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该研究预测并验证骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSCs)中mi R-181a对靶分子骨保护素(osteoprotegerin,OPG)m RNA水平的调控作用,探讨其在骨质疏松发病过程中对破骨细胞活性的影响。通过生物信息学预测mi R-181a可能调控的靶基因OPG,构建含mi R-181a结合位点的OPG m RNA 3′UTR(3′untranslated regions)荧光素酶报告载体。通过双荧光素酶载体系统检测mi R-181a与OPG m RNA 3′UTR相互作用对荧光素酶活性的影响;转染mi R-181a进入BMMSCs并与破骨细胞共培养,TRAP(tartrate resistant acid phosphatase)染色检测共培养体系中破骨细胞数目的改变。结果表明,双荧光素酶报告提示,mi R-181a的靶分子为OPG m RNA,上调mi R-181a(mimics组)水平后,破骨细胞数目较对照组显著增多(P<0.05);下调mi R-181a(inhibitor组)水平后,破骨细胞数目较对照组显著降低(P<0.05)。研究结果显示,mi R-181a可以负调控OPG的蛋白水平,进而影响破骨细胞活性。
This study predicts and validates the regulatory effect of mi R-181a on the mRNA level of osteoprotegerin (OPG) in bone marrow mesenchymal stem cells (BMMSCs), and explores its role in the pathogenesis of osteoporosis On the activity of osteoclasts. Bioinformatics was used to predict OPG, a target gene possibly regulated by mi R-181a, to construct an OPG mRNA 3’UTR-linked luciferase reporter vector containing mi R-181a binding site. Luciferase system was used to detect the effect of mi R-181a and OPG m RNA 3’UTR on luciferase activity; mi R-181a was transfected into BMMSCs and co-cultured with osteoclasts, TRAP (tartrate resistant acid phosphatase staining to detect changes in the number of osteoclasts in the co-culture system. The results showed that dual luciferase reporter suggested that the target molecule of mi R-181a was OPG m RNA. The number of osteoclasts increased significantly (P <0.05) after upregulation of mi R-181a (mimics group) Compared with control group, the number of osteoclasts was significantly decreased after mi R-181a (inhibitor group) level (P <0.05). The results show that mi R-181a can negatively regulate the protein level of OPG, thereby affecting the activity of osteoclasts.