miR-424在人脂肪来源间充质干细胞成骨分化中的调控作用

Regulatory effect of miR-424 on the osteogenic differentiation of human adipose derived mesenchymal stem cells

目的探讨miR-424在人脂肪来源间充质干细胞(h ASC)成骨分化中的表达及miR-424对h ASC体外成骨分化的生物学作用。方法 q PCR检测miR-424在h ASC成骨分化不同阶段的表达水平。通过慢病毒颗粒在体外感染h ASC,使miR-424过表达或抑制其表达,进行Western blotting、细胞免疫荧光试验及碱性磷酸酶(ALP)和茜素红(ARS)染色试验,研究miR-424对骨形态发生蛋白2(BMP-2)及成骨诱导液诱导h ASC成骨分化过程的影响。预测miR-424的靶基因,并利用q PCR及Western blotting验证。结果 q PCR结果显示miR-424在h ASC的表达量随着成骨诱导时间延长逐渐下降(P<0.05)。在BMP-2诱导下,过表达组的碱性磷酸酶(ALP)和骨桥蛋白(OPN)蛋白水平均下降,而抑制组的表达增加(P<0.05)。细胞免疫荧光显示,过表达miR-424的细胞骨唾液酸蛋白(BSP)阳性率显著下降(P<0.05)。ALP和ARS染色显示,过表达组ALP活性降低,钙盐沉积下降,而抑制组ALP活性升高,矿化能力增强。过表达miR-424能降低h ASC中Smad5的mRNA和蛋白水平,而抑制miR-424则使其水平升高(P<0.05)。结论 miR-424在h ASC成骨分化中表达下调,其在hASC体外成骨分化过程中起抑制作用。

Objective To explore the expression and biological effect of mLR-424 during osteogenic differentiation of human adipose derived mesenchymal stem cells （ hASC）. Methods The expressions of miR- 424 at different stages of osteogenic differentiation of hASC were detected with real-time PGR. miR-424 was over-expressed or miR-424 expression was inhibited vhz infection of hASCs with lentivirus particles in vitro. Western blotting, immunocytochemistry, and alkaline phosphatase （ALP） staining and alizarin red （ARS） staining were performed. The effects of miR-424 on osteogenic differentiation of hASGs induced with BMP-2 and osteogenic differentiation medium were investigated. The target gene of miR-424 was predicted and was verified with qPGR and Western blotting. Results The results of qPGR revealed that the expression of miR-424 declined with the time of osteogenic differentiation （P 〈 0.05）. The protein levels of ALP and osteopontin （OPN） decreased in the over-expression group and increased in the inhibition group with the induction of bone morphogenetic protein 2（BMP-2） （P〈0.05） . The results of immunocytochemistry revealed that over-expression of miR-424 significantly reduced the cell positive rate of bone sialoprotein （BSP） （P〈0.05）. ALP and ARS staining showed that the activity of ALP and capacity of mineralization in the over-expression group decreased, while the activity of ALP and capacity of mineralization increased in the inhibition group. Over-expression of miR- 424 could decrease the mRNA and protein expression levels of Smad5 in hASCs and inhibition of miR-424 could increase the mRNA and protein expression levels of Smad5 （P 〈 0.05）. Conclusion The expression of miR- 424 declines during the osteogenic differentiation of hASCs and miR- 424 inhibits the osteogenic differentiation of hASCs.