Experimental Study On The Mechantism Of MicroRNAs As In The Process Of Chondrocytes Differentiation From Human Bone Marrow Mesenchymal Stem Cells

[Backgroud]With the social ageing, morbidity of osteoarthritis(OA) is higher, which seriously affects social labor. At present there is no specific drug treatment and artificial joints bring many complications.So the treatment of OA is a problem difficult to deal with in Department of rheumatology and Department of orthopedics. Tissue engineering is potential treatment to cure osteoarthritis. At present, origins of chondrocyte, include cultured autologous chondrocytes and chondrocytes from mesenchymal stem cells(MSCs) chondrogenesis in vitro. Bone marrow-derived mesenchymal stem cells had the ability to self-renew, which make it more promising compared with autologous chondrocytes.However, the mechanism of MSCs chongdrogenesis is not clear and the stability of chondrocyte phenotype is not saticfied. MicroRNAs are a group of small molecules with identified roles in tumorigenesis. Studies have shown that microRNA play a regulatory role in bone and cartilage cell differentiation. But the research of microRNA target gene and the regulation mechanism of microRNA in cartilage differentiation of deficiency is needed. The research is mainly to solve the scientific problems, which include "how to establish the stable method of chondrogenesis from MSCs?"and To investigate the mechanism of regulation of microRNA during induced differentiation." The final purpose is to optimize the method of chondrogenesis from MSCs and to provide stable and reliable source of chondrocytes for transplantation, so we can ultimately solve the problem with treatment of osteoarthritis.[Objective]1、To establish the method for human bone marrow mesenchymal stem cells to differentiation into chondrocytes,providing stable cell resource for the research.2、To detect expression changes of microRNAs in the process of chondrocytes differentiation from human bone marrow mesenchymal stem cells,and verify the changes.3、To find the microRNAs which play a role in the process of chondrocytes differentiation from human bone marrow mesenchymal stem cells,and explore the mechanisms of microRNAs in the process. [Methods]1、The human bone marrow mesenchymal stem cells were divided into two group,the non-induced group cultured with DSCM and the induced group cultured with MCDM.During the process of cells culture,we observed the changes of cells morphology,detected the type Ⅱ collagen expression by immunofluorescence staining,immunohistochemical stainig and Western Blot,and detected the proteoglycan expression by alcian blue staining.2、Human bone marrow mesenchymal stem cells were cultured with MCDM in three-dimensional lattice in vitro.The total RNAs extracted were used to detect the small RNA expression by hign-throughoutput analysis.Quantitative real-time PCR was used to confirm the high-throughput sequencing results.3、RUNX3played a role in the process of chondrocyte differentiation.Bioinformatics softwares were used to predict target genes.Dual luciferase report gene was used to verify the interraction of microRNA and RUNX3mRNA3’UTR.Recombinant adenovirus were used to overexpress hsa-mir-210or inhibit the expression of hsa-mir-210in cells.After48hours of infection, Western Blot was used to detect the expression of RUNX3protein.[Results]1、Cells in the both groups adhered well to the plastic,while cells in the induced groups gradually became polygan with pseudopodia in morphology.Type Ⅱ collagen were detected by immunofluorescence staining,immunohistochemistry stainining and Western Blot.Alcin blue staining was positive in cells of induced group.2、Compared the higan-throughput results of the two samples, we found28kinds of microRNA upregulated significantly and43kinds of microRNA donwregulated in the process of chondrocyte differentiation.Quantitative real-time PCR detected significant difference of hsa-mir-210expression between the two group(p<0.05),but no significant difference of hsa-mir-130a expression(p>0.05).3、According to the results of bioniformatics software,such as TargetScan,we chose hsa-mir-19a-19b-27a-27b-210-214-330-495as our research subject to the dual luciferase report genn assay.The interatction of hsa-mir-210with RUNX3mRNA3’UTR was confirmed. After48hours of adenovirus infection,the RUNX3expression upregulated in AD-hsa-mir-210inhibitor group compared with control group,while RUNX3expression downregulated in AD-hsa-mir-210group compared with control group.[Conclusions] 1、By inducing differerntion culture,human bone marrow mesenchymal stem cells became cells which were morphologically similar to chondrocyte and able to produce the component of articular cartilage,including typeⅡ collagen and proteoglycan. The induction method provided stable cells sources for the experiment.2、In the process of human bone marrow mesenchymal stem cells differentating to chondrocytes,28kinds of microRNA expression upregulated to more than double and in the while43kinds of microRNA expression downregulated to below50%.Quantitative real-time PCR confirmed the high-throughput sequencing results,including the downregulation of hsa-mir-210.However there was no significant change in hsa-mir-130a confirmed by qRT-PCR.3、By binding site predicted,hsa-mir-210targeted RUNX3mRNA3’UTR, inhibited posttranslation.Overexprssion of hsa-mir-210reduced the production of RUNX3protein in cells and vice versa.