摘要
目的明确萎缩性骨不连组织水平表达上调的hsa-miR-654-5p在人骨髓基质干细胞(hBMSCs)中对其预测靶基因骨形态发生蛋白2(BMP2)mRNA和蛋白的抑制作用,探索其在成骨分化过程中的生物学调控功能。方法分离培养hBMSCs,将第4代hBMSCs培养16 h后分别按相应体系转染细胞,再培养48 h后取六孔板内细胞提取总RNA和总蛋白,进行实时定量PCR(qRT-PCR)和Western blotting,取24孔板内细胞进行双荧光素酶报告基因检测。结果当hBMSCs中hsa-miR-654-5p过表达时,BMP2的mRNA和蛋白表达水平均发生明显下调;双荧光素酶报告基因检测提示,BMP2的预测靶位点直接受hsa-miR-654-5p的抑制调控,该靶位点被突变后hsa-miR-654-5p对BMP2的抑制作用消失。结论 hsa-miR-654-5p可通过作用于BMP2的特定靶位点而直接抑制BMP2的mRNA和蛋白表达。hsa-miR-654-5p的变化在成骨分化调控过程中具有重要作用。
Objective To study the effect of hsa-miR-654-5p in repressing bone morphogenetic protein 2（BMP2） mRNA and protein in human bone marrow mesenchymal stem cells（hBMSCs）,and explore its regulatory role in osteogenic differentiation of hBMSCs.Methods hBMSCs in the 4th passage were cultured for 16 h and transfected with hsa-miR-654-5p followed by further culture for 48 h.qRT-PCR and Western blotting were performed to detect the expressions of BMP2 mRNA and protein.Dual-luciferase reporter gene assay was employed to examine the repression of the BMP2 gene.Results BMP2 mRNA and protein expressions were significantly down-regulated in hBMSCs with hsa-miR-654-5p overexpression.Dualluci-ferase reporter gene assay indicated that the predicted target site of BMP2 was repressed directly by hsa-miR-654-5p,but this repression did not occur at the mutant predicted target site of BMP2.Conclusion hsa-miR-654-5p can directly repress the mRNA and protein expressions of BMP2 by binding to a specific target site.The changes in hsa-miR-654-5p can play an important role in osteogenic differentiation regulation of hBMSCs.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2012年第3期291-295,共5页
Journal of Southern Medical University
基金
国家自然科学青年基金(81000803)
北京市自然科学基金(7083114)~~
