Chang Tingjie, Xie Jie, Li Hongzhuo, Li Dong, Liu Ping, Hu Yihe
Department of Orthopedics, Xiangya Hospital of Central South University, Changsha, Hunan, 410008, China.
Department of Orthopedics, Peace Hospital of Changzhi Medical College, Changzhi, Shanxi, 046000, China.
Cell Prolif. 2016 Apr;49(2):207-18. doi: 10.1111/cpr.12246. Epub 2016 Mar 10.
Sox9 has recently been reported to be a key mediator during cartilage degradation in osteoarthritis (OA). Our aim was to clarify the role of microRNA-30a (miR-30a) and its target gene Sox9 in regulation of extracellular matrix (ECM) degradation in OA.
Expression of miR-30a in cartilage tissues and in primary chondrocytes from healthy and OA donors, was determined by real-time PCR, and levels of Sox9 mRNA and protein were analyzed by real-time PCR and western blotting, respectively. Subsequently, the target of miR-30a was predicted by bioinformatics and confirmed using a luciferase assay. Expression of ECM-related genes was determined by tissue-specific staining, immunofluorescence, real-time PCR, and western blotting. The role of miR-30a in OA was examined in vivo using a collagenase-induced OA rat model.
miR-30a was significantly upregulated and Sox9 was downregulated in primary chondrocytes from cartilage taken from OA donors compared to healthy controls. We showed that miR-30a specifically bound to the 3' UTR of Sox9, and overexpression of miR-30a downregulated expression levels of Sox9, proteoglycan aggrecan, and Col II compared to those induced by small interfering RNA transfection to knockdown Sox9. miR-30a inhibition reversed the effects of ECM degradation in vitro and in vivo.
miR-30a acts as a virulence MRA in OA, promoting ECM degradation by targeting Sox9 and by modulating activity of its downstream effectors Col II and proteoglycan aggrecan.
最近有报道称，Sox9是骨关节炎（OA）软骨降解过程中的关键介质。我们的目的是阐明微小RNA - 30a（miR - 30a）及其靶基因Sox9在OA细胞外基质（ECM）降解调节中的作用。
通过实时PCR测定miR - 30a在健康和OA供体的软骨组织及原代软骨细胞中的表达，分别通过实时PCR和蛋白质印迹分析Sox9 mRNA和蛋白质水平。随后，通过生物信息学预测miR - 30a的靶标，并使用荧光素酶测定法进行验证。通过组织特异性染色、免疫荧光、实时PCR和蛋白质印迹测定ECM相关基因的表达。使用胶原酶诱导的OA大鼠模型在体内研究miR - 30a在OA中的作用。
与健康对照相比，来自OA供体软骨的原代软骨细胞中miR - 30a显著上调，Sox9下调。我们表明，miR - 30a特异性结合Sox9的3'UTR，与小干扰RNA转染敲低Sox9相比，miR - 30a过表达下调了Sox9、蛋白聚糖聚集蛋白聚糖和Col II的表达水平。miR - 30a抑制在体外和体内逆转了ECM降解的作用。
miR - 30a在OA中作为一种致病微小RNA发挥作用，通过靶向Sox9并调节其下游效应物Col II和蛋白聚糖聚集蛋白聚糖的活性来促进ECM降解。
