KR102256680B1 - Composition using mirna-101 for differntiation of mesenchymal stem cell into hepatocyte-like cell and composition using mirna-101 for diagnosis, prevention or treatment of hepatic fibrosis - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for hepatocyte differentiation and liver fibrosis treatment comprising miR-101-3p as an active ingredient and, more specifically, to a pharmaceutical composition for hepatocyte differentiation and liver fibrosis treatment, which can exhibit an excellent effect in the prevention or treatment of liver fibrosis and liver disease induced thereby by inhibiting the activation of hepatocytes as well as promoting the differentiation of stem cells into hepatocytes at the same time. In addition, the present invention relates to a therapeutic effect monitoring biomarker that can predict the therapeutic effect of liver fibrosis, including the treatment of liver disease using stem cells.

Description

A composition for differentiation of similar hepatocytes using MIR-101 and a composition for diagnosing, preventing or treating liver fibrosis {COMPOSITION USING MIRNA-101 FOR DIFFERNTIATION OF MESENCHYMAL STEM CELL INTO HEPATOCYTE-LIKE CELL AND COMPOSITION USING MIRNA-101 FOR DIAGNOSIS, PREVENTION OR TREATMENT OF HEPATIC FIBROSIS}

The present invention relates to a composition for differentiation of hepatocytes comprising miR-101, a pharmaceutical composition for preventing or treating liver fibrosis comprising miR-101, a composition for diagnosing liver fibrosis using miR-101, and a method for diagnosing liver fibrosis using the same.

Liver fibrosis refers to a condition in which damaged liver tissue is transformed into fibrous tissue such as collagen without being restored to normal hepatocytes, and generally starts from damage to liver cells due to various causes such as viral, alcoholic, drug toxicity, and autoimmunity. do. When such damage is not cured initially and becomes chronic, hepatic stellate cells (HSC) are formed by factors such as platform-derived growth factor (PDGF) and TGF-β secreted by various inflammatory cells gathered around damaged hepatocytes. Activated and activated hepatic stellate cells are differentiated into proliferative myofibroblasts that express α-SMA (alpha smooth muscle actin), and differentiated myofibroblasts are various cells such as collagen and elastin. The secretion of the extracellular matrix (ECM) leads to liver fibrosis. In addition, according to a recent report, liver fibrosis is also induced through epithelial mesenchymal transition (EMT), a new mechanism related to liver fibrosis.

Liver fibrosis is an inevitable pathological result of the liver accompanying chronic liver damage, and a decrease in liver function inevitably appears in that the liver is replaced by fibrous tissue that cannot perform its own functions, such as metabolism of biological substances or secretion of bile. Liver fibrosis is reversible, consists of thin fibril, and is known to have no nodule formation, so if the cause of liver damage is lost, it may be possible to return to normal. However, if the fibrosis process continues repeatedly, exchange and binding between ECMs may increase, leading to irreversible liver cirrhosis in which thick fibril and regenerated nodules are generated. Liver cirrhosis is a disease with a high mortality rate because it is difficult to treat when complications are involved. In addition, liver fibrosis and cirrhosis are important risk factors for liver cancer, and about 80% of liver cancers occur after progression to cirrhosis, and research results have reported that the incidence of cancer increases by up to 6 times or more depending on the degree of fibrosis progression.

In Korea, liver disease is classified as a refractory disease, which is the fourth largest death factor excluding cancer. The most effective treatment method for severe liver disease is liver transplantation, but due to problems such as scarcity of liver donors and high cost of surgery, there are very limited cases in which liver transplantation is actually performed. Allogeneic hepatocyte transplantation has been suggested as an effective treatment for acute liver failure, but this also has a difficult problem in obtaining human hepatocytes. Accordingly, there is a need for other effective alternatives for the prevention or treatment of liver disease.

Recently, studies on stem cell transplantation have been actively conducted as a more fundamental treatment method involved in the regeneration and maintenance of human tissues by restoring the function of damaged cells or tissues. Treatment using stem cells is expected not only to supply large amounts of hepatocytes to liver tissues in which liver disease occurred, but also to be involved in the regeneration and maintenance of liver tissues. The importance of research on cell function-specific microRNA is also emerging.

KR 101671776 B1 KR 101263760 B1

M. F. Mahmoud, et al, European Journal of Pharmacology, vol. 742, pp. 118-124, 2014 J. H. Jun, et al., Clinical and Molecular Hepatology, vol. 22, no. 3, pp. 372-381, 2016 M. J. Lee et al., Journal of Cellular Biochemistry, vol. 111, no. 6, pp. 1453-1463, 2010 J. Jung, et al., International Journal of Stem Cells, vol. 8, no. 1, pp. 79-89, 2015

An object of the present invention is to provide a composition for inducing or promoting differentiation of hepatocytes comprising a miRNA of a specific sequence.

Another object is to provide a differentiation method for differentiating stem cells into hepatocytes by processing miRNAs of a specific sequence.

In addition, it is another object to provide a pharmaceutical composition for preventing or treating liver fibrosis and diseases induced therefrom comprising miRNA of a specific sequence.

In addition, it is another object to provide a composition for diagnosing liver fibrosis or predicting a therapeutic effect that detects miRNA of a specific sequence.

In addition, it is another object of the present invention to provide a method of providing information on diagnosing liver fibrosis or predicting therapeutic effects by comparing the levels of miRNA expression of a specific sequence.

According to an aspect of the present invention, a composition for inducing differentiation from stem cells into hepatocytes containing miR-101-3p as an active ingredient is provided.

In addition, the miR-101-3p may consist of the nucleotide sequence of SEQ ID NO: 1.

In addition, the miR-101-3p is a form including at least partially a phosphorothiolate structure; DNA, peptide nucleic acids (PNA), or locked nucleic acid (LNA) molecules instead of RNA, at least partially substituted; And it may be contained in at least one form selected from the group consisting of a form in which the 2'hydroxyl group of the RNA sugar is substituted with another functional group.

In addition, the miR-101-3p is a miRNA precursor form; Mature miRNA form; Plasmid miRNA precursor form; And it may be contained in at least one form selected from the group consisting of the form introduced into the virus.

In addition, the stem cells may be mesenchymal stem cells.

In addition, the mesenchymal stem cells may be derived from bone marrow.

According to another aspect of the present invention, there is provided a method for differentiating from stem cells into hepatocytes, comprising the step of treating the composition to stem cells.

In addition, the treatment may be performed by culturing the stem cells in a medium containing the composition or by introducing the composition into the stem cells.

In addition, miR-101-3p may be overexpressed in the stem cells by the introduction.

According to another aspect of the present invention, there is provided a composition for preventing or treating liver disease containing the composition and mesenchymal stem cells as an active ingredient.

According to another aspect of the present invention, there is provided a composition for preventing or treating liver fibrosis or cirrhosis containing miR-101-3p as an active ingredient.

In addition, the miR-101-3p may consist of the nucleotide sequence of SEQ ID NO: 1.

In addition, the miR-101-3p is a form including at least partially a phosphorothiolate structure; DNA, peptide nucleic acids (PNA), or locked nucleic acid (LNA) molecules instead of RNA, at least partially substituted; And it may be at least one form selected from the group consisting of a form in which the 2'hydroxyl group of the RNA sugar is substituted with another functional group.

In addition, the miR-101-3p is a miRNA precursor form; Mature miRNA form; Plasmid miRNA precursor form; And it may be at least one form selected from the group consisting of a form introduced into a virus.

In addition, it is possible to inhibit the activation of hepatic stellate cells.

According to another aspect of the present invention, there is provided a composition for diagnosing or analyzing the prognosis of treatment for liver fibrosis or cirrhosis comprising an agent capable of detecting miR-101-3p.

In addition, the agent may be at least one selected from the group consisting of primers, probes, and antibodies having a sequence complementary to miR-101-3p.

According to another aspect of the present invention, a method of providing information on diagnosis or treatment prognosis of liver fibrosis or cirrhosis, comprising measuring the expression level of miR-101-3p in a sample isolated from a subject Is provided.

In addition, the step of comparing the expression level of miR-101-3p of the measured sample with the expression level of the normal control may be further included.

In addition, the sample may be at least one selected from the group consisting of tissue, cells, blood, plasma, serum, saliva, and urine.

The composition according to an aspect of the present invention may increase the number of hepatocytes by inducing or promoting differentiation from stem cells into hepatocytes. Through this, it can be usefully used as a cell therapy treated with stem cells for the treatment or prevention of various liver diseases caused by liver damage.

In addition, stem cells can be used to induce or promote differentiation of hepatocytes from mesenchymal stem cells (MSCs), which have superior stability compared to other stem cells, and are advantageous in mass production due to standardized isolation and culture techniques. It can be applied as an effective treatment for liver disease. In addition, since it has the effect of making it possible to mass-produce patient-specific hepatocytes, it can be used to build a model for predicting patient-specific treatment or treatment effect through in vitro experiments.

In addition, since it can inhibit the activity of hepatic stellate cells (HSC), which induce liver fibrosis, it can be used as a stable pharmaceutical composition with excellent effect for the treatment of liver fibrosis.

In addition, since it exhibits an effect of inhibiting liver fibrosis and differentiation of liver cells at the same time, it can be used as a pharmaceutical composition for an efficient and fundamental treatment for liver fibrosis.

The composition according to another aspect of the present invention may be used as a biomarker for monitoring the therapeutic effect capable of predicting or diagnosing the therapeutic effect of liver fibrosis.

1 shows the function of miR-101-3p on the liver,
Figure 2 shows the expression of miR-101-3p in hepatocytes and differentiated hepatocytes based on mesenchymal stem cells through Next Generation Sequencing (NGS analysis) in fibrotic liver tissues based on normal liver tissues. ,
Figure 3 is a quantitative real-time PCR (Quantitative real-time PCR; qRT-PCR) confirming the expression of miR-101-3p and markers over time inducing differentiation of mesenchymal stem cells into hepatocytes,
Fig. 4 shows the differentiation of miR-101-3p overexpressing mesenchymal stem cells cultured for 7 days into hepatocytes.
Figure 5 is a human hepatic stellate cell (Human Hepatic Stellate Cell), confirming the expression of miR-101-3p in the cell in the activated state of LX2,
6 and 7 confirming the effect of miR-101-3p on inhibiting fibrosis on activated LX2 cells,
FIG. 8 is a view confirming the therapeutic effect of stem cell transplantation enhanced with stem cell transplantation and PRL-1 overexpression in an animal model of liver fibrosis,
Figure 9 shows the expression of miR-101-3p in a liver fibrosis animal model confirming the therapeutic effect after stem cell transplantation.

The present invention is capable of inducing or promoting differentiation of stem cells into hepatocytes, as well as inhibiting the activation of hepatic stellate cells at the same time, thereby preventing or treating liver fibrosis and liver diseases induced by multiple pathways. It was conceived by discovering miRNA that can exhibit excellent effects and confirming its efficacy.

Hereinafter, the present invention will be described in more detail.

The term miRNA used in the present invention refers to a non-coding RNA that regulates gene expression after transcription by promoting degradation of target RNA or inhibiting their translation. In general, microRNA is transcribed into a precursor of about 70-80 nt (nucleotide) length with a hairpin structure called pre-miRNA, and then cut by the RNAse III enzyme Dicer to produce a mature form, and this mature miRNA is It binds to RISC (RNA induced silencing complex) to show its function. In the present invention, miRNA may be mixed and used as miR, microRNA, or microRNA.

The term hepatocyte as used in the present invention means a major constituent cell of a liver tissue. Hepatocytes produce various serum proteins such as albumin, blood coagulation factor, and complement, and bilirubin, and exhibit various functions including metabolism of amino acids, glucose, fatty acids, cholesterol, decomposition of drugs and harmful substances, and detoxification. The hepatocytes of the present invention include hepatocyte-like cells that function similarly or have the same function as hepatocytes.

The miRNA included as an active ingredient in the present invention may be miR-101-3p. Specifically, it may be a polynucleotide comprising the sequence of SEQ ID NO: 1. The miR-101-3p may be a mammal-derived, preferably human-derived sequence.

In the present invention, miR-101-3p may be used in the form of a miRNA precursor or a mature miRNA. Specifically, miR-101-3p used in the present invention may include miRNA precursors such as pri-miR-101 and pre-miR-101, mature miR-101 and miR-101-3p. For example, it can be used in the form of mature miR-101 consisting of the sequence of SEQ ID NO: 2. In addition, it may include isoforms and fragments thereof that can substantially exhibit the biological effect of miR-101-3p according to the present invention.

The miRNA of the present invention may exist in the form of a heteromorph. Here, the miRNA isoform refers to a sequence having a mutation with respect to the reference miRNA sequence. Specifically, it means that the core sequence of the miRNA is the same, but a part of the nucleotide sequence before or after the core sequence has been added or deleted. It is known that miRNA sequences can be obtained from miRbase (www.mirbase.org/), and the miRNA heterolog expression profile can show race, population, and gender dependence.

Further, in the present invention, a derivative of miR-101-3p or a mimic thereof may be used in place of miR-101-3p. The derivative may be one containing one or more chemical modifications in miR-101-3p. The derivative is in the form of a miRNA precursor; Plasmid miRNA precursor form; A form containing at least partially a phosphorothiolate structure; DNA, peptide nucleic acids (PNA), and locked nucleic acid (LNA) molecules instead of RNA, at least partially substituted; And at least one selected from the group consisting of a form in which the 2'hydroxyl group of the RNA sugar is substituted with another functional group, but is not limited thereto. The 2'hydroxyl group of the RNA sugar may be substituted with, for example, a methyl, methoxy, or fluorine-containing functional group, but is not limited thereto.

The miRNA of the present invention may include not only the miRNA sequence described in each of the above sequence numbers, but also a miRNA sequence having 80% or more, specifically, 90% or more, more specifically, 95% or more homology with the sequence. As a sequence having such homology, any sequence representing a miRNA that is substantially the same as each of the miRNAs or exhibits a corresponding efficacy is included without limitation. In addition, in the case of a miRNA sequence having such homology, it is obvious that some of the sequences are deleted, modified, substituted, or added miRNA sequences are also included within the scope of the present invention.

The miRNA of the present invention can be used in the composition as such or in the form of a pharmaceutically acceptable salt. Herein, the pharmaceutically acceptable salt means that the biological activity of the miRNA of interest in the present invention is maintained while undesired toxicity is minimized. Such salts are, for example, base addition salts formed with metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, etc. and salts formed with organic amino acids, or ammonia, N, N-dibenzylethylene-diamine, D-glucosamine, tetraethylammonium, or a salt formed with a cation derived from ethylenediamine, but is not limited thereto.

The miRNA according to the present invention is synthesized as an oligonucleotide using a nucleic acid synthesizer, or is constructed as a part of an expression plasmid or vector containing a polynucleotide encoding it under a promoter, and is used in vivo or in vitro. It can be used so that it can be transferred. In the latter case, the expression plasmid itself may be used, or the miRNA transcribed therefrom may be separated and used. The oligonucleotide, polynucleotide, expression plasmid or vector may be calcium phosphate method, DEAE-dextran method, cationic lipid mediated liposome delivery method, infection or transduction using virus or cells, electroporation, or transduction or It can be delivered to the cells, tissues or organs of interest through methods such as microinjection.

According to an aspect of the present invention, a composition comprising miR-101-3p or a derivative thereof is provided. The composition according to an aspect of the present invention may induce or promote differentiation of stem cells into hepatocytes in vivo or in vitro, and a hepatocyte culture may be obtained through this. The obtained hepatocyte culture may be transplanted into a problem tissue and used for treatment for restoring liver function or for research for treating liver disease. Here, the stem cells may be adult stem cells, preferably mesenchymal stem cells. The mesenchymal stem cells may be of human origin, and may be isolated from bone marrow, umbilical cord, placenta, adipose tissue, molar tooth protrusion, skin, muscle, nerve, amniotic fluid, blood, or umbilical cord blood, but is not limited thereto.

According to another aspect of the present invention, a method of differentiating stem cells into hepatocytes using miR-101-3p or a derivative thereof is provided. The composition according to an aspect of the present invention may be treated to stem cells, or may be performed by introducing the composition according to an aspect of the present invention into stem cells. Specifically, stem cells by overexpressing miR-101-3p in stem cells while culturing stem cells in a medium containing the composition according to an aspect of the present invention or introducing and culturing miR-101-3p or a derivative thereof into stem cells It can induce or promote differentiation into hepatocytes. Through this, it is possible to obtain a hepatocyte culture. In this case, the culture period for inducing differentiation may be 5 to 30 days, preferably 5 to 10 days, more preferably 6 to 8 days, but is not limited thereto. Here, the introduction may be performed through the method of delivering the nucleotide polymer, expression vector, or plasmid described above, but is not limited thereto.

The composition according to an aspect of the present invention may be provided as a pharmaceutical composition for preventing or treating liver fibrosis or cirrhosis. The miRNA of the present invention can prevent, ameliorate or treat fibrotic symptoms by inhibiting or inhibiting the activation of hepatic stellate cells. At the same time, differentiation into hepatocytes can be induced or promoted, and thus, when treated with stem cells, more efficient and stable preventive, improved, or therapeutic effects can be exhibited against liver fibrosis and cirrhosis.

According to another aspect of the present invention, the composition according to an aspect of the present invention may be provided as a pharmaceutical composition for cell therapy for the prevention, improvement or treatment of liver disease together with stem cells. Here, the liver disease that the pharmaceutical composition for cell therapy is the target of prevention, improvement or treatment may be caused by reduction of hepatocytes or decreased function of hepatocytes, or may be accompanied by a decrease in liver function, and the liver fibrosis and Liver disease resulting therefrom. The liver disease includes acute liver disease, chronic liver disease or genetic liver function impairment, for example, hepatitis, hepatitis C, sarcoidosis, drug allergy, fatty liver, alcoholic liver disease, liver cancer, Liver cirrhosis, pregnancy toxicosis, non-Hodgkin's lymphoma, Reye's syndrome, other neonatal jaundice, Kawasaki disease, lupus, candidiasis, malaria, leptospirosis, dengue fever, hepatic flukes, acute liver cirrhosis, chronic cirrhosis, inherited metabolic diseases, and bile duct disease. duct disease), but is not limited thereto.

The composition according to an aspect of the present invention may further include an active ingredient exhibiting the same or similar function in relation to the treatment of liver diseases, and a compound for maintaining or increasing the solubility or absorption of the active ingredient. If necessary, at least one selected from the group consisting of chemotherapeutic agents, anti-inflammatory agents, antiviral agents, and immunomodulatory agents may be further included. In addition, it may further include or be administered with a pharmaceutically acceptable carrier. The carrier may be selected and used appropriately depending on the dosage form, and may be used alone or in combination. As a carrier, for example, when administered orally, a conjugate, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, a coloring agent or a fragrance may be used, and in the case of an injection, a buffering agent, a preservative, and painless An agent, a solubilizing agent, an isotonic agent, and a stabilizer may be used, and in the case of topical administration, a base agent, an excipient, a lubricant, and a preservative may be used, but the present invention is not limited thereto.

The composition according to an aspect of the present invention may be used in the form of a general pharmaceutical formulation, and may be prepared in an appropriate formulation including an acceptable carrier according to an administration method. For example, it can be used in the form of a sterilized aqueous solution, non-aqueous solvent, suspension, emulsion or lyophilized formulation as a parenteral formulation, and in the form of tablets, troches, capsules, elixirs, suspensions, syrups or wafers when administered orally, It can be used as injection in unit dosage ampoules or in multiple dosage forms.

A method of treating liver disease using the composition according to an aspect of the present invention may include administration through a general route through which a predetermined substance is introduced to a patient in an appropriate manner. The administration method includes intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration, but is not limited thereto.

If desired, the active substance can be administered by any device capable of moving to the target cell. Preferred modes of administration and dosage forms include, for example, intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, or drop injections, but are not limited thereto.

For the treatment of liver disease, the composition according to an aspect of the present invention may be administered in a pharmaceutically effective amount. The pharmaceutically effective amount is known in the medical field, such as the type of disease, the patient's age, weight, health, sex, sensitivity to the patient's drug, the route of administration, the method of administration, the number of administrations, the duration of treatment, and drugs used in combination or simultaneously. It can be easily determined by a person skilled in the art depending on the factors. For example, a dose of 0.1 to 1000 mg/kg, preferably 10 to 100 mg/kg per day may be administered to an adult once to several times a day, but is not limited thereto.

According to another aspect of the present invention, a composition for diagnosing or analyzing the prognosis of treatment for liver fibrosis or cirrhosis comprising an agent capable of detecting the miRNA of the present invention may be provided. The detection may be for confirming the expression level of the miRNA of the present invention. Here, the expression level means the level of expression, which is the degree of quantitative expression. The miRNA of the present invention is expressed at a high level in normal hepatocytes, but its expression is reduced in hepatocytes undergoing fibrosis, and thus it appears to have a significant correlation with the occurrence of hepatic fibrosis. Therefore, the miRNA of the present invention can safely and easily predict the occurrence of liver fibrosis or the therapeutic effect of liver fibrosis without using an invasive method of biopsy of liver tissue, so that the biomarker for diagnosing liver fibrosis or monitoring the therapeutic effect It can be applied usefully. The agent capable of detecting the miRNA of the present invention refers to a substance capable of specifically binding to the miRNA of the present invention, for example, primers, probes and antibodies having a sequence complementary to the miRNA nucleotide sequence of the present invention. It may be at least one selected from the group consisting of, but is not limited thereto.

According to another aspect of the present invention, a method of diagnosing liver fibrosis or cirrhosis or analyzing prognosis using the miRNA of the present invention may be provided. This can be performed using the diagnostic composition according to an aspect of the present invention. Specifically, the diagnosis of liver fibrosis or cirrhosis by treating a sample isolated from a subject by treating the diagnostic composition to measure the expression level of miRNA of the present invention, and comparing the expression level of the measured sample with the expression level of a normal control group Alternatively, prognostic analysis can be performed. As an example, after detecting the expression level of the miRNA biomarker of the present invention in a sample derived from a subject requiring diagnosis of liver fibrosis or cirrhosis, the expression level of the miRNA biomarker of the present invention is compared with the result of the normal control. If it is found to have decreased, diagnosis of liver fibrosis or cirrhosis of the liver can provide diagnostic information for liver fibrosis for the subject. As another example, in order to monitor the therapeutic effect after treatment of stem cells for treatment of liver fibrosis in a subject in need of treatment for liver fibrosis, expression of miRNA of the present invention targets the subject-derived sample before and after stem cell treatment. Prognostic information for stem cell therapy can be provided by measuring and comparing the level and predicting the therapeutic effect that the treatment is doing well when the expression increases. The sample may be at least one selected from the group consisting of tissue, cells, blood, plasma, serum, saliva, and urine, but is not limited thereto. The measurement of the miRNA expression level consists of reverse transcription polymerase chain reaction, real-time polymerase chain reaction, northern blotting, and transcriptome analysis. It may be achieved through at least one selected from the group, but is not limited thereto.

According to another aspect of the present invention, a method for screening a therapeutic substance having a therapeutic effect or an inducing substance causing liver fibrosis or cirrhosis by using the miRNA of the present invention may be provided. This can be performed using the diagnostic composition according to an aspect of the present invention. Treatment of the test substance to be analyzed in the normal experimental group, and measuring the expression level of miRNA of the present invention before and after treatment in the test group treated with the test substance, and when the expression level after treatment decreases than before treatment of the test substance, causes liver fibrosis or cirrhosis. By judging as a substance, the inducing substance can be screened. Alternatively, if the test material to be analyzed is treated in the liver fibrosis test group, and the expression level of miRNA of the present invention is measured before and after treatment in the test material-treated test group, the expression level is increased compared to before treatment of the test material. By judging as a therapeutic substance having a therapeutic effect, selection of a therapeutic substance can be performed.

Hereinafter, preferred embodiments are presented to aid in understanding of the present invention, but these examples are only illustrative of the present invention and do not limit the scope of the appended claims, and examples within the scope and spirit of the present invention It is obvious to those skilled in the art that various changes and modifications are possible, and it is natural that such modifications and modifications fall within the appended claims.

Example

<Analyzing differences in miRNA according to differentiation of mesenchymal stem cells into hepatocytes in vitro>

Human Bone Marrow-derived Mesenchymal Stem Cells (hBM-MSCs, Catholic Master Cells, Catholic Cell Therapy Corporation, Korea) were used. Freeze-stored Catholic master cells (passage 2) were distributed and DMEM-LG medium (Dulbecco's modified Eagle medium low) supplemented with 20% fetal bovine serum (FBS; Gibco) and 1% antibiotic (Antibiotic antimycotic, Gibco). Glucose, Hyclone) in a 5% CO ₂ incubator at 37°C. In describing the examples in the present invention, the percentage may be understood as w/v%.

In order to differentiate the bone marrow-derived mesenchymal stem cells into hepatocytes, a two-step differentiation process was performed. Mesenchymal stem cells were treated with 60% Dulbecco's modified Eagle's medium-Low Glucose (DMEM-LG; Gibco) to which 10 ng/mL of bFGF (basic fibroblast growth factor, Peprotech) and 20 ng/mL of EGF (epidermal growth factor, Peprotech) were added. ) And 40% MCDB-201 MEDIUM (Sigma) were pre-cultured for 2 days in a mixed medium. After 2 days had elapsed, the cells were treated with 10 ng/mL of bFGF and 20 ng/mL of HGF (hepatocyte growth factor, Peprotech) in step 1, and cultured for an additional week. Then, in a second step, the cells were treated with 20 ng/mL of oncostatin M (OSM; Peprotech), 1 μmol/L of dexamethasone (DEXA; Peprotech) and 50 mg/mL of insulin-transferrin-selenium (ITS; Gibco). Hepatocyte differentiation was induced by culturing for an additional 2 weeks. The basic medium composition was 60% DMEM-LG, 40% MCDB201, 0% FBS and 1% Antibiotic antimycotic conditions. Only passage 5 or less was used for Catholic master cells.

hBM-MSCs, hBM-MSCs differentiated into hepatocytes, hepatocytes (Zenbio), liver tissue obtained from a normal person without liver disease (Invitrogen), liver tissue obtained from a person with liver fibrosis disease in stage 1, and a liver obtained from a person with liver fibrosis disease in stage 3 Each total RNA was isolated from the tissue and miRNA next generation sequencing (NGS) was performed. The liver tissue obtained from the above-described stage 1 and stage 3 liver fibrosis patients received written consent from the patient before participating in the trial, and was approved by the Institutional Review Board of Seoul St. Mary's Hospital. In addition, the stages of liver fibrosis were classified according to the definition of METAVIR liver fibrosis stage, and the degree of fibrosis was evaluated by histological analysis by a pathologist. Library protocol preparation was performed using the Illumina TruSeq small RNA preparation kit (Illumina, USA)/TruSeq Small RNA Library Prep Guide according to the manufacturer's protocol. Most mature miRNAs have 5-phosphate and 3-hydroxyl groups and are the result of the cellular pathway used to generate them. Because of this, the kit's Illumina adapters are directly linked to miRNAs. A reverse transcription reaction and short PCR were performed to obtain sufficient cDNA for sequence analysis, and a size of 145 bp to 160 bp was fractionated through polyacrylamide gel electrophoresis. Finally, an Agilent 2100 bio analyzer (Agilent) was used to evaluate the quality and band size of the library. The library was quantified by qPCR using the CFX96 real-time system (Biorad). After quantification, sequence analysis of the library prepared in the Nextseq 500 system (Illumina) with a 75 bp single end read was performed. The miRNA was compared and analyzed with the control using the RPM (Read Per Million) value.

In order to verify the expression of miR-101-3p and to confirm the differentiation of mesenchymal stem cells, the expression levels of liver-specific genes and mesenchymal markers were measured in quantitative real-time PCR (Quantitative real-time). It was confirmed by quantification through time PCR; qRT-PCR).

1 ml of TRIZOL reagent (Invitrogen) was added to the cultured cells, mixed, and allowed to stand at room temperature for 5 minutes. 0.2 ml of chloroform was added thereto, mixed well, left at room temperature for 2 to 3 minutes, centrifuged for 15 minutes at 4°C and 12,000 rpm, and the colorless supernatant was transferred to a new tube. 0.5 ml of isopropanol was mixed with the supernatant, left to stand at room temperature for 1 hour, and then centrifuged at 4° C. and 12,000 rpm for 10 minutes to precipitate RNA and remove the upper layer. 1 ml of 75% ethanol was well mixed with the precipitated RNA, and then centrifuged at 4° C. and 12,000 rpm for 10 minutes to remove the upper layer and dried at room temperature for 10 minutes. After dissolving the dried RNA by adding 20 µl of UltraPure DNase/Rnase free water (ambion), 1 µl of the mixture was taken and the absorbance was measured using a spectrometer.

For mRNA expression analysis, 1 μg of RNA was reverse transcribed using an RNA polymerase chain reaction kit (QuantiTect Reverse Transcription kit, QIAGEN), and then cDNAs were transferred to qRT using TaqMan probe (Applied Biosystems) and LightCycler480 master mix (Roche). -PCR (Roche, light cycler480) was performed. The expression levels of all genes were normalized to GAPDH. The probe used (mRNA qRT-PCR TaqMan probe) is shown in Table 1 below, the qRT-PCR reaction solution composition is shown in Table 2 below, and the qRT-PCR performance conditions are shown in Table 3 below. In Table 2 below, the content of DNase/Rnase free water (Roche) and cDNA was adjusted within the stated range for each gene to be amplified, and the total volume of the reaction solution was 20 μl. In Table 3 below, the reaction temperature is largely divided into three ranges of 95°C, Tm°C and 40°C, but Tm°C is set differently to 60°C for amplification and 72°C for dissolution. Denaturation, amplification, dissolution, and final reaction were carried out in the order of the above-described reaction temperature at the time and number of cycles shown in Table 3 below.

division Probe
(Probe) Gene ID
(Gene ID) Catalog number
(Catalog Number) Reference
(Reference) Control
(Loading control) hGAPDH 2597 #4331182 Hs02786624_g1 Liver specific gene 1:
hepatic phenotypes
(Hepatocyte) hAlb 213 #4331182 Hs00910225_m1 Liver specific gene 2:
hepatic phenotypes C/EBPβ 1051 #4331182 Hs002709237_s1 Liver specific gene 3:
hepatic phenotypes
(Hepatocyte precursor) hHNF4α 3172 #4331182 Hs00230853_m1 Mesenchymal marker
(Mesenchymal marker) hSnail1 6615 #4331182 Hs00195591_m1 Mesenchymal marker
(Mesenchymal marker) hEZH2 2146 #4331182 Hs00544830_m1 Mesenchymal marker hCDH2 1000 #4331182 Hs00983056_m1

PCR reaction solution volume LightCycler®480 Probes Master 2x 10µl Taqman mRNA Assays (20x) 1 μl DNase/Rnase free water (Roche) 5~7 µl cDNA 2~4 μl Total amount 20 μl

95 ℃ Tm ℃ 40 ℃ cycle denaturalization 10 minutes - - One Amplification 10 seconds 30 seconds (60 ℃) - 55 Dissolution - 1 second (72 ℃) - One final - - 30 seconds One

For miRNA expression analysis, 10 ng of RNA was reverse transcribed using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) according to the “TaqMan Micro Assays” protocol. cDNAs were subjected to qRT-PCR (Roche, light cycler480) using TaqMan probe (Applied Biosystems) and LightCycler480 master mix (Roche). U6 small nuclear RNA was used as a control gene, and the CT value of miRNA was expressed as the ratio divided by the CT value of U6 small nuclear RNA. The probe used (mRNA qRT-PCR TaqMan probe) is shown in Table 4 below, the qRT-PCR reaction solution composition is shown in Table 5 below, and the qRT-PCR performance conditions are shown in Table 6 below. The interpretation of Table 6 below is the same as that of Table 3.

division
(Assay name) Gene ID
(Gene ID) Catalog number
(Cat.No) miRBase ID U6 snRNA 001973 #4427975 - hsa-miR-101 002253 #4427975 hsa-miR-101-3p

PCR reaction solution volume LightCycler®480 Probes Master 2x 10µl Taqman MicroRNA Assays (20x) 1 μl DNase/Rnase free water (Roche) 7 μl cDNA 2 μl Total amount 20 μl

95 ℃ Tm ℃ 40 ℃ cycle denaturalization 10 minutes - - One Amplification 15 seconds 60 seconds (60 ℃) - 40 final - - 30 seconds One

Using the NGS analysis results for each experimental group, it was confirmed that among the increased miRNAs after they were differentiated into hepatocytes, miRNAs whose expression decreased as the stage of liver fibrosis progressed, among which, as shown in FIG. 2(A), inducing liver cell differentiation. It was confirmed that miR-101-3p increased fold change by more than 2 times. In Fig. 2(B), hepatocytes obtained from a normal person without liver disease in order from the left, hBM-MSCs differentiated into hepatocytes (hp BM-MSC), liver tissue obtained from a patient with liver fibrosis disease in stage 1 (Fibrosis1) And miR-101-3p expression in liver tissue (Fibrosis3) obtained from a patient with hepatic fibrosis in stage 3. Referring to FIG. 2(B), it can be seen that miR-101-3p is expressed at a high level in normal hepatocytes and differentiated hepatocytes, but its expression is reduced in hepatocytes undergoing fibrosis.

In order to confirm the expression level of miR-101-3p over time when inducing differentiation of mesenchymal stem cells into hepatocytes, bone marrow-derived mesenchymal stem cells are expressed every week while inducing differentiation into hepatocytes in the same manner as described above. The amount was confirmed. While confirming the expression level of miR-101-3p, the expression levels of genes specifically expressed in the liver (liver-specific genes) and mesenchymal markers were also confirmed, and the results are shown in FIG. 3. In FIG. 3, the expression levels of each miRNA, gene, and marker were expressed as relative expression levels over time after that based on the expression levels in bone marrow-derived mesenchymal stem cells before induction of differentiation.

Referring to FIGS. 3(A) and 3(B), it can be seen that the expression of liver-specific genes increased and the expression of mesenchymal markers decreased during induction of differentiation into hepatocytes. Referring to FIG. 3(C), it can be seen that the expression level of miR-101-3p gradually increases with the passage of time up to 3 weeks during the induction of differentiation into hepatocytes. In the case of differentiation by applying the method used for differentiation of mesenchymal stem cells into hepatocytes described above, considering that it is considered that differentiated hepatocytes can be obtained only after induction for a period of 3 weeks, miR-101-3p is It can be considered to be a liver-specific nucleotide polymer that has a significant relationship with differentiation into hepatocytes.

<Confirmation of the effect of inducing and promoting differentiation into hepatocytes by miR-101-3p>

To find out whether miR-101-3p affects the differentiation of mesenchymal stem cells into hepatocytes, it was confirmed by overexpressing miR-101-3p in human bone marrow-derived mesenchymal stem cells. miR-101-3p overexpression was performed by transformation, and overexpression was first quantified and confirmed through quantitative real-time PCR (qRT-PCR). Transformation was performed by transfection in a serum free medium containing no antibiotics using lipofectamine RNAiMAX. Mimics for human miRNA 101-3p (GenePharma, Cat. No. B02003) and GMR-miR microRNA mimics double-stranded negative control (GenePharma, Cat. No. B04002) were purchased and used from Bionics. TRIzol reagent for untransformed bone marrow-derived mesenchymal stem cells (BM-MSC), mimics negative control as a control, and bone marrow-derived mesenchymal stem cells transformed with miR-101-3p (miR-101-3p mimic), respectively. (Invitrogen) was used to isolate total RNA. Thereafter, cDNA was amplified by PCR using the following conditions to confirm the relative expression level of miR-101-3p for each group. 10 ng of RNA was reverse transcribed using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) according to the “TaqMan Micro Assays” protocol. cDNAs were subjected to qRT-PCR (Roche, light cycler480) using TaqMan probe (Applied Biosystems) and LightCycler480 master mix (Roche). U6 small nuclear RNA was used as a control gene, and the CT value of miRNA was expressed as the ratio divided by the CT value of U6 small nuclear RNA. The probes used and the qRT-PCR conditions were carried out in the same manner as described in Tables 4 to 6.

Quantification results using qRT-PCR are shown in Fig. 4(B). Referring to FIG. 4(B), it can be seen that miR-101-3p is overexpressed in an experimental group of mesenchymal stem cells transformed with miR-101-3p (miR-101-3p mimic).

While culturing miR-101-3p overexpressing mesenchymal stem cells in a serum free medium, the morphological appearance was observed, and the degree of differentiation from mesenchymal stem cells to hepatocytes was confirmed while confirming the expression level of hepatocyte differentiation markers. Bone marrow-derived mesenchymal stem cells (BM-MSC), mimics NC bone marrow-derived mesenchymal stem cells (mimic NC) and miR-101-3p overexpressing bone marrow-derived mesenchymal stem cells (miR-101-3p mimic) were After culturing in a serum free medium for 7 days, the degree of differentiation was confirmed through a change in shape and a change in the expression level of a marker, and the result of confirming the shape of the cultured cells with an optical microscope is shown in FIG. 4(A).

In Fig. 4(A), bone marrow-derived mesenchymal stem cells (BM-MSC), mimics NC bone marrow-derived mesenchymal stem cells (mimic NC), and miR-101-3p overexpression bone marrow-derived mesenchymal stem cells that were not induce differentiation in order from the left. Stem cells (miR-101-3p mimic) were cultured in serum-free medium for 7 days. Referring to FIG. 4(A), it can be seen that differentiation accompanied by a morphological change occurs in miR-101-3p overexpressing mesenchymal stem cells cultured for 7 days.

The expression levels of hepatocyte differentiation genes and mesenchymal markers were confirmed using qRT-PCR. The expression levels of hepatocyte differentiation markers ALB (albumin), HNF4α (Hepatocyte nuclear factor 4 alpha), HGF (hepatocyte growth factor) and CEBPB (CCAAT/enhancer binding protein beta) and mesenchymal marker SNAI1 (snail family transcriptional repressor 1) The expression levels were compared, and the results are shown in Figs. 4(C) and 4(D). During qRT-PCR, the reaction solution composition and performance conditions were the same as those in Tables 2 and 3, and the probes used (mRNA qRT-PCR TaqMan probe) are shown in Table 7 below.

division Probe
(Probe) Gene ID
(Gene ID) Catalog number
(Catalog Number) Reference
(Reference) Control (Loading control) hGAPDH 2597 #4331182 Hs02786624_g1 Hepatocyte differentiation gene 1:
hepatic phenotypes
(Hepatocyte) hAlb 213 #4331182 Hs00910225_m1 Hepatocyte differentiation gene 2:
hepatic phenotypes
(Hepatocyte precursor) hHNF4α 3172 #4331182 Hs00230853_m1 Hepatocyte differentiation gene 3:
hepatic phenotypes hHGF 3082 #4331182 Hs00300159_m1 Hepatocyte differentiation gene 4:
hepatic phenotypes C/EBPβ 1051 #4331182 Hs002709237_s1 Mesenchymal marker hSnail1 6615 #4331182 Hs00195591_m1

4(C) and 4(D), miR-101-3p overexpressing mesenchymal stem cells (miR-101-3p) are added with other cytokines necessary for inducing differentiation of hepatocytes or the above-described two-step differentiation induction. Even without changing the medium according to the method, the expression of the hepatocyte differentiation-specific gene remarkably increased and the expression of the mesenchymal marker decreased within 7 days, confirming that the differentiation into functional mature hepatocytes. In addition, it can be seen that the culture period required for differentiation into hepatocytes is significantly reduced from 21 days to 7 days. From this, it can be seen that miR-101-3p can not only induce differentiation into hepatocytes, but also shorten the time required for differentiation to promote differentiation.

It was confirmed that by using miR-101-3p when differentiating human mesenchymal stem cells into hepatocytes, hepatocytes can be obtained in a short time by a simple method compared to the prior art. If stem cells are not used as a cell therapy agent and are treated by inducing differentiation into similar hepatocytes in vitro, the complexity of the differentiation induction steps necessary to differentiate into hepatocytes when inducing differentiation using miR-101-3p Because this is reduced, the probability of cell contamination during the differentiation induction step can also be reduced. Through this, it is possible to shorten the period required for induction of differentiation and improve the stability of obtaining while reducing the cost.

<Confirmation of the effect of miR-101-3p on inhibiting liver fibrosis>

It was confirmed whether there was any change in the expression of miR-101-3p under liver fibrosis conditions. To confirm the expression of liver fibrosis, human-derived hepatic stellate cells LX2 (Sigma-Aldrich) were added to DMEM- supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% antibiotic (Antibiotic antimycotic, Gibco). HG medium (Dulbecco's modified Eagle medium High Glucose, Hyclone) was prepared by culturing in _{a 5% CO 2 incubator at 37°C.} LX2 cells were cultured in serum free medium for 24 hours, treated with TGF-β1 at a content of 2.5 ng/ml, and cultured for 24 hours to induce LX2 activation. Alpha smooth muscle actin (α-SMA) and COL1α1 (Collagen Type I Alpha 1) were selected as markers for confirming fibrosis in LX2 before and after activation, and expression together with miR-101-3p was confirmed. Expression was confirmed by performing qRT-PCR, and the results are shown in FIG. 5. During qRT-PCR, the reaction solution composition and performance conditions were the same as those in Tables 2 and 3, and the probes used (mRNA qRT-PCR TaqMan probe) are shown in Table 8 below.

division Probe
(Probe) Gene ID
(Gene ID) Catalog number
(Catalog Number) Reference
(Reference) Control (Loading control) hGAPDH 2597 #4331182 Hs02786624_g1 Fibrosis marker
(fibrosis marker) hα-SMA 59 #4331182 Hs00426835_g1 Fibrosis marker hCol1α1 1277 #4331182 Hs00164004_m1

Referring to FIG. 5(A), it can be seen that LX2 was activated by the addition of TGF-β1, and referring to FIG. 5(B), it was found that miR-101-3p expression was decreased in the activated hepatic stellate cells. .

In order to confirm the effect of miR-101-3p on liver fibrosis, miR-101-3p was overexpressed in LX2 cells, treated with TGF-β1 under the same conditions as described above, and cultured to induce LX2 activation. As controls, LX2 (con) that was not overexpressed and activated and LX2 (TGF-β1) that only induced activation without overexpressing were prepared, respectively.

For each experimental group and a control group, cDNA was amplified by PCR to confirm the relative expression level of miR-101-3p for each group. In addition, α-SMA, COL1α1, TGF-β1, EZH2 (Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit), ZEB1 (Zinc Finger E-Box Binding Homeobox 1), MCL-1 (Myeloid cell leukemia 1) and HGF (Hepatocyte Growth Factor) was confirmed through qRT-PCR. qRT-PCR was performed under the same conditions as described above, and the results are shown in FIG. 6. The probe used (mRNA qRT-PCR TaqMan probe) is shown in Table 9 below.

division Probe
(Probe) Gene ID
(Gene ID) Catalog number
(Catalog Number) Reference
(Reference) Control
(Loading control) hGAPDH 2597 #4331182 Hs02786624_g1 Fibrosis marker
(fibrosis marker) hα-SMA 59 #4331182 Hs00426835_g1 Fibrosis marker hCol1α1 1277 #4331182 Hs00164004_m1 Fibrosis marker
(fibrosis marker) hTGFβ1 7040 #4331182 Hs00998133_m1 Mesenchymal marker hEZH2 2146 #4331182 Hs00544830_m1 Mesenchymal marker hZEB1 6935 #4331182 Hs00232783_m1 Cell death inhibitor
(apoptosis inhibitor) hMCL1 4170 #4331182 Hs03043899_m1 Antifibrotic marker
(Anti-fibrosis marker) hHGF 3082 #4331182 Hs00300159_m1

Referring to FIG. 6, it can be seen that miR-101-3p is overexpressed in the LX2 (miR-101-3p, TGF-β1) experimental group transformed with miR-101-3p. In addition, when miR-101-3p was overexpressed, the expression of fibrosis, mesenchymal markers, and apoptosis inhibitors by activation was reduced, and the expression of HGF, an antifibrotic and cytoprotective factor, was increased. Through this, it can be confirmed that the activation of LX2 by TGF-β1 is inhibited by miR-101-3p.

Western blot was performed to confirm the effect of miR-101-3p on liver fibrosis at the protein level. Α-tubulin (sigma-aldrich) was used as an internal standard, α-SMA (sigma-aldrich), CDH2 (Cadherin 2m, BD bioscience), EZH2 (cell signaling technology), BCL -xL (B-cell lymphoma-extra large, cell signaling technology) and PCNA (Proliferating cell nuclear antigen, abcam) were used. Culture and activation of LX2 cells were prepared by performing the same conditions as described above, and proteins were extracted using PRO-PREP protein extraction buffer (iNtRON Biotechnology), and the same amount of each sample was added to 10% SDS-PAGE gel (gel). Electrophoresis was performed at. The protein of the gel after electrophoresis was transferred to a polyvinyldene Fluoride membrane (PVDF, Millipore) through an electric flow, and the PVDF membrane was blocked at room temperature for 1 hour with a TBS/T buffer containing 5% skim milk powder. After blocking was completed, the membrane to which the proteins were transferred was reacted with the primary antibody overnight at 4°C. After reacting with HRP-conjugated secondary antibody at room temperature for 1 hour, expression of each protein was detected using EZ-Western Lumi La kit (enhanced chemiluminesecent system, DoGen) and Las-4000 Plus (Fujifilm corporation). Concentration analysis was performed with Image Gauge 4.0 software. The results of confirming the expression are shown in FIG. 7. In FIG. 7, FIG. 7(B) is an illustration of the results of FIG. 7(A) being numerically converted into relative values.

Referring to FIG. 7, it was found that the expression of the fibrotic marker, mesenchymal marker, and apoptosis inhibitor was reduced when LX2 overexpressing miR-101-3p was activated, similar to the results of the previous qRT-PCR. Even at the protein level, it was found that the activation of LX2 by TGF-β1 was inhibited by miR-101-3p overexpression, and it was confirmed that miR-101-3p exhibited an effect of inhibiting fibrosis by inhibiting hepatic stellate cell activation.

<Confirmation of increased expression of miR-101-3p in a liver fibrosis model in which the therapeutic effect of stem cell transplantation was confirmed>

Prepare human bone marrow-derived mesenchymal stem cells (BM-MSCs) and human bone marrow-derived mesenchymal stem cells (BM-MSC ^PRL-1 ) with enhanced stem cell function by overexpressing PRL-1 that enhances stem cell function. Expressions of miR-101-3p, α-SMA, SNAI1, COL1A, E-cadherin, and ALB were confirmed by qRT-PCR in each transplanted bile duct ligation (BDL) rat model.

Seven-week-old male Sprague-Dawley rats (Orient Bio Inc., Seongnam, Korea) were maintained in an air-conditioned animal facility. Previously reported methods (MF Mahmoud, et al, European Journal of Pharmacology, vol. 742, pp. 118-124, 2014; and JH Jun, et al., Clinical and Molecular Hepatology, vol. 22, no. 3, pp.372-381, 2016), the common bile duct was ligated under general anesthesia using 75 mg/ml avertin (Avertin, 2,2,2 tribromoethanol, Sigma-Aldrich). After 10 days of bile duct ligation (BDL), BM-MSCs (2×10 ⁶ cells, passages 8-10) or BM-MSC ^PRL-1 were injected intravenously through the tail vein of the transplant group. The number of BM-MSCs is determined by conventional dose-determining tests (MJ Lee et al., Journal of Cellular Biochemistry, vol. 111, no. 6, pp. 1453-1463, 2010; J. Jung, et al., International Journal of Stem Cells, vol. 8, no. 1, pp. 79-89, 2015). Liver tissue and blood samples were collected at 1, 2, 3, and 5 weeks after transplantation in the transplant group (Tx), and at 1, 2, 3, and 5 weeks after BDL in the non-transplant group (NTx). The experimental protocol was approved by the Institutional Animal Care and Use Committee of CHA University. Rat liver tissue was homogenized and lysed, and total RNA was isolated using TRIzol reagent (Invitrogen). Reverse transcription was performed using 500 ng of total RNA and Superscript III reverse transcriptase (Invitrogen). Real-time polymerase chain reaction (PCR) was performed using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Thereafter, cDNA was amplified by PCR using the following conditions: 40 cycles at 95°C for 5 minutes, 95°C for 5 minutes and 60°C for 30 minutes. The primer sequence, reaction solution composition, and amplification method used when performing PCR are shown in Tables 10, 11, and 12, respectively. In Table 11, DEPC means diethylpyrocarbonate. GAPDH was used as an internal control for normalization, and the results of confirming the expression in rats are shown in FIGS. 8 and 9. 8 shows the expression levels of α-SMA, SNAI1, COL1A, E-cadherin and ALB, and FIG. 9 shows the expression levels of miR-101-3p. 8 and 9, samples obtained from non-BDL normal rats were used as controls (N or con).

gene Primer classification Primer sequence (5'-3') Junction temperature(℃) a-SMA F SEQ ID NO: 3 60 R SEQ ID NO: 4 Snail1 F SEQ ID NO: 5 55 R SEQ ID NO: 6 Collagen type 1 F SEQ ID NO: 7 60 R SEQ ID NO: 8 E-cadherin F SEQ ID NO: 9 60 R SEQ ID NO: 10 Albumin F SEQ ID NO: 11 55 R SEQ ID NO: 12 GAPDH F SEQ ID NO: 13 60 R SEQ ID NO: 14

PCR reaction solution volume cDNA 5 μl Forward primer 0.5 μl Reverse primer 0.5 μl Enzyme (FasStart universal SYBR Green master mix) 12.5 μl DEPC-treated water* 6.5 μl Total amount 25 μl

95 ℃ Tm ℃ 20 ℃ cycle denaturalization 10 minutes One Amplification 10 seconds 30 seconds (55 ℃) 40 Dissolution 1 second (60-94 ℃)(melt curve 65-95℃, increasing 1℃) One final 1 minute One

In the present invention, all tests were performed twice or three times, and the data are expressed as mean ± standard deviation. For comparison between groups, Student's t-tests were performed, and P<0.05 was measured by multiple comparison as a significance level. Statistical analysis was performed using PASW version 22.0 (SPSS Inc., Chicago, IL, USA).

8, the expression of fibrosis-related genes and mesenchymal markers decreased, the expression of liver function-related markers was increased in the experimental group in which stem cells were transplanted, and these effects were more pronounced in the experimental group in which PRL-1 overexpressing stem cells were transplanted. It has been shown to be enhanced. Through this, it can be seen that the transplantation of stem cells shows an effect on the treatment of liver fibrosis, and when PRL-1 is overexpressed, an improved therapeutic effect is shown.

Referring to FIG. 9, the expression of miR-101-3p was increased 2 and 3 weeks after transplantation in the experimental group in which stem cells were transplanted, and miR-101 in the experimental group in which stem cells enhanced by PRL-1 were transplanted. It was found that the expression of -3p was significantly increased. Through this, it can be confirmed that miR-101-3p is suitable to be applied as a therapeutic effect monitoring biomarker capable of predicting the effect of liver fibrosis treatment, including the treatment of liver diseases using stem cells.

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> COMPOSITION USING MIRNA-101 FOR DIFFERNTIATION OF MESENCHYMAL STEM CELL INTO HEPATOCYTE-LIKE CELL AND COMPOSITION USING MIRNA-101 FOR DIAGNOSIS, PREVENTION OR TREATMENT OF HEPATIC FIBROSIS <130> NP-2020-0068 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> RNA <213> Homo sapiens <400> 1 uacaguacug ugauaacuga a 21 <210> 2 <211> 75 <212> RNA <213> Homo sapiens <400> 2 ugcccuggcu caguuaucac agugcugaug cugucuauuc uaaagguaca guacugugau 60 aacugaagga uggca 75 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> a-SMA F primer <400> 3 aactggtatt gtgctggact ctg 23 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> a-SMA R primer <400> 4 ctcagcagta gtcacgaagg aata 24 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Snail1 F primer <400> 5 gaagcccaac tatagcgagc 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Snail1 R primer <400> 6 agagtcccag atgagggtg 19 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Collagen type 1 F primer <400> 7 catgttcagc tttgtggacc t 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Collagen type 1 R primer <400> 8 gcagctgact tcagggatgt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> E-cadherin F primer <400> 9 ccaccagatg acgatacccg 20 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> E-cadherin R primer <400> 10 gaatcacttc cgtctggca 19 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Albumin F primer <400> 11 cttcaagcct gggcagtag 19 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Albumin R primer <400> 12 gcactggctt atcacagcaa 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH F primer <400> 13 tccctcaaga ttgtcagcaa 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH R primer <400> 14 agatccacaa cggatacatt 20

Claims (20)

A composition for inducing differentiation from stem cells into hepatocytes containing miR-101-3p as an active ingredient.
The method according to claim 1,
The miR-101-3p is composed of the nucleotide sequence of SEQ ID NO: 1, a composition for inducing differentiation from stem cells into hepatocytes.
The method according to claim 1,
The miR-101-3p is a form including at least partially a phosphorothiolate structure; DNA, peptide nucleic acids (PNA), or locked nucleic acid (LNA) molecules instead of RNA, at least partially substituted; And at least one form selected from the group consisting of a form in which the 2'hydroxyl group of the RNA sugar is substituted with another functional group, a composition for inducing differentiation from stem cells into hepatocytes.
The method according to claim 1,
The miR-101-3p is a miRNA precursor form; Mature miRNA form; Plasmid miRNA precursor form; And at least one form selected from the group consisting of a form introduced into a virus, a composition for inducing differentiation from stem cells into hepatocytes.
The method according to claim 1,
The stem cells are mesenchymal stem cells, a composition for inducing differentiation from stem cells into hepatocytes.
The method of claim 5,
The mesenchymal stem cells are derived from bone marrow, a composition for inducing differentiation from stem cells into hepatocytes.
A method for differentiation from stem cells into hepatocytes comprising the step of treating the composition according to any one of claims 1 to 6 to stem cells in mammals other than humans or in vitro.
The method of claim 7,
The treatment is performed by culturing the stem cells in a medium containing the composition or by introducing the composition into the stem cells.
The method of claim 8,
A method for differentiation from stem cells into hepatocytes, wherein miR-101-3p is overexpressed in the stem cells by the introduction.
The composition for inducing differentiation from stem cells into hepatocytes according to any one of claims 1 to 6, and a composition for preventing or treating liver disease, containing mesenchymal stem cells as an active ingredient.
The method of claim 10,
The liver disease is a composition for preventing or treating liver disease, including liver fibrosis or cirrhosis.
The method of claim 10,
A composition for preventing or treating liver disease, in the form of a unit dosage ampoule or injection.
delete delete The method of claim 11,
A composition for preventing or treating liver disease, which inhibits the activation of hepatic stellate cells. delete delete delete delete delete

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