miR-186靶向调控骨桥蛋白基因表达对骨髓间充质干细胞成骨分化的影响

摘要

Objective:To explore the effects of the miR-186 target-regulating the osteopontin(OPN) expression on the osteogenic differentiation of bone marrow mesenchymal stem cells(bMSCs). Methods:The bMSCs were isolated from bone marrow,induced with BMP 2,and identified using the Alkaline Phosphatase( ALP) and Von-kossa staining. Targeting pairing relationship between miR-186 and OPN was predicted by bioinformatics,and identified with luciferase assay. After transfecting miR-186 mimics and siRNA interfering OPN gene into cells,the expressions of miR-186 and OPN were determined by real-time PCR,and the protein expression of OPN was detected using western blot. Results:The uniform distribution of bMSCs was found under light microscope,and the induced cells could be stained blue by ALP. MiRanda and TargetScan showed that miR-186 was well complementary with OPN gene. The result of luciferase assay showed that miR-186 could inhibit the expression of OPN mRNA. The results of real-time PCR and western blot showed that the over-expression of miR-186 could downregulate the expressions of OPN mRNA and protein. Conclusions:MiR-186 can inhibit the osteogenic differentiation of bMSCs by downregulating the OPN gene expression.%目的:探讨miR-186对骨髓间充质干细胞(bMSCs)成骨向诱导分化过程中骨桥蛋白(OPN)基因的靶向作用及其对bMSCs成骨分化的影响.方法:分离培养bMSCs,应用骨形成蛋白2成骨向诱导分化并采用碱性磷酸酶染色和Von-kossa染色鉴定;运用生物信息学方法对miR-186和OPN基因的靶向配对关系进行预测,采用荧光素酶报告系统鉴定;脂质体2000转染miR-186模拟物以及干扰OPN基因的siRNA后,Real-time PCR检测miR-186和OPN mRNA的表达,Western blot检测OPN蛋白的表达.结果:光镜下可见bMSCs均匀分布,诱导分化后的细胞碱性磷酸酶染色呈蓝色.生物信息学软件TargetScan和miRanda显示miR-186和OPN基因二者靶向配对良好,荧光素酶报告系统进一步验证发现,miR-186能够抑制OPN mRNA表达.Real-time PCR和Western blot检测结果表明过表达miR-186能够降低OPN mRNA和蛋白的表达.结论:miR-186通过下调bMSCs成骨向诱导分化过程中OPN基因表达进而抑制bMSCs的成骨分化.