miR-150下调Notch3抑制人脂肪干细胞成骨分化

MicroRNA-150 inhibts osteogenic differentiation of human adipose-derived stem cells by downregulating Notch3

目的:探讨miR-150调控Notch3对人脂肪干细胞(hADSCs)成骨分化的影响。方法:体外分离和培养hADSCs,将其分为空白对照组(NC组)、绿色荧光蛋白对照组(GFP组)和过表达miR-150组(miR-150组)。实时荧光定量PCR法检测各组hADSCs miR-150的表达,免疫印迹检测hADSCs中成骨标志物RUNX-2、Notch3的表达。构建Notch3敲低的hADSCs组(Notch3-siR组)、GFP组和NC组,免疫印迹检测3组hADSCs Notch3的表达。利用成骨细胞诱导液定向诱导各组hADSCs向成骨细胞分化,免疫印迹检测各组hADSCs RUNX-2的表达。结果:hADSCs成骨诱导14 d期间,miR-150表达逐渐下降。miR-150组的Notch3表达明显低于GFP组和NC组。经体外成骨诱导后,miR-150组RUNX-2蛋白表达水平低于GFP组和NC组。敲低Notch3后体外诱导hADSCs,Notch3-siR组RUNX-2蛋白的表达水平低于GFP组和NC组。结论:miR-150通过下调Notch3表达抑制hADSCs成骨分化。

Objective:To investigate the effect of miR-150 on osteogenic differentiation of human adipose stem cells(hADSCs)by regulating Notch3.Methods:hADSCs in vitro were isolated and cultured and divided into normal control(NC)group,green fluorescent protein control group(GFP group),and miR-150 overexpressing group(miR-150 group).Real-time PCR was used to detect the expression of miR-150 in cells of each group,and Western blotting was used to detect the expression level of osteogenic marker RUNX-2,Notch3.The hADSCs of Notch3 knockdown group(Notch3-siR group),GFP group and NC group were cultured.The expression level of Notch3 was detected by Western blotting.Osteoinductive fluid were used to induce hADSCs to differentiate into osteoblasts.The expression level of RUNX-2 was detected by Western blotting.Results:During 14 d osteogenic induction,miR-150 expression in hADSCs gradually decreased.Notch3 expression was statistically downregulated in miR-150 group compared to GFP group and NC group.And hADSCs overexpressing miR-150 caused statistical downregulation of RUNX-2 protein compared to those control cells and cells treated with GFP.Transfection with siRNA targeting Notch3 produced statistical decrease in RUNX-2 expression in hADSCs cultured in osteogenic medium.Conclusion:miR-150 inhibits osteogenic differentiation of hADSCs by downregulating Notch3 expression.