miR-195对人骨髓间充质干细胞成骨分化的影响

miR-195 effect on osteogenic differentiation of human bone marrow mesenchymal stem cells

背景:既往研究发现,miR-195在人骨髓间充质干细胞成骨分化过程中,表达量显著增加,但是其作用及其机制尚不明确。目的:探索mi R-195对人骨髓间充质干细胞成骨分化的影响及其机制。方法:体外分离、培养人骨髓间充质干细胞,通过碱性磷酸酶活性测定试剂盒、蛋白免疫和实时定量反转录PCR(q RT-PCR)检测在人骨髓间充质干细胞诱导分化过程中,碱性磷酸酶活性和骨分化特异基因Runx2和osteopontin表达水平的变化,以及miR-195和它的潜在靶SMAD7表达量的变化;通过脂质体转染构建miR-195低表达的人骨髓间充质干细胞,研究miR-195对人骨髓间充质干细胞成骨分化的影响。利用荧光素酶报告基因实验检测miR-195对SMAD7的靶向作用。结果与结论:(1)分离培养的人骨髓间充质干细胞具有优良的体外成骨分化能力;(2)miR-195表达量随人骨髓间充质干细胞诱导分化时间的增加而升高,SMAD7则相反。(3)mi R-195能够促进人骨髓间充质干细胞成骨分化;(4)荧光素酶实验证实SMAD7是miR-195的直接靶,SMAD7过表达抑制人骨髓间充质干细胞成骨分化;(5)结果提示,miR-195通过靶向SMAD7促进人骨髓间充质干细胞成骨分化。

BACKGROUND：Previous studies have found that the expression level of miR-195 in differentiated human bone marrow mesenchymal stem cel s （hBMMSCs） is significantly higher than that in undifferentiated hBMMSCs. However, miR-195 effect during this differentiation process and possible mechanism remain unclear. OBJECTIVE：To explore the effect of miR-195 on osteogenic differentiation of hBMMSCs and possible mechanism. METHODS：hBMMSCs were isolated and cultured in vitro. Alkaline phosphatase activity, Runx2, osteopontin and SMAD7 protein expression and miR-195 expression level during osteogenic differentiation of hBMMSCs were determined by alkaline phosphatase kit, western blot and real-time PCR, respectively. miR-195-downexpressed hBMMSCs were constructed by lipofection transfection, and were used to investigate the effect of miR-195 was on osteogenic differentiation of hBMMSCs. Dual luciferase reporter assay was used to identify whether the 3’UTR of SMAD7 mRNA was a binding target of miR-195. In addition, we transfected hBMMSCs with SMAD7 cDNA （pcDNA-SMAD7）, and investigated the effect of SMAD7 on osteogenic differentiation of hBMMSCs. RESULTS AND CONCLUSION：The isolated and cultured hBMMSCs had good osteogenic differentiation ability in vitro. Expression level of miR-195 was increased with the increasing of induction time, and the expression level of SMAD7 was reversed. miR-195 promoted osteogenic differentiation of hBMMSCs. Luciferase assay confirmed that miR-195 targeted SMAD7 directly, and overexpression of SMAD7 inhibited the osteogenic differentiation of hBMMSCs. Taken together, miR-195 promotes osteogenic differentiation of hBMMSCs by targeting SMAD7.