KR20150069375A - A Composition for Promoting Chondrogenesis from Stem Cells - Google Patents

Abstract

The present invention relates to a stem cell, more specifically, a composition for promoting differentiation of mesenchymal stem cells into chondrocytes, a composition for preventing or treating cartilage damage disease, and a screening method for the same.
The present invention newly identified miR-495 as an miRNA that inhibits the differentiation of stem cells into chondrocytes, and confirmed that it is possible to efficiently differentiate pluripotent stem cells into cartilage cells through inhibition thereof.
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.

Description

[0001] The present invention relates to a composition for promoting differentiation from stem cells to chondrocytes,

The present invention relates to a composition for promoting the differentiation of stem cells into chondrocytes comprising an inhibitor of microRNAs.

A stem cell is a cell capable of differentiating organisms that constitute a biological tissue into various cells, collectively referred to as undifferentiated cells at the stage before differentiation, which can be obtained from each tissue of the embryo, fetus and adult body . Stem cells are differentiated into specific cells by differentiation stimuli (environment). Unlike the differentiated cells in which cell division is stopped, proliferation (proliferation) of cells can be produced by dividing cells by self-renewal. expansion, and can be differentiated into other cells by different environments or differentiation stimuli, which is characterized by plasticity in differentiation.

Stem cells are largely derived from pluripotency embryonic stem cells (ES cells) obtained from embryos and totipotent to differentiate into all cells and multipotent stem cells obtained from each tissue ) Adult stem cells are divided into. Embryonic stem cells are undifferentiated, undifferentiated cells that can differentiate into all cells. Unlike adult stem cells, they can also produce germ cells, which can be inherited to the next generation. However, in spite of these advantages, there are many difficulties in practical use such as cancer formation, immune rejection reaction and ethical legal constraint in using embryonic stem cells as a cell therapy agent. Recently, mesenchymal stem cells have been suggested as an alternative to overcome these problems. The mesenchymal stem cells are pluripotent cells capable of differentiating into adipocytes, osteocytes, chondrocytes, muscle cells, neurons, and cardiomyocytes and have been reported to have the function of regulating the immune response.

Human bone marrow-derived mesenchymal stem cells have the ability to differentiate into various tissues such as adipocytes, chondrocytes, and osteocytes according to culture conditions. Stem cells are processed through several stages, each stage being made by various factors and their interactions, but its exact path has not yet been elucidated. Recently, microRNA (microRNA) has been discovered as a substance that regulates cartilage differentiation and is being studied extensively. The microRNA regulates the target gene by degrading the target mRNA with a small RNA of about 20 to 24 nucleotides or by inhibiting the post-transcriptional process. In many studies, microRNAs have been reported to play an important role in various biological processes including cell proliferation, apoptosis, and differentiation.

Arthritis is a multiple disease with the highest prevalence among diseases suffered by mankind. Of these, degenerative arthritis is a representative geriatric disease, with 70% to 80% of people aged 65 years or older being 75 years old or older. Currently, more than 10% of the total population in Korea has degenerative arthritis, and this prevalence is rapidly increasing due to the rapid aging of Korean society. In addition, it is estimated that there are more than 500 million degenerative arthritis patients worldwide, and as the average life span of human life and the duration of social activity increase, degenerative arthritis is expected to be overcome in terms of quality improvement of human life It is emerging as a disease.

The cartilage tissue, which acts like a membrane around the end of the bone in the joint tissue, acts as a structural buffer to prevent pain and bone wear that may occur when the bones are in direct contact with each other. Chondrocyte is the only cellular component in cartilage tissue. It plays a role of synthesizing, secreting and decomposing matrix of collagen and proteoglycan which is essential for maintaining cartilage tissue's normal function, so as to maintain the functional homeostasis of articular cartilage tissue It plays an essential role in giving. Thus, maintaining the activity of chondrocytes is directly related to preservation of the structural function of the joint tissue.

In order for chondrocyte tissue to function normally, chondrocyte production and differentiation must be appropriately performed, the survival of the chondrocyte should be well protected, and the calcification of the chondrocyte that is present is continuously suppressed, Should be prevented. The most prevalent cause of degenerative arthritis is that the essential function of these articular cartilage cells is lost as a result of aging

In the present invention, microRNAs involved in the cartilage differentiation process of human stem cells are identified, and the expression of the microRNA is induced to induce the differentiation specificity of the stem cells to the cartilage, thereby ultimately regenerating the lost cartilage tissue and regenerating the cells of diseases such as degenerative arthritis .

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have tried to find a factor that specifically differentiates multipotency stem cells into chondrocytes. As a result, microRNA-495 (miR-495) inhibits cartilage differentiation of human stem cells. When the expression of the miRNA-495 is inhibited, a specific differentiation into chondrocytes is induced and the cartilage tissue is regenerated The present invention can be applied to diseases such as arthritis, degenerative arthritis and the like.

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 is to provide a pharmaceutical composition for preventing or treating cartilage damage diseases.

It is still another object of the present invention to provide a method for screening a composition for promoting differentiation from stem cells to cartilage cells.

It is another object of the present invention to provide a method for screening a pharmaceutical composition for preventing or treating cartilage damage.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a composition for promoting the differentiation of stem cells into chondrocytes comprising a nucleic acid molecule which inhibits the expression of microRNA-495 as an active ingredient.

The present inventors have tried to find a factor that specifically differentiates multipotency stem cells into chondrocytes. As a result, microRNA-495 (miR-495) inhibits cartilage differentiation of human stem cells. When the expression of the miRNA-495 is inhibited, a specific differentiation into chondrocytes is induced and the cartilage tissue is regenerated And 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 a biological tissue, and refers to undifferentiated cells capable of regenerating unrestrictedly to form specialized cells of tissues and organs . Stem cells are pluripotent or pluripotent cells that can develop. Stem cells can divide into two daughter stem cells, or one daughter stem cell and one derived (transit) cell, and then multiply into the mature and complete form of the tissue. Specifically, stem cells capable of promoting differentiation into chondrocytes by 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 " means a multipotent stem cell capable of differentiating into adipocytes, bone cells, chondrocytes, muscle cells, neurons, and cardiomyocytes. Mesenchymal stem cells are identified by their vortex shape and the degree of expression of the basic cell surface markers CD73 (+), CD105 (+), CD34 (-), and CD45 (-).

In the present specification, the term " nucleic acid molecule " has the meaning inclusive of DNA (gDNA and cDNA) and RNA molecules, and the nucleotide, which is a basic constituent unit in the nucleic acid molecule, includes not only natural nucleotides, (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 miRNA of interest, miR-495, and that can form a duplex with the miRNA . Thus, the term " nucleic acid molecule that inhibits expression " herein may be referred to 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, PNA (peptide nucleic acids) or antisense oligonucleotide.

As used herein, the term " siRNA " means a short double-stranded RNA capable of inducing RNAi (RNA interference) phenomenon through cleavage of a specific mRNA. A sense RNA strand having a sequence homologous to the mRNA of the target gene and an antisense RNA strand having a sequence complementary thereto. Since siRNA can inhibit the expression of a target gene, it is provided as an efficient gene knockdown method or as a method of gene therapy.

The siRNA is not limited to a complete pair of double-stranded RNA portions that are paired with each other, but is paired by a mismatch (the corresponding base is not complementary), a bulge (no base corresponding to one chain) May be included. The total length is 10 to 100 bases, preferably 15 to 80 bases, and most preferably 20 to 70 bases. The siRNA terminal structure is capable of blunt or cohesive termini as long as it can inhibit the expression of the target gene by the RNAi effect. The sticky end structure can have both a 3-terminal protruding structure and a 5-terminal protruding structure. The number of protruding bases is not limited. For example, the base water may be from 1 to 8 bases, preferably from 2 to 6 bases. In addition, the siRNA can be added to a protruding portion at one end in a range that can maintain the effect of suppressing the expression of a target gene, for example, a low molecular RNA (for example, a natural RNA molecule such as a tRNA, Molecules). The siRNA terminal structure does not need to have a cleavage structure on both sides, and a stem loop structure in which one terminal region of double-stranded RNA is connected by linker RNA may be used. The length of the linker is not particularly limited as long as it does not interfere with the pairing of the stem portions.

As used herein, the term " shRNA " refers to a nucleotide consisting of 50-70 single strands and forms a stem-loop structure in vivo . A long RNA of 19-29 nucleotides complementary to both the loop region of 5-10 nucleotides forms a double stranded stem with base pairs.

As used herein, the term " miRNA (microRNA) " regulates gene expression and refers to a nucleic acid molecule having a total length of 20-50 nucleotides, preferably 20-45 nucleotides, more preferably 20-40 nucleotides, Refers to a single stranded RNA molecule consisting of 30 nucleotides, most preferably 21-23 nucleotides. miRNAs are oligonucleotides that are not expressed in cells and have a short stem-loop structure. A miRNA has total or partial homology with one or more mRNA (messenger RNA) and inhibits target gene expression through a complementary binding to the mRNA.

As used herein, the term " ribozyme " refers to an RNA molecule that has the same function as an enzyme that recognizes a nucleotide sequence of a specific RNA and cleaves the nucleotide sequence of the specific RNA. The ribozyme is a region that specifically binds with a complementary base sequence of the target messenger RNA strand and cleaves the target RNA.

As used herein, the term " PNA (peptide nucleic acid) " means a molecule having both nucleic acid and protein properties, and capable of complementarily binding with DNA or RNA. PNA was first reported in 1999 as a pseudo-DNA in which a nucleobase is linked by a peptide bond (Nielsen PE, Egholm M, Berg RH, Buchardt O, "Sequence-selective recognition of DNA by strand displacement with a thymine- substituted polyamide ", Science 1991, Vol. 254: pp1497-1500). PNAs are synthesized by artificial chemical methods, not found in nature. PNA forms a double strand by causing a hybridization reaction with a natural nucleic acid of a complementary base sequence. When the length is the same, the PNA / DNA double strand is more stable than the DNA / DNA double strand, and the PNA / RNA double strand is more stable than the DNA / RNA double strand. The peptide backbone, which is repeatedly linked by N- (2-aminoethyl) glycine to amide bond, is most commonly used as the peptide backbone. In this case, unlike the basic structure of a negatively charged natural nucleic acid, Is neutral. The four nucleic acid bases present in the PNA have a spatial size and a distance between the nucleotide bases is almost the same as that of the native nucleic acid. PNA is chemically more stable than natural nucleic acid, and biologically stable because it is not degraded by nuclease or protease.

The term " antisense oligonucleotide " as used herein refers to DNA or RNA or a derivative thereof containing a nucleotide sequence complementary to the sequence of a specific mRNA, and binds to a complementary sequence in mRNA to inhibit translation of mRNA into a protein The antisense nucleotide sequence of the present invention means a DNA or RNA sequence complementary to the mRNA of the target gene and capable of binding to the mRNA of the target gene, and the translation of the mRNA of the target gene, the transposition into the cytoplasm ), Maturation, or any other overall biological function. The length of the antisense oligonucleotide is 6 to 100 bases, preferably 40 bases in 10.

The antisense oligonucleotides may be modified at one or more base, sugar or backbone sites to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol. , 5 (3): 343-55, 1995 ). The oligonucleotide backbone can be modified with phosphorothioate, phosphotriester, methylphosphonate, short chain alkyl, cycloalkyl, short chain heteroatomic, heterocyclic biantennary bond, and the like. In addition, the antisense nucleic acid may comprise one or more substituted sugar moieties. Antisense oligonucleotides may comprise modified bases. Modified bases include hypoxanthane, 6-methyladenine, 5-methylpyrimidine (especially 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, genentioyl HMC, -Thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diaminopurine .

In accordance with 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 antisense oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 2.

Most specifically, the antisense oligonucleotide comprises a sequence complementary to the 15th to 21st nucleotide sequence of the first sequence of the sequence listing.

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, miR-495 inhibits the expression of Sox9, an important transcription factor in the overall process of cartilage differentiation, and the composition of the present invention, which inhibits miR-495, restores the expression of Sox9, Promotes differentiation into cells.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating 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 damage disease prevented or treated with the composition of the present invention is degenerative arthritis.

As used herein, the term " osteoarthritis " refers to a disease in which joint tissue required for joint exercise is injured 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 cartilage cells. Thus, unlike conventional therapies such as inflammatory control, which are merely for the treatment of rheumatoid arthritis in which joint tissue is destroyed by an inflammatory reaction due to immune dysfunction, the composition of the present invention provides a fundamental treatment method for degenerative arthritis .

According to another aspect of the present invention, there is provided a method for screening a composition for promoting differentiation from stem cells to chondrocytes, comprising the steps of:

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

(b) measuring the expression level of microRNA-495 in the stem cell, and when the expression level of microRNA-495 is decreased, the sample is determined as a composition for promoting differentiation from stem cells to chondrocytes.

According to the present invention, the first sequence of the sequence listing is the nucleotide sequence of miR-495.

According to the method of the present invention, the test material is contacted with the undifferentiated stem cell. Specifically, the undifferentiated stem cells are mesenchymal stem cells. The term " test substance " used in reference to the screening method of the present invention means an unknown substance used in screening to check whether or not it affects the expression level of miR-495. Such test materials include, but are not limited to, chemicals, nucleotides, antisense-RNA, small interference RNA (siRNA) and natural product extracts. Next, the expression level of miR-495 is measured in the sample-treated cells. Measurement of the expression level can be performed through various methods known in the art and can be measured, for example, through a microarray or the like. As a result of the measurement, when the expression of miR-495 is inhibited, the test substance can be determined as a composition for promoting differentiation into chondrocytes.

According to another aspect of the present invention, there is provided a method for screening a composition for preventing or treating a cartilage damage disease comprising the steps of:

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

(b) measuring the expression level of microRNA-495 in the stem cell, and when the expression level of microRNA-495 is decreased, the sample is determined as a composition for preventing or treating cartilage damage disease.

Since the stem cells, nucleotides, specific screening methods and procedures used in the present invention have already been described above, the description thereof is omitted in order to avoid excessive duplication. Since the substance discovered through the expression of miR-495 expression using the method of the present invention promotes the differentiation of endogenous stem cells or transplanted therapeutic stem cells into chondrocytes, the degenerative arthritis And the like.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides stem cells, more specifically, a composition for promoting differentiation of mesenchymal stem cells into chondrocytes, a composition for preventing or treating cartilage damage diseases, and a screening method for the same.

(b) The present invention newly identified miR-495 as a miRNA which inhibits the differentiation of stem cells into chondrocytes, and it has been confirmed that it is possible to efficiently differentiate pluripotent stem cells into chondrocytes through 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 the lost cartilage tissue.

FIG. 1 is a graph showing the expression patterns of Sox9 and miRNA during differentiation of chondrocytes. FIG. 1A is a graph showing that mRNA expression of Sox9 is gradually increased as mesenchymal stem cells differentiate into cartilage. FIG. 1B is a diagram showing a microarray microRNA array using a one-way ANOVA as a hierarchical clustering map showing microRNAs showing a difference of 1.5 times or more from that of the control group.
FIG. 2 is a graph showing the expression patterns of miR-495 and miR-431 during differentiation of cartilage cells. miR-495 and miR-431 were expected to bind to the Sox9 3'UTR (Fig. 2a), and the expression of miR-495 and miR-431 decreased during chondrogenesis of mesenchymal stem cells was confirmed by real-time PCR (Fig. 2B).
FIG. 3 shows the effect of miR-495 on the expression of Sox9 in human chondrosarcoma cell SW1353. FIG. 3 is a graph showing the results obtained at the level of mRNA (FIG. 3A) and protein (FIG.
FIG. 4 is a graph showing the results of confirming that miR-495 specifically binds to 3'UTR of Sox9. 4A is a view showing the 3'UTR region of Sox9 to which miR-495 binds. 4B is a schematic diagram of a vector structure including Sox9 3'UTR. FIG. 4C is a graph showing the result of performing a luciferase assay after a vector including miR-495 and Sox9 3'UTR was introduced into cells together. FIG. 4D is a graph showing the results of performing a luciferase assay after miR-495 mutated the 15-21st nucleotide sequence capable of binding to Sox9 3'UTR, wherein the 15-21th nucleotide sequence of miR-495 Lt; RTI ID = 0.0 > Sox9 < / RTI >
FIG. 5 shows the expression levels of Sox9 by increasing the expression of Sox9 using TGF-β3 after treating miR-495 with mesenchymal stem cells, Western blotting (FIG. 5A) and quantifying Western blotting results FIG. 5B). FIG.
Figure 6 shows the effect of miR-495 on the expression of cartilage cell markers. After culturing the mesenchymal stem cells with miR-sc and miR-495, they induced the differentiation of cartilage with TGF-β3. Then, mRNAs of Col2 (FIG. 6a) and aggran (FIG. 6b) And immunological fluorescent staining was used to observe protein expression of Col2 (Fig. 6c) and aggran (Fig. 6d), respectively.
FIG. 7 is a graph showing the effect of overexpression of miR-495 on cartilage differentiation of human mesenchymal stem cells. FIG. 7A is a graph showing the results of GAG (glycosaminoglycan) measurement on the 10th day after induction of cartilage differentiation after treatment of miR-sc and miR-495 on mesenchymal stem cells. Fig. 7b shows the results of the sapranin O staining of the miR-sc and miR-495 treated groups.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

Culturing of bone marrow-derived human mesenchymal stem cell (hMSC) and chondrosarcoma cell line

BM aspirates were obtained in the posterior iliac crest of 12 adult donors aged 19 to 63 years with the approval of the Institutional 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 mixed with DMEM-LG (low-glucose Dulbecco's modified Eagle's medium; Welgene, Daegu, USA) supplemented with 10% FBS (Gibco, Grand Island, NY, USA) and 1% antibiotic- Korea) for 7 days. The human chondrosarcoma cell line, SW1353, was cultured in DMEM-HG (DMEM-high glucose, Welgene) supplemented with 10% FBS and 1% antibiotic-antifungal agent solution.

In vitro chondrogenesis of hMSC using micromass culture

In vitro, a micromass culture method was used to differentiate hMSC into cartilage. The trypsinized hMSCs were resuspended at a concentration of 1 x 10 ⁷ cells / mL in DMEM-LG containing 10% FBS and 10 μL of them were dotted into each well of a 24-well plate (1 × 10 ⁵ cells per well) gave. Cells were allowed to adsorb for 2 hours at 37 ° C and then treated with 1% antibiotic-antifungal agent solution, 1% insulin transferrin selenium-A (ITS; Invitrogen), 50 μg / ml ascorbic acid (Invitrogen) and 10 ng / ml TGF- R & D systems, Minneapolis, MN, USA) supplemented with DMEM-HG. During the in vitro differentiation phase, the cartilage differentiation medium containing 10 ng / mL TGF-β3 was replaced every 2 days.

Quantitative real-time PCR

Total RNA from hMSCs and SW1353 cells was isolated using RNA iso Plus (Takara, Shiga, Japan) reagent according to the manufacturer's instructions. Total RNA was reverse transcribed using the Omniscript kit (Qiagen, Venlo, Netherlands) for cDNA synthesis. Real-time PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems; ABI, Carlsbad, Calif., USA). Real-time PCR was performed using an ABI7500 real-time instrument (Applied Biosystems, ABI, Carlsbad, Calif.). Primer sets were purchased from Bioneer (Daejeon, South Korea) and the primers used were: GAPDH (P267613), Sox9 (P232240). No proven primers for Col2a1 and aggrecnan were designed as follows: Col2a1, 5'-GTCCTCTCCCAAGTCCACACAG-3 '(sense) and 5'-GGGCACGAAGGCTCATCATTC-3'(antisense); aggrecan , 5'-CCACTGTTACCGCCACTT-3 '(sense) and 5' GTAGTCTTGGGCATTGTTGT 3 '(anceptense). The PCR process was initiated at 95 ° C for 30 seconds, followed by 40 cycles of thermal cycling at 95 ° C for 5 seconds and again at 20 ° C 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. Measurements of each sample were normalized to the internal control GAPDH.

For the precursor-microRNA reverse transcription, the Mir-X microRNA First-Strand Synthesis kit (Clontech, Mountain View, CA, USA) was used according to the manufacturer's instructions and cDNA synthesized from microRNA was quantified with SYBRqRT-PCR Respectively. A list of primer sets used for specific mature-microRNA amplification was obtained from Genolution (Genolution, Seoul, South Korea) and Bioneer Inc. The designed primers are 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 an ABI7500 real-time instrument. The PCR process was initiated at 95 ° C for 10 seconds, followed by 40 cycles of thermal cycling at 95 ° C for 5 seconds and again at 20 ° C at 60 ° C. U6 snRNA (small nuclear RNA) was used to normalize the relative expression of all precursor-microRNAs.

MicroRNA Array Analysis

Total RNA of undifferentiated hMSCs (0-10 days incubation) and cartilage-derived hMSCs derived from the same donor were separated using RNA iso Plus (Takara) according to the manufacturer's instructions. The overall quality of the total RNA was confirmed using a spectrophotometer. The expression pattern of each microRNA was examined with GenechipmiRNA 1.0 array (Affymetrix, Santa Clara, CA, USA). Pearson correlation analysis was performed to identify microRNAs specifically expressed in cartilage differentiation - induced mesenchymal stem cells. Briefly, genes expressed differentially were selected using routine mathematical equations that subtracted the control values from the differentiated groups: a strict cut-off reference point with a minimum expression difference of 1.5-fold for gene screening was applied Respectively. Data were obtained by analyzing two donors and crossing the results of two donors.

MicroRNA Transformation

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

Anti-miR-SC and anti-miR-495 were purchased from Bioneer Inc. and were designed with the following sequences: anti-miR-495: 5'-AAGAAGUGCACCAUGUUUGUUU-3 ''-UCACAACCUCCUAGAAAGAGUAGA-3' (sense). The constructed microRNA was transfected into SW1353 and hMSC according to the manual using Lipofectamin LTX & Plus reagent (Invitrogen).

Western blotting analysis

Cells were disrupted in a passive lysis buffer (Passive Lysis Buffer, Promega, Madison, WI, USA) containing 10 [mu] g / ml protease and phosphatase inhibitor and harvested using Pierce ™ BCA Protein Assay Kit (Thermo Scientific, Logan, UT) Respectively. Samples were separated by 10% SDS-PAGE and transferred to PVDF membranes (Amersham, Pharmacia, Piscataway, NJ, USA). Briefly, membranes were incubated with Sox9 antibody (Millipore, Billerica, MA, USA) or GAPDH antibody (Santa Cruz) at a 1: 3000 concentration for 4 hours at room temperature. Membranes were washed repeatedly with 1X TBST and incubated with appropriate HRP (horseradish peroxidase) -conjugated secondary antibody (Amersham Pharmacia) for 1 hour. GAPDH was used as an internal control, and the intensity of the band was measured by analyzing an image file obtained through an Epson image scanner with an Image J program.

Luciferase reporter analysis

The pEZX-SOX9 3'UTR and pEZX-MT01 vectors were purchased from Genecopoeia (Rockville, MD, USA) to determine whether miR-495 binds to Sox9 3'UTR. pEZX-SOX9 3'UTR contains firefly luciferase gene and Renilla luciferase gene with SOX9 3'UTR, whereas pEZX-MT01 contains firefly luciferase and Renilla luciferase without SOX9 3'UTR Lt; / RTI > gene. PEZX-SOX9 3'UTR or pEZX-MT01 was co-transformed with miR-495 or negative microRNA and HeLa cells using Lipofectamin LTX & Plus reagent (Invitrogen). Firefly and Renilla luciferase activities were measured 24 hours after dual-luciferase microRNA transformation using a reporter assay system (Promega, Madison, Wisconsin, USA), and firefly luciferase activity was measured in Renilla expression And three times.

Immunohistochemistry

Paraffin-embedded sections were deparaffinized, rehydrated and washed twice with PBS. To reduce nonspecific background staining by endogenous peroxides, the sections were incubated in a hydrogen peroxide block for 10 minutes and washed twice with PBS. The sections were incubated overnight at 4 [deg.] C with rabbit anti-CTLA2 (Santa Cruz) or mouse anti-aggrecan (Santa Cruz) and washed with PBS. For visualization of the primary antibody, PE (Phycoerythrin) conjugated goat anti-rabbit secondary antibody (Santa Cruz) and GFP (green fluorescent protein) conjugated anti-mouse secondary antibody were used. The nuclei were stained with DAPI (4,6-diamidino-2-phenyindole, Sigma) and images were captured with an inverted fluorescence microscope (IX-71, Olympus).

Saprinin O staining

The mass pellet was washed twice with PBS and fixed in 10% formalin solution for 24 hours. After fixing, the pellets were dehydrated in ethanol and the dehydrated pellets were embedded in paraffin and sectioned. For the sapranin O staining, the segmented micromass pellet was deparaffinized and rehydrated. The rehydrated pellet was washed with PBS and stained with 1% sapranin O solution (Sigma, St. Louis, MO, USA) in 1% acetic acid for 30 min. A hematoxylin solution (Sigma) was used to stain the background of the sapranin O-stained pellet. The stained pellet sections were observed under a microscope.

GAG content analysis

The amount of sulfated GAG in the cultures obtained from the microRNAs-transformed chondrogenic differentiation micromass was determined according to the manufacturer's instructions using the Blyscan kit (Biocolor, Newtownabbey, Northern Ireland, UK). Briefly, a total of 500 μL of the culture is mixed with 1 ml Blyscan staining reagent for 30 minutes by gentle shaking to complete the binding of the GAG-dye. After centrifugation, the dye bound to the 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 tested three times.

Statistical analysis

Statistical analysis of the results obtained in hBM-MSCs, SW1353, and HeLa cell lines was performed using Student's t -test and the data are expressed as mean ± SD. P <0.05 or 0.01 was considered statistically significant.

Experiment result

Sox9 and miRNA expression patterns during chondrocyte differentiation

MicroRNA microarrays were performed to select microRNAs that showed an opposite pattern of expression of Sox9 when mesenchymal stem cells induced cartilage differentiation. The microRNA microarray was performed using CT10 and TGF-β3, which did not induce differentiation of mesenchymal stem cells, and CT10, which did not treat CH10 and TGF-β3, which induces cartilage differentiation for 10 days. Sox9, an important transcription factor for cellular cartilage differentiation, increased expression during chondrogenic differentiation (Fig. La). The microRNA microarrays were analyzed using one-way ANOVA and microRNAs with a 1.5-fold difference in expression compared to the control group were shown as a hierarchical clustering map (Fig. 1B).

Expression patterns of miR-495 and miR-431 during chondrogenic differentiation

It was predicted that miR-495 and miR-431 among the microRNAs showing a pattern of decrease in microRNA microarray analysis using target scan and miRanda would have a sequence that binds to Sox9 3'UTR (Fig. 2a). In addition, it was confirmed that the expression of miR-495 and miR-431 decreased during the chondrogenic differentiation of mesenchymal stem cells using real-time PCR (Fig. 2B).

Effect of miR-495 on the expression of Sox9 in SW1353

As a result of observing mRNA and protein expression changes after treating miR-495 and miR-431 in human chondrosarcoma cell line SW1353, miR-495 decreased expression of Sox9 mRNA and protein (Fig. 3a) Sox9 protein expression (Fig. 3B). Subsequent experiments proceeded to miR-495.

Luciferase

Luciferase assay using a luciferase vector with Sox9 3'UTR inserted resulted in a decrease in luciferase activity in proportion to the increased concentration of miR-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 mutated with the seed sequence predicted to bind miR-495 was changed ( 4d). It was confirmed that miR-495 directly binds to Sox9.

Effect of miR-495 on the expression of endogenous Sox9 in human mesenchymal stem cells

After the treatment of miR-495 with mesenchymal stem cells, the expression of Sox9 was observed after the increase of Sox9 expression using TGF-β3. By observing that the expression of Sox9 was significantly reduced in the miR-495 treated samples compared to the negative control miR-sc treated samples (Fig. 5A and 5B), miR-495 was found in the differentiation of mesenchymal stem cells Of the cells.

Effect of miR-495 on the expression of cartilage cell markers

After treatment of miR-sc and miR-495 with mesenchymal stem cells, TGF-β3 was used to induce cartilage differentiation for 5 days, and collagen type 2 (Col2) and aggrecan aggrecan) expression was observed by real time PCR. it was confirmed that the expression of Col2 and aggrecan in miR-495-treated samples was reduced compared to the control (Figs. 6A and 6B). In addition, protein expression of Col2 and aggrecan was measured using immunofluorescence staining, and the expression of Col2 and aggrecan was decreased by miR-495 (FIGS. 6C and 6D). Thus, it was confirmed that the expression of Col2 and aggrecan, which are cartilage differentiation markers, was reduced by suppression of Sox9 by miR-495.

Effect of overexpression of miR-495 on cartilage differentiation of human mesenchymal stem cells

After treatment of miR-sc and miR-495 with mesenchymal stem cells, GAG (glycosaminoglycan) content was measured 10 days after induction of cartilage differentiation (Fig. 7a) (Fig. 7B). As a result, it was confirmed that miR-495 inhibited the chondrogenic differentiation of mesenchymal stem cells.

Induction of cartilage differentiation by anti-miR-495

In order to confirm whether the inhibition of miR-495 expression actually induced chondrogenesis of stem cells, the changes of Sox9 expression in human mesenchymal stem cells and SW1353 after treatment with miR-495 and anti-miR-495 were confirmed by Western blotting As a result, it was confirmed that Sox9 expression was significantly increased by anti-miR-495 (FIG. 8A). In addition, when human mesenchymal stem cells were treated with anti-miR-495 to induce cartilage differentiation, the expression of Col2 and aggrecan, which are chondrogenic differentiation markers, was confirmed by real time PCR. As a result, the expression of Col2 and aggrecan (Fig. 8B).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Industry-Academic Cooperation Foundation Yonsei University <120> A Composition for Promoting Chondrogenesis from Stem Cells <130> PN130606 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> RNA <213> homo sapiens <400> 1 aaacaaacau ggugcacuuc uu 22 <210> 2 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> Anti-miR-495 <400> 2 aagaagugca ccauguuugu uu 22

Claims (15)

A composition for promoting the differentiation of stem cells into chondrocytes comprising a nucleic acid molecule inhibiting the expression of microRNA-495 as an active ingredient.
2. The composition of claim 1, wherein the nucleic acid molecule is an siRNA, shRNA, miRNA, ribozyme, PNA (peptide nucleic acids) or antisense oligonucleotide.
3. The composition of claim 2, wherein the nucleic acid molecule is an antisense oligonucleotide.
4. The composition of claim 3, wherein the antisense oligonucleotide has a length of 15 to 40 nucleotides.
4. The composition of claim 3, wherein said antisense oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 2.
2. The composition of claim 1, wherein the composition increases expression of Sox9 in stem cells.
The composition according to claim 1, wherein the stem cells are mesenchymal stem cells.
8. The composition of claim 7, wherein the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells.
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.
10. The composition of claim 9, wherein the cartilage damage disorder is degenerative arthritis.
A method for screening a composition for promoting differentiation from stem cells to chondrocytes comprising the steps of:
(a) contacting a stem cell with a sample to be analyzed; And
(b) measuring the expression level of microRNA-495 in the stem cell, and when the expression level of microRNA-495 is decreased, the sample is determined as a composition for promoting differentiation from stem cells to chondrocytes.
12. The method according to claim 11, wherein the stem cells are mesenchymal stem cells.
A method for screening a composition for preventing or treating cartilage damage disease comprising the steps of:
(a) contacting a stem cell with a sample to be analyzed; And
(b) measuring the expression level of microRNA-495 in the stem cell, and when the expression level of microRNA-495 is decreased, the sample is determined as a composition for preventing or treating cartilage damage disease.
14. The method according to claim 13, wherein the stem cells are mesenchymal stem cells.
14. The method of claim 13, wherein the cartilage damage disorder is degenerative arthritis.

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