MicroRNA-30a通过Sox9调控骨髓间充质干细胞软骨分化的研究  

MicroRNA-30a regulating chondrogenic differentiation of human mesenchymal stem cells by targeting Sox9

作　　者：王运佳[1] 张宏其[1] 余洪贵 周振海[1] 杨冠腾 高琪乐[1] WANG Yun-jia;ZHANG Hong-qi;YU Hong-gui;ZHOU Zhen-hai;YANG Guan-teng;GAO Qi-le(Xiangya Hospital,Southeast University,Changsha 410012,China)

机构地区：[1]中南大学湘雅医院,湖南省长沙市湘雅路87号410012

出　　处：《中国矫形外科杂志》2018年第20期1887-1892,共6页Orthopedic Journal of China

基　　金：国家自然科学基金资助项目(编号:81472145;81772298);中南大学研究生自主探索创新资助项目(编号:2017zzts212)

摘　　要：[目的]筛选在骨髓间充质干细胞(MSC)软骨分化过程中调控Sox9表达的microRNA (miRNA),探讨软骨形成过程的基因干预靶点和手段。[方法]经过鉴定的MSC行软骨分化,监测分化过程中候选miRNAs的表达趋势,研究差异表达miRNA对MSC软骨分化的影响及其与Sox9的相互作用关系。[结果]在筛选的4条具有调控Sox9能力的miRNA中,miRNA-30a和miRNA-195在MSC软骨分化过程中呈递增趋势。其中,miRNA-30a mimic转染MSC后细胞存活率无明显变化,在软骨分化21 d后,转染组甲苯胺蓝染色较对照组减弱。荧光素酶报告基因结果显示野生型Sox9 3'UTR报告质粒荧光释放被miRNA-30a mimic转染显著抑制,而突变体无此效果,同时,Western blot结果显示转染miRNA-30a mimic的MSC中,Sox9表达量较对照组降低。[结论] miRNA-30a在MSC软骨分化过程中表达逐渐上升并通过Sox9完成其负调节作用。此研究表明miRNA-30a作为调控MSC软骨分化的潜在靶点,对于软骨再生和软骨发育相关疾病的应用和防治具有重要意义。[Objective] To screen microRNAs（miRNAs） that regulate chondrogenic differentiation of bone marrow mesenchymal stem cells（MSC） by targeting Sox9, and to explore the target and approache of gene intervention during cartilage formation. [Methods] The MSC was characterized by flow cytometry and induced to chondrogenic differentiation. The expression levels of candidate miRNAs during the differentiation were monitored, while the effects of differentially expressed miRNAs on MSC chondrogenic differentiation and their interaction with Sox9 were studied. [Results] Four microRNAs that had the potential ability to regulate Sox9 were selected.Of them, microRNA-30a and microRNA-195 revealed a tendency of increasing expression levels during MSC chondrogenic differentiation. However, the cell viability of MSCs transfected by microRNA-30a mimic had no significantly change, associated with weaker toluidine blue staining in the transfection group than the control group after 21 days of chondrogenic differentiation.The luciferase reporter gene showed that the fluorescent release of the wild-type Sox9 3＇UTR reporter plasmid was significantly inhibited by microRNA-30a mimic transfection, whereas the mutant did not have this effect. In addition, western blot also showed that Sox9 expression was inhibited by microRNA-30a mimic transfection. [Conclusion] MicroRNA-30a is gradually increased during MSC chondrogenic differentiation and its negative regulation is achieved by targeting Sox9. This study demonstrates that miR-30a is a potential target for regulation of MSC chondrogenic differentiation, which might be of great significance for the application of cartilage regeneration and prevention of cartilage development-related diseases.

关 键 词：骨髓间充质干细胞 MICRORNA SOX9 软骨分化 

分 类 号：R318[医药卫生—生物医学工程]