed that the inflammation was not exacerbated.
In addition, miR-29a was administered based on the results of counterstaining with Sirius red and fast green under FIGS. 1B, 1C, 2A and 2B, and the graphs of FIGS. 1D and 2C. In the mouse liver, it was revealed that the area of the fiber decreased.

(4) Hydroxypurine analysis Hydroxypurine contained in the liver was quantified using a part of the liver extracted in (3). The method for quantifying hydroxypurine was performed using a hydroxypurine assay kit (manufactured by Biovision) according to the instruction manual. Hydroxypurine is the main component of collagen, and the degree of fibrosis can be evaluated by quantifying hydroxypurine. The results are shown in FIGS. 1E and 2D.

  From FIG. 1E and FIG. 2D, it can be inferred that in any of the chronic hepatitis model mice, the amount of hydroxypurine is decreased and fibrosis is improved in the liver of mice administered with miR-29a.

(5) RNA extraction from liver and expression analysis of mRNA and miRNA Using the RNeasy (registered trademark) mini kit (manufactured by Qiagen), using the part of liver extracted in (3), instruction manual Extracted according to the instructions. CDNA was synthesized from the obtained RNA using a transcripter first strand cDNA synthesis kit (Roche). Real-time PCR was performed using Fast Start Universal SYBR Green Master Mix (Roche). The primer sequences used are as shown in Table 2 below. The reaction was measured in triplicate. Subsequently, using TaqMan® microRNA assay (Thermo Fisher Scientific), relative to hsa-miR-29a (Assay ID.002447) and U18 (Assay ID.001204) as a control. Expression levels were quantified. cDNA was synthesized using TaqMan (registered trademark) microRNA reverse transcription kit (manufactured by Thermo Fisher Scientific). Real-time PCR analysis was performed using a Fast Start Universal Probe Master (Roche). Co1a1 mRNA expression analysis was performed using the primers shown in Table 2 below and FastStart Universal SYBR Green Master mix (Roche). MRNA and microRNA were detected using a Step One Plus real-time PCR system (manufactured by Thermo Fisher Scientific). The reaction was measured in triplicate. The results are shown in FIGS. 1F, 1G, 2E and 2F.

  From FIG. 1F, FIG. 1G, FIG. 2E and FIG. 2F, the expression of collagen 1α1 (Col1a1) is decreased in the liver of mice treated with miR-29a for 1 week in any chronic hepatitis model mouse However, mice treated with miR-29a for 2 weeks showed a tendency to decrease although no statistical difference was observed. Moreover, the expression of miR-29a in liver tissue did not change with the administration of miR-29a.

  Furthermore, in order to elucidate the mechanism of fibrosis by miR-29a, the target gene of miR-29a was transferred to Targetscan, mirtarbase and microRNA. predicted using org's online search algorithm. The results are shown in FIG.

From FIG. 3, as target genes of miR-29a, disintegrin and metallopeptidase domain 19 (Adam19) gene, fibrinogen-like protein 2 (Fgl2) gene, PDGF gene, collagen IV type α4c4ol4p Col4a4) gene, Map4k4 (mitogen-activated protein kinase kinase kinase 4) gene, methyltransferase 3B (methyltransferase 3B; DNmt3b) gene and Col1a1 gene were identified.
It was confirmed that at least the base sequence (5′-UAGCACC-3 ′) represented by SEQ ID NO: 1 in miR-29a hybridizes to any target gene. Therefore, in miR-29a, it is presumed that the base sequence represented by SEQ ID NO: 1 is a seed sequence.

[Example 2]
(1) Culture of LX-2 cells Scott L., a liver disease specialist at Mount Sinai Hospital. Human HSC cell line LX-2 donated by Friedman was prepared using 10% fetal bovine serum (FBS), 100 U / mL penicillin, 100 μg / mL streptomycin (manufactured by Thermo Fisher Scientific) and non-essential amino acids ( The culture was performed using Dulbecco's modified medium (manufactured by Wako) containing Thermo Fisher Scientific). Subsequently, LX-2 cells were seeded in a 6-well plate (manufactured by Thermo Fisher Scientific) so as to be 1 × 10 ⁵ cells / well.

(2) Treatment with transforming growth factor-β1 (Transforming Growth Factor-β1: TGFβ1) 2.5 ng / mL Transforming growth factor-β1 (Transforming Growth Factor-β1: TGFβ1) was added to the cells to induce inflammation of the cells. Induced (see FIG. 4A).

(3) Transfection of microRNA into LX-2 cells Subsequently, 40 μM of miR-29a or NC miRNA was added to the cells one day after the TGFβ1 treatment in (2), and Lipofectamine RNAiMAX Trans Reagent (Thermo Fisher Scientific). LX-2 cells were transfected using the cell line, and the cells were further cultured for 2 days (see FIG. 4A).

(4) RNA extraction from LX-2 cells and expression analysis of mRNA and miRNA Subsequently, cells two days after transfection in (3) were collected, and total RNA was RNeasy (registered trademark) mini kit (manufactured by Qiagen). ) And extracted according to the instruction manual. Using the same method as in Example 1 (5), the relative expression level of miR-29a, the relative expression level of mRNA of Col1a1, PDGFC and matrix metalloproteinase 2 (Matrix metalloproteinase; MMP2) were measured. MMP2 mRNA expression analysis was performed using the primers shown in Table 3 below and FastStart Universal SYBR Green Master mix (Roche). The results are shown in FIGS. 4B and 4C.

  4B and 4C, it was confirmed that the expression of Col1a1 and MMP2 mRNA was suppressed by administering miR-29a in LX-2 cells in which inflammation was induced by treatment with TGFβ1. Although MMP2 has the effect | action which absorbs a fiber, since fiber expression does not rise by administering miR-29a, it is thought that the fiber absorption function was also suppressed. The expression of PDGFC mRNA tended to decrease with the administration of miR-29a, although no statistical difference was observed.

(5) Evaluation of the amount of Col1a1 protein Subsequently, a part of the cells 2 days after the transfection of (3) was subjected to 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% sodium dodecyl sulfate (sodium dodecyl). sulfate; SDS), 0.5% deoxycholic acid and 1% NP-40. Subsequently, 20% glycerol containing 125 mM Tris-HCl (pH 6.8), 4% SDS, 10% 2-mercaptoethanol and 0.01% bromophenol blue is added to the quantified supernatant (20 μg / well). Add amount and boil. Subsequently, SDS polyacrylamide gel electrophoresis was performed using PAGEL (registered trademark) (5 to 20%) (manufactured by ATTO). It was transferred to a polyvinylidene fluoride membrane (Millipore) by submarine blotting. Subsequently, the obtained membrane was incubated with 5% skim milk and Tween 20-containing Tris buffered saline to perform blocking. Subsequently, incubation was performed using an anti-COLA1 antibody (sc-8784, manufactured by Santa Cruz) as a primary antibody or an anti-β-actin antibody (sc-69879, manufactured by Santa Cruz) as a control. Subsequently, as a secondary antibody, a horseradish peroxidase-conjugated goat anti-rabbit antibody or a horseradish peroxidase-conjugated goat anti-mouse antibody (manufactured by Dako) was used for incubation. Immunoreactivity was visualized by chemiluminescence (manufactured by GE Healthcare). The result is shown in FIG. 4D.

  From FIG. 4D, it was revealed that the amount of Col1a1 protein was decreased by administering miR-29a.

(6) Cell viability analysis LX-2 cells were seeded in a 96-well plate so as to be 1 × 10 ⁴ cells / well. Cell proliferation kit II (XTT) (Roche) was used according to the instruction manual. Specifically, first, 2.5 ng / mL transforming growth factor-β1 (Transforming Growth Factor-β1: TGFβ1) was added to the seeded cells. Subsequently, 40 μM miR-29a or NC miRNA was transfected into the cells one day after TGFβ1 treatment using Lipofectamine RNAiMAX Trans Reagent (Thermo Fisher Scientific). Subsequently, 50 μL of XTT reagent was added to the cells 48 hours after transfection and cultured at 37 ° C. for 6 hours. Subsequently, the absorbance at 450 nm was measured. The result is shown in FIG. 4E.

  FIG. 4E revealed that the cell viability was slightly reduced in LX-2 cells in which miR-29a was overexpressed regardless of the presence or absence of TGFβ1 treatment.

(7) Caspase 9 activity assay LX-2 cells were seeded in a 96-well plate at 1 × 10 ⁴ cells / well. Subsequently, 2.5 ng / mL transforming growth factor-β1 (Transforming Growth Factor-β1: TGFβ1) was added to the seeded cells. Subsequently, 40 μM miR-29a or NC miRNA was transfected into the cells one day after TGFβ1 treatment using Lipofectamine RNAiMAX Trans Reagent (Thermo Fisher Scientific). Subsequently, Caspase-Glo (registered trademark) 9 assay kit (manufactured by Promega) was used for the cells 48 hours after transfection according to the instruction manual. The result is shown in FIG. 4F.

  FIG. 4F revealed that Caspase 9 activity increased in LX-2 cells overexpressed with miR-29a compared to LX-2 cells transfected with NC miRNA.

[Example 3]
(1) Culture of LX-2 cells Using the same method as in (1) of Example 2, 6 well plates (Thermo Fisher Scientific) were prepared so that LX-2 cells would be 1 × 10 ⁵ cells / well. Sowing).

(2) Transfection of microRNA into LX-2 cells Subsequently, one day after cell seeding, the same method as (3) of Example 2 was applied to the LX-2 cells seeded in (1). 40 μM miR-29a or NC miRNA was transfected (see FIG. 5A).

(3) Treatment with TGFβ1 Subsequently, TGFβ1 was added to LX-2 cells 6 hours after transfection in (2) using the same method as in Example 2 (2) to induce cell inflammation. (See FIG. 5A).

(4) RNA extraction from LX-2 cells and mRNA and miRNA expression analysis Subsequently, cells were collected one or two days after the transfection of (2), and the same method as in (4) of Example 2 Was used to collect total RNA, and the relative expression levels of mRNA of Col1a1, PDGFC and MMP2 were measured. The result is shown in FIG. 5B.

  From FIG. 5B, it was confirmed that expression of Col1a1 and MMP2 mRNA was suppressed by administering miR-29a in advance. Although MMP2 has the effect | action which absorbs a fiber, since fiber expression does not rise by administering miR-29a, it is thought that the fiber absorption function was also suppressed. Moreover, about the expression of mRNA of PDGFC, the inhibitory effect by administration of miR-29a was not seen.

(5) Evaluation of the amount of Col1a1 protein Subsequently, using a part of cells two days after the transfection of (2), the Col1a1 protein was immunostained using the same method as in (5) of Example 2 . The result is shown in FIG. 5C.

  From FIG. 5C, it became clear that the amount of Col1a1 protein decreases by administering miR-29a.

  Moreover, FIG. 6A and FIG. 6B are figures which show the suppression or inhibition mechanism of liver fibrosis by miR-29a. RhoA, MAPK8, RAF1, SRF, IL-1b, NDRG1 and BCL2 shown in FIGS. 6A and 6B are genes shown in Table 4 below. Moreover, the graph which shows the relative expression level of mRNA of said 7 types of genes is using the part of a cell 2 days after transfection of (2), SurePrint G3 Mouse Gene Expression 8x60k Microarray Kit and the following. It is the result detected using the probe shown in Table 5.

  From Examples 1 to 3, it was revealed that miR-29a directly hybridized to each of the Col1a1 gene and the PDGFC gene and suppressed the expression of these genes. As a result, the production of collagen which is an extracellular matrix is suppressed. Furthermore, from FIG. 6A, the expression of PDGFC that induces inflammation is suppressed, and cell migration or cell proliferation is suppressed by suppressing the downstream genes RhoA, MAPK8, RAF, and SRF. Inferred.

  Further, from FIG. 6B, it was revealed that administration of miR-29a decreases the IL-1b gene, the NDRG1 gene, and the BCL gene, although not the target gene. By suppressing the IL-1 gene, inflammation is suppressed, and by suppressing the NDRG1 gene, it is presumed that blood vessel formation is suppressed, and further, apoptosis is promoted by suppressing the BCL2 gene.

  ADVANTAGE OF THE INVENTION According to this invention, absorption of a liver fiber can be accelerated | stimulated, liver fibrosis can be suppressed or inhibited, and hepatic fibrosis can be treated safely and effectively. Further, since the degree of liver fibrosis is closely related to the liver carcinogenesis rate, it is possible to suppress carcinogenesis in the liver by suppressing or inhibiting liver fibrosis.

Claims (5)

  A preventive / therapeutic agent for hepatic fibrosis, comprising, as an active ingredient, microRNA (miRNA) comprising a sequence comprising the base sequence represented by SEQ ID NO: 1.   A preventive / therapeutic agent for hepatic fibrosis, comprising as an active ingredient a vector capable of expressing a microRNA comprising a sequence comprising the base sequence represented by SEQ ID NO: 1.   The preventive / therapeutic agent for liver fibrosis according to claim 1 or 2, wherein the miRNA is miR-29a or a precursor thereof.   A method for suppressing activation of hepatic stellate cells, comprising a step of introducing miRNA comprising a sequence containing the base sequence represented by SEQ ID NO: 1 into hepatic stellate cells in vitro.   A method for inhibiting the production of an extracellular matrix, comprising a step of introducing miRNA comprising a sequence containing the base sequence represented by SEQ ID NO: 1 into a cell in vitro.