The Mechanism Of MicroRNA-29b Involved In Hypertrophic Chondrocyte Differentiation

ObjectiveArticular cartilage injury can be caused by trauma and diseases with irreversible degenerative pathological change and eventually lead to osteoarthritis(OA). According to the World Health Organization(WHO)statistics report, the occurrence of OA reached to 50% among population over 60 and 80% among population over 75. A total of 7.5 billion people were diagnosed of OA currently and the number is 0.12 billion in China. The cost on OA world-wide was estimated to be 0.546 billion dollars per year with more than 0.5 million arthroplasty. Pain is a main complication of OA, which severely affected the life quality of patients. In addition, there is a 53% risk of disability leading effect of OA. Due to the complex structure of articular cartilage without blood vessels and nerves, it is difficult for self-repairing. In this consideration, it is worthy to make deep investigation on the mechanism of cartilage repair.In recent years, due to the rapid development of tissue engineering technology and innovation, tissue engineered cartilage gradually become an ideal way to repair cartilage damage. Among them, cell source for tissue engineered cartilage and inducing method has been the key research in the field of cartilage repair. Mesenchymal stem cells(MSCs) are considered to be the seed cell of tissue-engineered cartilage in the construction. Among a class of seed cells, MSCs were known for its low immunogenicity, and a high degree of pluripotency and keep proliferation in vitro and ability to maintain the original cell morphology and physiological function. However, studies have found that chondrocytes derived from MSCs will not secrete large amounts of extracellular matrix in late differentiation stage. The chondrocytes become hypertrophic chondrocytes and eventually the repair graft will form a non-functional fibrous cartilage or calcification. Therefore, how to inhibit the differentiation of MSCs derived chondrocytes to hypertrophic chondrocytes and to keep the appropriate phenotype and characteristics of chondrocytes has become an important issue in the cartilage tissue engineering. The process in the formation of cartilage and cartilage biological functions involved a variety of factors proteins and related transcription factors and signaling pathways. The process is associated with a complex regulatory network with bone formation and interaction. Recent studies suggested that small non-coding RNA(micro RNA, abbreviated as mi RNA) plays a key role in the regulation of chondrocyte differentiation and biological function. Among many of the mi RNAs, mi R-29 b can positively regulate bone marrow mesenchymal stem cells(BMSCs) osteogenic differentiation. Studies have demonstrated that histone deacetylase 4(HDAC4) is one of downstream target genes of mi R-29 b. Moreover, HDAC4 is a negative regulator for chondrocyte hypertrophy by inhibiting Runx2, an important transcription factor for chondrocyte hypertrophy. Thus, it is assumed that the mi R-29 b promotes the differentiation transition from chondrocytes to hypertrophy probably by HDAC4. In this study, overexpression of mi R-29 b and mi R-29 b suppression technology were used to explore the potential regulatory processes and mechanisms of mi R-29 b.MethodsBased on our previously established MSCs chondrocytes hypertrophy inducing system, this study focused the role of mi R-29 b and its mechanism of the process for MSCs differentiation into chondrocytes.The induced system of MSCs differentiation into chondrocyte hypertrophy wasadopted. Safranin O-fast green staining of cell pellets of chondroitin sulfate, is determinedin vitro into chondrocyte hypertrophy-inducing success. And cell pellets were taken 3, 7,14, 21, 28 d, using RT-PCR to detect Col II, expression Col X, Runx2, Sox9, HDAC4 andmi R-29 b of m RNA level.mi R-29 b intervention experiments of MSCs into chondrocyte hypertrophy based onthe above results. Experimental groups include: â‘  overexpression group, transfectedwith pre-mi R-29 b mimics; â‘¡ overexpression group, transfected with negative control;â‘¢suppression group, transfected with mi R-29 b inhibitor(antagomi R-29b); â‘£ inhibitioncontrol group, transfected with antagomi R-29 b control. All experimental groups weretransfected the first time at induced 10 days, followed by supplementary transfection everythree days. The expression of mi R-29 b and chondrocyte hypertrophy associated genes, ColII, Col X, Sox9, MMP13, Runx2 m RNA, were detected by RT-PCR method. SafraninO-fast green staining method was used to detect the inhibition effects of mi R-29 b expression. The role of mi R-29 b in the regulation of chondrocytes was analysed from the protein level by immunohistochemistry and western blot.ResultsSafranin O-fast green staining showed that, compare with the control group, chondroitin sulfate chondrocyte hypertrophy group reduced secretion, especially when induced 28 d, in the success of chondrocyte hypertrophy in vitro display. Col II m RNA cartilage gene expression signature: a cartilage phase into an upward trend, the enlarged stage tended to decrease(P <0.05); hypertrophy related genes Col X m RNA expression: a lower expression of cartilage phase, significantly increased hypertrophy stage(P <0.05). The expression of Runx2 and mi R-29 b was in a lower level of cartilage stage, in the stage of chondrocyte hypertrophy was significantly increased(P <0.05); In contrast, the expression of Sox9 in chondrocytes stage rise, while at the stage mast cells was decreased(P <0.05). Accordingly, we believe that lower level of expression of mi R-29 b can delay or block the process of hypertrophic chondrocytes derived MSCs.mi R-29 b intervention MSCs into chondrocytes hypertrophy of differentiation, results showed that: in the mi R-29 b overexpression group, cartilage differentiation associated gene Col II, m RNA expression of HDAC4 and Sox9 compared with the control group was significantly lower(P <0.05) and hypertrophy of cartilage-related genes Col X, m RNA expression of MMP13 and Runx2 were significantly increased(P <0.05); in inhibiting the expression of mi R-29 b in the opposite group, chondrocyte hypertrophy related gene expression than the control group significantly lower(P <0.05), but the expression of cartilage-related genes is significantly increased(P <0.05). In safranin O-fast green staining experiments, inhibition than the control group significantly increased secretion of extracellular matrix cells mi R-29 b. In immunohistochemistry, the inhibition expression of mi R-29 b higher in the experimental group X collagen. Western blot assay showed that: mi R-29 b over Col II expression group, HDAC4 and Sox9 protein levels were significantly increased(P <0.05), Col X, m RNA expression of Runx2 was significantly decreased(P <0.05); and inhibition of mi R-29 b expression group, the expression of each protein expression and over-the opposite group.Resultsmi R-29 b was a positive regulatory factor of chondrocyte hypertrophy, and promotes the process of chondrocyte hypertrophy. Inhibition of mi R-29 b can delay the process of chondrocyte hypertrophy.