微小RNA-30d-5p通过葡萄糖调节蛋白78调控骨髓基质细胞成骨分化的机制

Mechanism of microRNA-30d-5p regulating osteogenic differentiation of bone marrow stromal cells through glucose-regulated 78

目的探讨微小RNA(miR)-30d-5p对骨髓基质细胞成骨分化和凋亡的影响及其作用机制。方法将骨髓基质细胞分为miR-30d-5p过表达阴性对照组、miR-30d-5p过表达组、miR-30d-5p抑制阴性对照组和miR-30d-5p抑制组。碱性磷酸酶(ALP)染色鉴定成骨效果,茜素红染色检测钙结节沉淀情况,TUNEL检测凋亡水平。Real-time PCR、Western blotting检测mRNA和蛋白表达水平,生物信息学分析网站Targetscan 7.1预测miR-30d-5p的潜在结合位点。结果MiR-30d-5p过表达后,细胞成骨分化能力和矿化能力均降低(P<0.05),而细胞凋亡水平升高(P<0.05)。葡萄糖调节蛋白78(GRP78)和成骨特异性Runt相关转录因子2(RUNX2)蛋白表达均显著降低(P<0.05)。但是,miR-30d-5p抑制剂处理细胞后,细胞成骨分化能力和矿化能力均有提升(P<0.05),细胞凋亡水平降低(P<0.05)。GRP78和RUNX2蛋白水平均升高(P<0.05)。MiR-30d-5p结合位点位于GRP78基因3’UTR的142~148 bp处。结论MiR-30d-5p通过下调GRP78蛋白的表达抑制骨髓基质细胞成骨分化,并且促进细胞凋亡。

Objective To investigate the effect of microRNA(miR)-30d-5p on osteogenic differentiation and apoptosis of bone marrow stromal cells and its mechanism.Methods Bone marrow stromal cells were divided into miR30d-5p overexpression negative control group,miR-30d-5p overexpression group,miR-30d-5p inhibition negative control group and miR-30d-5p inhibition group.Alkaline phosphatase(ALP)staining was used to identify osteogenesis,alizarin red staining was used to detect calcium nodules precipitation,and TUNEL was used to detect apoptosis.mRNA and protein expression levels were detected by Real-time PCR and Western blotting,and the potential binding sites of miR-30d-5p were predicted by the bioinformatics analysis website Targetscan 7.1.Results After miR-30d-5p overexpression,osteogenic differentiation ability,and mineralization ability of the cells decreased(P<0.05),while apoptosis level increased(P<0.05).The expression of glucoregulatory protein 78(GRP78)and osteogenic specific transcription factor Runt related transcription factor 2(RUNX2)decreased significantly(P<0.05).However,miR-30d-5p inhibitor-treated the cells with increased osteogenic differentiation and mineralization ability(P<0.05),and apoptosis level decreased(P<0.05).GRP78 and RUNX2 protein levels increased(P<0.05).The miR-30d-5p binding site was located at 142-148 bp of the 3’UTR of the GRP78 gene.Conclusion MiR-30d-5p inhibits osteogenic differentiation and promotes apoptosis of bone marrow stromal cells by down-regulating the expression of GRP78 protein.