WO2015088162A1 - Composition for promoting differentiation from stem cell to chondrocyte - Google Patents

Abstract

The present invention relates to a composition for promoting differentiation from stem cells, more specifically mesenchymal stem cells to chondrocytes, a composition for prevention or treatment of osteochondral injury disease, and a method for screening the same. The present invention newly identified miR-495 as miRNA to inhibit differentiation from stem cells to chondrocytes and it was confirmed that pluripotent stem cells can be effectively differentiated to chondrocytes by the inhibition of miR-495. The present invention can be used usefully for essential prevention or treatment of diseases associated with osteochondral injury as well as osteoarthritis through the reproduction of the lost cartilage tissue.

Description

 【Specification】

 [Name of invention]

 Composition for promoting differentiation from stem cells to chondrocytes

 This patent application claims priority to Korean Patent Application No. 10-2013-0155690 filed with the Korean Patent Office on December 13, 2013, the disclosure of which is hereby incorporated by reference.

 The present invention relates to a composition for promoting differentiation from stem cells to chondrocytes comprising an inhibitor of microRNA.

[Technique to become background of invention]

 Stem cells are cells that can differentiate the organisms that make up the tissue into a variety of cells. These stem cells are used to differentiate the undifferentiated cells of the embryonic and prenatal stages. Collectively. Stem cells are differentiated into specific cells by differentiation stimulation (environment), and unlike the differentiated cells in which cell division is stopped, they can proliferate because they can produce the same cells as themselves by cell division (proli ferat). It has the property of ion expansion, and can be differentiated into other cells by different environment or differentiation stimulus, so it has plasticity in differentiation.

Stem cells are largely derived from embryos and have itipotent embryonic stem cells (ES cells) with the potential to differentiate into all cells and multipotents obtained from each tissue ( It is divided into adult stem cells of mult ipotency. Embryonic stem cells are undifferentiated cells capable of unlimited proliferation and can be differentiated into all cells. Unlike adult stem cells, embryonic stem cells can be generated and can be inherited to the next generation. Despite these advantages, however, there are many difficulties in using embryonic stem cells as cell therapeutics, such as cancer formation, immune rejection, and ethical and legal constraints. Recently, mesenchymal stem cells have been proposed as an alternative to overcome these problems. Mesenchyme Stem cells are pluripotent cells capable of differentiation into adipocytes, bone cells, chondrocytes, muscle cells, neurons, and cardiomyocytes, and have been reported to have a function of regulating immune response.

 Human bone marrow-derived mesenchymal stem cells have the ability to differentiate into various tissues, such as adipocytes, chondrocytes, and bone cells, depending on the culture conditions. It proceeds through several stages of stem cells, each of which is driven by a variety of factors and their interactions, but the exact route has not yet been identified. Recently, micro RNA (m i croRNA) has been discovered as a substance for controlling cartilage differentiation and has been studied in depth. mi croRNA regulates target genes by digesting the target mRNA with a small NA of 20-24 nucleotides or by inhibiting post-transcriptional processes. Many studies have reported that mi croRNA plays an important role in various biological processes including cell proliferation, death and differentiation.

 Arthritis is a multiple disease with the highest prevalence among human diseases. In particular, degenerative arthritis is a representative senile disease that nearly 100% of people over 65 years of age are 70-80%. At present, more than 10% of the population in Korea suffers from degenerative arthritis, and the prevalence is increasing very rapidly due to the rapid aging of Korean society. It is also estimated that there are more than 500 million patients with degenerative arthritis worldwide, and as the duration of social activities increases along with the average life expectancy of humans, degenerative arthritis needs to be overcome in terms of improving the quality of human life. It is emerging as a disease.

The cartilaginous tissue, which is a structural complete layer, is surrounded by a membrane at the end of the joint within the joint. It prevents pain and bone abrasion that may occur when the bones directly contact each other. Chondrocytes are the only cellular components in cartilage tissues, which are responsible for synthesizing and secreting collagen and proteoglycan matrices, which are essential for the normal function of cartilage tissues, and decomposing them at appropriate rates, thereby maintaining functional homeostasis of articular cartilage tissue It plays an essential role in giving. Therefore, maintaining the activity of these chondrocytes is directly related to the structural functional preservation of joint tissue. In order for the function of chondrocytes in the chondrocytes to function normally, the chondrocytes should be properly formed and differentiated, the survival of the chondrocytes generated should be well protected, and the calcification of the chondrocytes existing is continuously suppressed. Hardening should be prevented. The most common cause of degenerative arthritis is the loss of the intrinsic functionality of these articular chondrocytes with age.

 In the present invention, the identification of mi croRNA involved in the cartilage differentiation process of human stem cells, and by regulating its expression to induce differentiation specificity of stem cells into cartilage, regenerates the cartilage tissue eventually lost, diseases such as degenerative arthritis It was intended to be applied to cell therapy of. Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

[Content of invention]

 Problem to be solved

 The present inventors made diligent research efforts to discover factors that specifically differentiate stem cells having mult i potency into chondrocytes. As a result, micro RNA-495 (mi R-495) plays a role in inhibiting cartilage differentiation of human stem cells, and when the expression thereof is inhibited, specific differentiation into chondrocytes is induced to treat cartilage tissue through regeneration. The present invention has been completed by discovering that the present invention can be usefully applied to diseases such as degenerative arthritis.

 Accordingly, an object of the present invention is to provide a composition for promoting differentiation from stem cells to chondrocytes.

 Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of cartilage damage disease.

Still another object of the present invention is to provide a method for screening a composition for promoting differentiation from stem cells to chondrocytes. Still another object of the present invention is to provide a method for screening a pharmaceutical composition for preventing or treating cartilage damage disease. Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

[Measures of problem]

 According to one aspect of the present invention, there is provided a composition for promoting differentiation from stem cells to chondrocytes comprising a nucleic acid molecule that inhibits the expression of micro R A-495. As an active ingredient.

 The present inventors have made diligent research efforts to discover factors that specifically differentiate stem cells with iiiultipotency into chondrocytes. As a result, micro RNA-495 (niiR-495) plays a role in inhibiting cartilage differentiation of human stem cells, and when inhibiting its expression, specific differentiation into chondrocytes is induced and can be treated through regeneration of cartilage tissue. It has been found that it can be usefully applied to diseases such as degenerative arthritis.

 As used herein, the term “stem cell” refers to a cell capable of differentiating into various cells constituting biological tissue, and refers to undifferentiated cells that can be reproduced without limitation to form specialized cells of tissues and organs. . Stem cells are developable pluripotent or pluripotent cells. Stem cells can divide into two daughter stem cells, or one daughter stem cell and one derived (trans) cell, and then proliferate into mature, fully formed cells of the tissue. Specifically, stem cells capable of promoting differentiation into chondrocytes with the composition of the present invention are mesenchymal stem cells. More specifically, the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells.

As used herein, the term "mesenchymal stem cell" refers to a stem cell having a multipotency capable of differentiation into adipocytes, bone cells, chondrocytes and muscle cells, neurons, and cardiomyocytes. Mesoderm stem cells are identified by their swirling morphology and expression of the basic cell surface markers CD73 (+), CD105 (+), CD34 (-), and CD45 (-). As used herein, the term “nucleic acid molecule” is meant to encompass DNMgDNA and cDNA) and RNA molecules inclusively, and the nucleotides that are the basic structural units in nucleic acid molecules are naturally occurring nucleotides, as well as analogs in which sugar or base sites are modified. also included (analogue) (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

As used herein, the term “nucleic acid molecule that inhibits expression” refers to any nucleic acid-based molecule that has a complementary sequence to the target miRNA, ie, the sequence of miR-495, and can form a duplex with niiRNA. Include. Thus, the term "nucleic acid molecule that inhibits expression" may also be expressed as "complementary nucleic acid-based inhibitor ^' '.

 According to a specific embodiment of the present invention, the nucleic acid molecule used in the present invention is siRNA, shRNA, miRNA, ribozyme (r ibozyme), peptide nucleic acids (PNA) or antisense oligonucleotides.

As used herein, the term "s ^' iRNA" refers to a short double-chain NA that can induce RNAKRNA interference through cleavage of specific mRNAs. It consists of a sense RNA strand having a sequence homologous to the mRNA of the other gene and an antisense RNA strand having a complementary sequence. siRNA is provided as an efficient gene knockdown method or gene therapy because it can suppress the expression of the target gene. siRNAs are not limited to fully paired double-stranded RNA portions of RNA, but are paired by mismatches (the corresponding bases are not complementary), bulges (there are no bases corresponding to one chain), etc. May be included. The total length is 10 to 100 bases, preferably 15 to 80 bases, most preferably 20 to 70 bases. The siRNA terminal structure can be either smooth or cohesive, as long as the expression of the target gene can be suppressed by the RNAi effect. The cohesive end structure is possible in both a three-terminal protruding structure and a five-terminal protruding structure. The number of protruding bases is not limited. For example, the number of bases may be 1 to 8 bases, preferably 2 to 6 bases. In addition, siRNA is, for example, one side to the extent that can maintain the expression inhibitory effect of the target gene The protruding portion of the terminal may include low molecular RNA (eg, natural RNA molecules such as tRNA, rRNA, viral RNA or artificial RNA molecules). The siRNA terminal structure does not need to have a cleavage structure at both sides, and may be a stem loop type structure in which one terminal portion of the double chain RNA is connected by a linker RNA. The length of the linker is not particularly limited as long as it does not interfere with pairing of stem portions.

 As used herein, the term "shRNA" refers to a nucleotide composed of 50-70 single strands, and forms a stem-loop structure in vivo. Long R A of 19-29 nucleotides complementary to both loop regions of 5-10 nucleotides form base pairs to form a double stranded stem.

As used herein, the term miRNA (micro NA) ^" modulates gene expression and includes full length 20-50 nucleotides, preferably 20-45 nucleotides, more preferably 20-40 nucleotides, even more preferably 20- Refers to a single stranded RNA molecule consisting of 30 nucleotides, most preferably 21-23 nucleotides miRNAs are oligonucleotides that are not expressed intracellularly and have a short stem-loop structure. homologous (messenger RNA) to the whole or in part, and by complementary binding to the mRNA inhibits the target gene expression.

 As used herein, the term "ribozyme" refers to an RNA molecule having a function such as an enzyme that recognizes and cleaves itself by a specific sequence of a base as a kind of RNA. Ribozyme is a complementary nucleotide sequence of a target messenger RNA strand consisting of a region that binds with specificity and a region that cleaves the target RNA.

As used herein, the term "PNA (Peptide nucleic acid)" is a molecule having both nucleic acid and protein properties, and means a molecule capable of complementarily binding to DNA or RNA. PNA was first reported in 1999 as analogous DNA with nucleobases linked by peptide bonds (Nielsen PE, Egholm M, Berg RH, Buchardt 0, “Sequence-select ive recognition of DNA by strand displacement with a thymine -subst i tuted polyamide ", Science 1991, Vol. 254: ppl497-1500]). PNA is not found in nature Artificially synthesized by chemical method. PNAs hybridize with native nucleic acids of complementary base sequences to form double strands. PNA / DNA double strands are more stable than DNA / DNA double strands and PNA / RNA double strands are more stable than DNA / RNA double strands. It is most commonly used as a peptide basic skeleton that N- (2-aminoethyl) glycine is repeatedly linked by amide bonds. In this case, the backbone of the peptide nucleic acid is electrically different from that of the negatively charged natural nucleic acid. As neutral. The four nucleic acid bases present in the PNA have approximately the same spatial size and distance between the nucleic acid bases as for natural nucleic acids. PNA is not only chemically stable than natural nucleic acids, but also biologically stable because it is not degraded by nucleases or proteases.

 As used herein, the term "antisense oligonucleotide" refers to DNA or RNA or derivatives thereof that contain a nucleotide sequence complementary to a sequence of a particular mRNA, which binds to a complementary sequence within the mRNA and translates the mRNA into a protein. Antisense nucleotide sequence of the present invention refers to a DNA or RNA sequence complementary to the mRNA of the target gene and capable of binding to the mWA of the target gene, and the translation of the target gene into mRNA and translocation into the cytoplasm. cation), maturation ions or any other essential biological activity can be inhibited. The antisense oligonucleotides are 6 to 100 bases in length, preferably 10 to 40 bases.

The antisense oligonucleotides can be modified at the position of one or more bases, sugars or backbones to enhance efficacy (De Mesmaeker et al., Curr Op in Struct Biol., 5 (3): 343-55, 1995). Oligonucleotide backbones can be modified with phosphorothioates, phosphoesters, methyl phosphonates, short chain alkyls, cycloalkyl short chain heteroatomics, heterocyclic intersaccharide bonds, and the like. In addition, antisense nucleic acids may include one or more substituted sugar moieties. Antisense oligonucleotides may comprise modified bases. Modified bases include hypoxanthine, 6-methyladenine, 5-methyl pyrimidine (particularly 5 ′ methylcytosine), 5-hydroxymethylcytosine (HMC). Glycosyl HMC, gentobiosil HMC, 2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminonucleosil) adenine, 2, 6-diaminopurine and the like.

 According to a more specific embodiment of the present invention, the nucleic acid molecule used in the present invention is an antisense oligonucleotide.

 According to a specific embodiment of the present invention, the antisense oligonucleotides used in the present invention have a length of 15 to 40 nucleotides.

 More specifically, the .1 sense oligonucleotide comprises a sequence complementary to the 15th to 21st nucleotide sequence of SEQ ID NO: 1.

 Most specifically, the antisense oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 2.

 According to a specific embodiment of the present invention, the composition of the present invention increases the expression of Sox9 in stem cells.

 According to the present invention, mi R-495 inhibits the expression of Sox9, which is an important transcription factor in the whole process of cartilage differentiation, and the composition of the present invention which inhibits mi R-495 inhibits the expression of Sox9 in stem cells. To promote differentiation into chondrocytes.

 According to another aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of cartilage damage diseases comprising the composition of the present invention as an active ingredient.

 According to a specific embodiment of the present invention, the cartilage injury disease prevented or treated with the composition of the present invention is degenerative arthritis.

As used herein, the term "ost eoar thr itis" means a disease in which joint tissue necessary for joint movement is damaged due to quantitative loss of cartilage tissue. According to the present invention, the composition of the present invention promotes regeneration of cartilage tissue in the joint by inducing specific differentiation of endogenous stem cells or transplanted therapeutic stem cells into chondrocytes. Thus, a symptomatic approach, such as conventional inflammation control, results in an inflammation of the joint tissue caused by abnormal immune function. In contrast to the treatment of disrupted rheumatoid arthritis, the compositions of the present invention provide a fundamental treatment for degenerative arthritis. According to another aspect of the invention, the invention provides a method for screening a composition for promoting differentiation from stem cells to chondrocytes comprising the following steps:

 (a) contacting a sample to be analyzed with stem cells; And

(b) measuring the expression level of the nucleotide of the sequence moktok first sequence in the stem cells. When the expression level of the nucleotide is reduced, the sample is determined as a composition for promoting differentiation from stem cells to chondrocytes. According to the present invention, the sequence listing first sequence is a nucleotide sequence of _m iR-495.

 According to the method of the present invention, first, a test substance is contacted with undifferentiated cells. Specifically, the undifferentiated stem cells are mesenchymal stem cells. As used to refer to the screening method of the present invention, the term "test material" refers to an unknown material used in screening to examine whether it affects the expression level of miR-495. Such test defects include, but are not limited to, chemicals, nucleotides, antisense-RNAs, small interference RNAs (siRNAs), and natural extracts. Next, the expression level of iiiiR-495 is measured in the cells treated with the sample. The measurement of the expression level can be made through various methods known in the art, for example, can be measured through a micro array or the like. As a result of the measurement, when the expression of niiR-495 is suppressed, the test substance may be determined as a composition for promoting differentiation into chondrocytes. According to another aspect of the present invention, the present invention provides a method for screening a composition for preventing or treating cartilage damage disease comprising the following steps:

 (a) contacting a sample to be analyzed with stem cells; And

(b) measuring the expression level of the nucleotides of the first sequence of the sequence listing in the stem cells, and when the expression level of the nucleotides decreases, the sample is determined as a composition for preventing or treating cartilage damage diseases. Stem cells, nucleotides, and specific screening methods and procedures used in the present invention have already been described above, so the description thereof is omitted to avoid excessive duplication. Substances discovered through the expression changes of mi R-495 using the method of the present invention promote differentiation of endogenous stem cells or transplanted therapeutic stem cells into chondrocytes, resulting in degeneration of cartilage tissues. It can be used as a therapeutic composition, such as arthritis. According to another aspect of the present invention, the present invention provides a method for promoting differentiation into chondrocytes comprising contacting stem cells with a composition comprising a nucleic acid molecule that inhibits the expression of microRNA-495 as an active ingredient.

 The method for promoting differentiation into chondrocytes of the present invention is achieved by contacting stem cells with a composition comprising a nucleic acid molecule that inhibits the expression of microRNA-495 as an active ingredient, which is another aspect of the present invention. The content will be omitted in order to avoid excessive complexity of the description. According to another aspect of the present invention, the present invention provides a method for preventing or treating cartilage damage disease comprising administering to a subject (subj ec t) a composition comprising a nucleic acid molecule that inhibits the expression of microRNA-495 as an active ingredient. Provide a method.

 In one embodiment of the invention, the cartilage damage disease is degenerative arthritis.

 The prophylactic or therapeutic method of the present invention is performed by administering to a subject a composition comprising a nucleic acid molecule that inhibits the expression of microRNA-495 as another active aspect of the present invention. Omitted to avoid excessive complexity of the description.

[effect】

The features and advantages of the present invention are summarized as follows: (a) The present invention provides a composition for promoting differentiation of stem cells, specifically, mesenchymal stem cells into cartilage cells, a composition for preventing or treating cartilage damage diseases and a screening method thereof.

 (b) The present invention newly identified miR-495 as a miRNA that inhibits the differentiation of stem cells into chondrocytes, and it was confirmed that the stem cells having pluripotency can be efficiently differentiated into chondrocytes through the inhibition thereof. .

 (c) The present invention can be usefully used for the fundamental prevention and treatment of cartilage damage-related diseases including degenerative arthritis through regeneration of lost cartilage tissue.

[Brief Description of Drawings]

1 is a diagram showing the expression patterns of Sox9 and miRNA during chondrocyte differentiation. La is a diagram showing that the mRNA expression of Sox9 gradually increases as the mesenchymal stem cells differentiate into cartilage. FIG. Lb is a diagram showing the microRNAs showing a difference of 1.5 times or more from the control group by microR A microarray analysis using a one-way AN0VA as a hierarchical clustering map ( ^D H).

 Figure 2 shows the expression patterns of miR-495 and niiR-431 during chondrocyte differentiation. miR-495 and miR-431 were expected to bind to Sox9 3 ′ UTR (FIG. 2A) and confirmed by real-time PCR that expression of miR-495 and miR-431 decreased during cartilage differentiation of mesenchymal stem cells. (FIG. 2B).

 FIG. 3 shows the results of miR-495 at the mRNA (FIG. 3A) and protein (FIG. 3B) levels, respectively, of the effect of miR-495 on Sox9 expression in human chondromacoma cell SW1353. FIG.

4 is a diagram showing the results of confirming that miR-495 specifically binds to the 3 'UTR of Sox9. Figure 4a shows the 3'UTR site of Sox9 to which miR-495 binds. 4B is a schematic diagram of a vector structure including Sox9 3'UTR. Figure 4c is a diagram showing the results of performing a luciferase analysis after the vector containing the miR-495 and Sox9 3'UTR introduced into the cell. Figure 4d shows the results of performing luciferase analysis after mutating the 15-21st nucleotide sequence that miR-495 can bind to Sox9 3 ^' UTR The figure shows that the 15-21st sequence of miR-495 is important for regulating Sox9 expression.

 Figure 5 is treated with miR-495 in mesenchymal stem cells after increasing the expression of Sox9 using TGF-I 3 and then quantification of Western blotting (Fig. 5a) and Western blotting results of the expression of Sox9, respectively Figure 5b shows the results of the investigation through.

 6 is a diagram showing the effect of miR-495 on the difference in the expression of chondrocyte markers. After treating miR-sc and miR-495 to mesenchymal stem cells, induce cartilage differentiation with TGF-p3, and then niRNAs of Col2 (Fig. 6a) and Aggrecan (Fig. 6b), which are the major markers of cartilage differentiation, by real time PCR Each was measured and the expression of Col2 (FIG. 6c) and aggrecan (FIG. 6d) protein was observed using immunofluorescence staining, respectively.

7 is a diagram showing the effect of ^' overexpression of miR-495 on the differentiation of human mesenchymal stem cells into cartilage. Figure 7a is a diagram showing the results of measuring the GAG (glycosaminoglycan) on the 10th day after induction of cartilage differentiation after treating miR-sc and miR-495 to mesenchymal stem cells. Figure 7b is a diagram showing the results of safranin ◦ staining of miR- sc and miR-495 treatment group.

[Specific contents to carry out invention]

 Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. . Example

Experiment method

Culture of Bone Marrow-Derived Human Mesenchymal Stem Cells (hMSCs) and Cartilage Sarcoma Cell Lines

BM aspirates were obtained from the posterior iliac crest of 12 adult donors aged 19-63 years with the approval of the Inst itut ional Review Board (IRB). Human bone marrow-derived mesenchymal stem cells (hMSCs) were selected based on their ability to adsorb to plastic cell culture flasks. BM aspirates were 10% FBSCGibco, Grand Island, NY, USA) and 1% Incubated for 7 days in DMEM-LG supplemented with antibiotic-antifungal solution (Gibco), low-glucose Dulbecco's modified Eagle's medium; Wei gene, Daegu, Korea. SW1353, a human chondrosarcoma cell line, was cultured in DMEM-HG (DMEM-high glucose, Welgene) supplemented with 10% FBS and 1% antibiotic-antifungal solution.

 ·

In vitro cartilage differentiation of SC using micromass culture method

Micromass culture methods were used to differentiate hMSCs into cartilage in vitro. Trypsinized hMSCs were resuspended in lMEMO ⁷ cells / inL concentration in DMEM-LG containing 10% FBS, of which only 1 μL was added to each well of 24-well pleated (lxl0 ⁵ cells per well). Dotting was done. Cells were allowed to adsorb for two hours at 37 ^° C., followed by 1% antibiotic-antifungal solution, 1% insulin transferrin selenium-ACITS; Cartilage differentiation medium consisting of DMEM-HG supplemented with Invitrogen), 50 y / mL ascorbic acid (Invitrogen) and 10 ng / mL TGF ^- P3 (R & D systems, Minneapolis, MN, USA) was added. Cartilage differentiation cultures containing 10 ng / mL TGF-3 were replaced every two days during the in vitro differentiation period. Quantitative Real-Time PCR

Species RNA was isolated from hMSCs and SW1353 cells using RNA iso Plus (Takara, Shiga, Japan) reagents according to the manufacturer's instructions. Total RNA was reverse transcribed using Omniscript kits (Qiagen, Venlo, Nether lands) for cDNA synthesis. The obtained cDNA was subjected to real-time PCR using SYBR® Green PCR Master Mix (Applied Biosystems; ABI, Carlsbad, CA, USA). Real time PCR was performed using an AB 17500 real time instrument (Applied Biosystems, ABI, Carlsbad, CA). The primer set was purchased from Bioneer (Dae j eon, South Korea), and the primers used were as follows: GAPDH (P267613), Sox9 (P232240). There were no proven primers for Col2al and aggr'ecnan ^' designed as follows: Col2al, 5'-GTCCTCTCCCAAGTCCACACAG-3' (sense) and 5 ¹ -GGGCACGAAGGCTCATCATTC-3 '(antisense); aggrecai 5'-CCACTGTTACCGCCACTT— 3 '(sense) and 5' GTAGTCTTGGGCATTGTTGT 3 '(antisense). PCR process initiated 30 sec at 95 ^° C After that, 40 cycles of heat cycle for 5 seconds at 95 ° C., followed by 20 seconds at 60 ° C. SYBR fluorescence was detected in the annealing / extension step, and all real time PCR products finally had a size of about 100 bp. The measurements of each sample were normalized to the internal control GAPDH.

Mir-X ^TM microRNA First-Strand for precursor-microRNA reverse transcription

Synthesis kit (Clontech, Mountain View, CA, USA) was used according to the manufacturer's instructions, and cDNA synthesized from _ni i _cro RNA was quantified by SYBR® qRT-PCR (Clontech). Specific Maturity—A list of primer sets used for microRNA amplification was obtained from Genolution (Genolu ion, Seoul, South Korea) and Bioneer Inc. Primers designed were as follows: 7: U6 snRNA, 5'-CTCGCTTCGGCAGCACA-3 '(sense) and 5'-AACGCTTCACGAATTTGCGT-3'(antisense); has-miR-495, 5'-AAACAAACATGGTGCACTTCTT-3 '(sense); has-miR-431, 5 *-TGTCTTGCAGGCCGTCATGCA-3 '(sense). Real time PCR was performed using AB 17500 real time instrument. After the PCR process was started in 10 seconds at 95 ° C, 40 cycles of 5 seconds at 95T: was performed 40 times, and 20 seconds at 60 ° C. U6 snRNACsmal 1 nuclear RNA) was used to standardize the relative expression of all precursor-microRNAs.

MicroRNA Array Analysis

Total RNA of undifferentiated hMSCs (0-10 days culture) and chondrogenic hMSCs derived from the same donor were isolated using RNA iso Plus (Takara) according to the manufacturer's instructions. The overall quality of total RNA was confirmed using a spectrophotometer. To 1.0 Genechip® miRNA array (Mfymetrix, Santa Clara, CA, USA) was ^{investigated,} the expression pattern of each microRNA. Pearson correlation was performed to identify microRNAs specifically expressed in cartilage differentiation-induced mesenchymal stem cells. Briefly, differently expressed genes were selected using routine equations subtracting control values from differentiated groups: applying a strict cut-off reference point with at least 1.5-fold difference in expression for screening genes. It was. Data was obtained by analyzing two donors, and the results of the two donors were crossed. MicroRNA transformation

 Selected microRNAs were purchased from Genolution Inc. to analyze the function of the screened microRNAs and their effect on Sox9 expression. The purchased microRNA mimics are prepared in the form of double strands, and once transformed, they undergo an internal mechanism of producing mature microRNAs. Since the anti-microRNA consists of a single strand that targets the complementary sequence of the miRNA, the scrambled oligonucleotide of the anti-microRNA was used as a negative control separately from the scramble oligonucleotide of the microRNA mimic.

 Anti-miR—SC and anti-miR-495 were purchased from Bioneer inc. And designed with the following sequence: anti-miR-495: 5′-AAGMGUGCACCAUGUUUGUUU-3 ′ (sense), anti-miR-SC: 5 '-UCACAACCUCCUAGA GAGUAGA-3' (sense). The constructed microRNA was transformed into SW1353 and hMSC using Lipofectamin LTX & Plus reagent (Invi trogen) according to the instructions. Western blotting analysis

Cells were disrupted in Passive Lysis Buffer (Passive Lysis Buffer, Pr omega, Madison, Wi, USA) containing 10 ug / iiiL protease and phosphatase inhibitors and Pierce ™ BCA Protein Assay Kit (Thermo Scientific, Logan, UT) Quantification using Samples were separated by 10% SDS ᅳ PAGE and transferred to PVDF membrane (Amershan Pharmacia, Piscata ay, NJ, USA). Briefly, membranes were incubated with 1: 3000 Sox9 antibody (Millipore® Billerica, Mass., USA) or GAPDH antibody (Santa Cruz) for 4 hours at room temperature. The membrane was washed repeatedly with IX TBST and then incubated with the appropriate HRP horseradish peroxidase-conjugated secondary antibody (Aniershani Pharmacia) for 1 hour. GAPDH was used as an internal control, and the intensity of the band was measured by analyzing the image file obtained through the Epson image scanner with the Image J program. Luciferase Reporter Assay PEZX-S0X9 3 'UTR and pEZX-MTOl vectors were purchased from Genecopoeia (Rockvi 1 le, MD, USA) to determine if miR-495 binds to Sox9 3' UTR. pEZX-S0X9 3 'UTR contains the firefly luciferase gene and the Renilla luciferase gene with S0X9 3' UTR, whereas pEZX-MTOl is the firefly luciferase and Renilla lucifer without S0X9 3 'UTR Laase gene. PEZX-S0X9 3 ^′ UTR or pEZX-MTOl was co-transformed into miR-495 or negative microRNA and HeLa cells using Lipofectaniin LTX & Plus reagent (Invi trogen). Firefly and Renilla luciferase activity was measured using a reporter assay system (Promega, Madison, Wisconsin, USA) 24 hours after the dual-luciferase microRNA transformation, and firefly psiferase activity was determined by Renilla expression. Standardized three times for. Immunohistochemistry

Paraffin embedded sections were deparaffinized, rehydrated and washed twice with PBS. To reduce nonspecific background staining by endogenous peroxides, sections were incubated in hydrogen peroxide blocks for 10 minutes and washed twice with PBS. Sections were incubated overnight at 4 ^° C with rabbit anti-C0L2A1 (Santa Cruz) or mouse anti-agrycan (Santa Cruz) and washed with PBS. For visualization of the primary antibody, PE (Phycoerythrin) conjugated goth anti-rabbit secondary antibody (Santa Cruz) and reen fluorescent protein (GFP) conjugated goth anti-mouse secondary antibody were used. Nuclei were stained with DAP I (4, 6-di am idi ηο-2-pheny i ndo 1 e, Sigma) and images were captured with an inverted fluorescence microscope (IX-71, Olympus). Safranin 0 Staining

The mass pellet was washed twice with PBS and fixed in 10% formalin solution for 24 hours. After fixation, the pellets were dehydrated in ethane and the dehydrated pellets were embedded in paraffin and sectioned. For safranin 0 staining, the sectioned micromass pellets were deparaffinized and rehydrated. Rehydrated pellets were washed with PBS and stained with 1% safranin 0 solution (Sigma, St. Louis, MO, USA) dissolved in 1% acetic acid for 30 minutes. Safranin 0-of dyed pellets Hematoxylin solution (Sigma) was used to stain the background. Stained pellet sections were observed under a microscope.

GAG Content Analysis

 The amount of sul fated GAG in culture obtained from niicroRNAs-transformed cartilage micromass was measured according to the manufacturer's instructions using Blyscan ¾ (Bioc () lor, New ownabbey, Northern Ireland, UK). Briefly, a total of 500 yL culture is gently shaken and mixed with 1 nil Blyscan staining reagent for 30 minutes to complete the binding of the GAG-dye. After centrifugation, the dye bound to GAG was dissolved in the separation reagent. The recovered dye concentration was measured at 656 nm and a standard curve was generated using Chondroitin 4-Sulfate Standard Solution (Biocolor). All samples were run three times. Statistical analysis

 Statistical analysis of the results obtained in hBM-MSCs, SW1353, and HeLa cell lines was performed using Student's ί-test, and the data are expressed as mean mean standard deviation. In case of ΡΟ.05 or 0.01, statistical significance was considered. Experiment result

Sox9 and niiRNA Expression in Chondrocyte Differentiation Period

It increases when mesenchymal derived enjoy cartilage differentiation of the cells to screen for microRNA expression patterns seen for Sox9 opposite ^'the microRNA microarray was carried out. MicroRNA microarrays were performed using CT10 and TGF-I33, which did not induce differentiation of mesenchymal stem cells, using CH10 and CT10 samples that did not induce cartilage differentiation for 10 days. Sox9, an important transcription factor for cell cartilage differentiation, increased expression during cartilage differentiation (FIG. La). The microRNA microarrays were analyzed using one-way AN0VA, and microRNAs showing 1.5 times or more expression difference compared to the control group were represented by a hierarchical clustering map (FIG. lb). Expression of iR-495 and miR-431 during Chondrocyte Differentiation Period

 It was predicted that miR-495 and miR-431 would bind to Sox9 3 ′ UTR in microRNAs with decreasing pattern in microRNA microarray analysis using target scan and miRanda (FIG. 2A). In addition, it was confirmed that expression of miR-495 and miR-431 decreased during chondrogenic differentiation of mesenchymal stem cells using real-time PCR (FIG. 2B). iniR-4957} Effect on the expression of SW1353 ^ Sox9

 After treatment with miR-495 and miR-431 in human cartilage sarcoma cell line SW1353, the expression of mRNA and protein was observed. MiR-495 decreased the expression of Sox9 mRNA and protein (FIG. 3a). It was found not to reduce the expression of Sox9 protein (FIG. 3B). Subsequent experiments were performed with miR-495. Luciferase Assay

Luciferase assays were performed using a luciferase vector incorporating Sox9 3 ^′ UTR, and the luciferase activity was reduced in proportion to the increased concentration of niiR-495 (FIG. 4C). On the other hand, the seed sequence of miR-495 was confirmed by confirming that luciferase activity did not change when mut-miR-495, which mutated the seed sequence (seed seqeunce) predicted to bind miR-495, did not change ( 4d). It was confirmed that miR-495 directly binds to Sox9 and affects it. miR-4957} Effect on endogenous Sox9 expression in human mesenchymal stem cells

After treating the mesenchymal stem cells with miR-495, the expression of Sox9 was observed after increasing the expression of Sox9 using TGF-P3. By observing a significant decrease in the expression of Sox9 in the miR-495-treated samples compared to the negative control miR-sc-treated samples (FIGS. 5A and 5B), miR-495 induced Sox9 in differentiation of mesenchymal stem cells. It was confirmed that suppresses the expression of. miR-495} Effect on chondrocyte marker expression After treating miR-sc and miR-495 to mesenchymal stem cells, induce cartilage differentiation for 5 days using TGF- | 33, followed by collagen type 2 (collagen type 2, Col 2) and aggrecan, the main markers of cartilage differentiation. (aggrecan) expression was observed by real time PCR. In the miR-495 treated samples, it was confirmed that the expression of Col2 and aggrecan was reduced compared to the control (FIGS. 6A and 6B). In addition, as a result of measuring protein expression of Col2 and aggrecan using immunofluorescence, it was observed that protein expression of Col2 and aggrecan was reduced by miR-495 (FIGS. 6C and 6D). As a result, it was confirmed that expression of Col2 and aggrecan, which are cartilage differentiation markers, was reduced by suppressing Sox9 by miR-495. Effect of overexpression of miR-495 on cartilage differentiation of human mesenchymal stem cells into cartilage and measuring the content of glycosaniinoglycan (GAG) on the 10th day after induction of cartilage differentiation after 7a) Paraffin sectioning of the micromass followed by safranin 0 staining (FIG. 7b). As a result, it was confirmed that miR-495 inhibits cartilage differentiation of mesenchymal stem cells. ai i—induced cartilage differentiation by miR-495

 In order to confirm whether miR-495 expression was actually induced to induce cartilage differentiation of stem cells, Western blotting was performed to confirm the change of Sox9 expression after treatment of miR-495 and anti-miR-495 to human mesenchymal stem cells and SW1353. As a result, it was confirmed that Sox9 expression was significantly increased by ant i -miR-495 (FIG. 8A). In addition, ant i -miR-495 was induced in human mesenchymal stem cells to induce cartilage differentiation, and the expression of cartilage differentiation markers Col2 and aggrecan was confirmed by real-time PCR. It was confirmed that the expression of (Figure 8b) increased. Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is merely a preferred embodiment and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the invention will be defined by the appended claims and equivalents thereof.

Claims

【Patent Claims】 [Claim 1] Nucleic acid molecules that inhibit the expression of micro RNA-495 A composition for promoting differentiation from stem cells to chondrocytes, comprising: [Claim 2] The composition according to claim 1, wherein the nucleic acid molecule is siRNA, shRNA, miRNA, ribozyme, PNA (pep tide nucleic acids), or antisense oligonucleotide. 【Claim 3】 The composition according to claim 2, wherein the nucleic acid molecule is an antisense oligonucleotide. 【Claim 4】 The composition of claim 3, wherein the antisense oligonucleotide has a length of 15 to 40 nucleotides. 【Claim 5】 The composition according to claim 3, wherein the antisense oligonucleotide comprises a nucleotide sequence in the second sequence of the sequence list. 【Claim 6】 The composition of claim 1, wherein the composition increases the expression of Sox9 in stem cells. 【Claim 7】 The composition according to claim 1, wherein the cells are mesenchymal stem cells. 【Claim 8】 The composition according to claim 7, wherein the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells. 【Claim 9】 A pharmaceutical composition for preventing or treating cartilage damage diseases comprising the composition of any one of claims 1 to 8 as an active ingredient. [Claim 10] The composition according to claim 9, wherein the cartilage damage disease is regressive arthritis. 【Claim 1U A screening method for a composition for promoting differentiation from stem cells to chondrocytes comprising the following steps: (a) contacting stem cells with a sample to be analyzed; and (b) Measuring the expression level of micro RNA-495 in the stem cells. If the expression level of micro RNA-95 decreases, the sample is determined to be a composition for promoting differentiation from stem cells to chondrocytes. 【Claim 12】 The method of claim 11, wherein the stem cells are mesenchymal stem cells. [Claim 13] A screening method for a composition for preventing or treating cartilage damage diseases comprising the following steps: (a) contacting stem cells with a sample to be analyzed; and (b) Measuring the expression level of micro RNA-495 in the stem cells. If the expression level of micro RNA-495 decreases, the sample is determined to be a composition for preventing or treating cartilage damage diseases. 【Claim 14】 The method of claim 13, wherein the stem cells are mesenchymal stem cells. 【Claim 15】 The method according to claim 13, wherein the cartilage damage disease is degenerative arthritis. 【Claim 16】 A method of promoting differentiation into chondrocytes comprising contacting stem cells with a composition containing a nucleic acid molecule that inhibits the expression of micro RNA-495 as an active ingredient. [Claim 17] Method for preventing or treating cartilage damage disease comprising administering to a subject (subj ect) a composition containing a nucleic acid molecule that inhibits the expression of micro RNA-495 as an active ingredient. [Claim 18] The method according to claim 17, wherein the cartilage damage disease is degenerative arthritis.