MiRNA-26a通过调控结缔组织生长因子缓解血管平滑肌细胞的钙化

miRNA-26a reduces vascular smooth muscle cell calcification by regulating connective tissue growth factor

目的探究miRNA-26a调控结缔组织生长因子对血管平滑肌细胞钙化的作用。方法将细胞分为对照组、模型组、模型+AR234960组、AR234960+miR-26a mimic组和miR-26a mimic组。对照组细胞正常培养基培养,模型组VSMC细胞诱导钙化,模型+AR234960组的VSMC细胞诱导钙化后加入AR234960激动剂干预,AR234960+miR-26a mimic组的VSMC细胞诱导钙化后用AR234960激动剂和miR-26a mimic处理,miR-26a mimic组的VSMC细胞诱导钙化后转染miR-26a mimic。茜素红染色测定各组细胞钙化沉积;试剂盒检测各组细胞碱性磷酸酶(ALP)活性;荧光定量PCR和Westernblot检测各组细胞miR-26a、OPG、OPN、BMP-2、Collagen II基因和蛋白表达情况。双荧光素酶基因验证miR-26a与CTGF的结合。结果平滑肌A7r5细胞钙化后,miR-26a的表达随着钙化时间的增加而降低(P<0.05),而CTGF的表达随钙化时间的增加而增加(P<0.05)。茜素红染色结果显示,AR234960能够增加细胞的钙化程度,而miR-26a mimic能够减轻细胞钙化。与对照组相比,模型组ALP活性、OPN、BMP-2、Collagen IImRNA和蛋白的表达显著升高(P<0.05),OPG mRNA和蛋白的表达显著降低(P<0.05);与模型组相比,模型+AR234960组上述指标均无显著差异(P>0.05),而miR-26a mimic组ALP活性、OPN、BMP-2、Collagen IImRNA和蛋白的表达显著降低(P<0.05),OPG mRNA和蛋白的表达显著升高(P<0.05);与miR-26a mimic组相比,AR234960+miR-26a mimic组ALP活性、OPN、BMP-2、Collagen IImRNA和蛋白的表达显著升高(P<0.05),OPG mRNA和蛋白的表达显著降低(P<0.05)。双荧光素酶基因检测结果显示,miR-26a-inhibitor组CTGF 3'-UTR野生型中相对荧光素酶活性较miR-26a-mimic组显著升高(P<0.05)。结论miRNA-26a能够有效降低细胞中ALP活性及OPN、BMP-2、Collagen II的表达,同时增加钙化细胞中OPG水平,为miRNA-26a通过调控CTGF水平进而对缓解血管平滑肌细胞钙化提供了理论依据。

Objective To investigate the regulatory role of miRNA-26a in vascular smooth muscle cell(VSMC)calcification by regulating connective tissue growth factor(CTGF).Methods Rat thoracic aorta VSMCs(A7r5 cells)with induced calcification were treated with AR234960 agonist or transfected with miR-26a mimic,or with both treatments.Alizarin red staining was used to determine calcium deposition,and phosphatase(ALP)activity in the cells was measured.The mRNA and protein expressions of miR-26a,OPG,OPN,BMP-2 and collagen II were detected using qPCR and Western blotting.The binding of miR-26a to CTGF was verified using dual luciferase reporter gene assay.Results After induced calcification,A7r5 cells showed gradually decreased miR-26a expression(P<0.05)and progressively increased CTGF expression(P<0.05)with the extension of induction time.Treatment of the cells with AR234960 obviously increased calcification in the cells,while transfection with miR 26a mimic significantly reduced cell calcification.The calcifying cells showed significantly increased ALP activity and expressions of OPN,BMP-2 and collagen II(P<0.05)and lowered OPG expression(P<0.05),and treatment with AR234960 did not produce obvious effects on these changes(P>0.05).Transfection with miR-26a mimic resulted in significantly decreased ALP activity and expressions OPN,BMP-2 and collagen II expression(P<0.05)and increased OPG expression(P<0.05)in the calcifying cells.These effects of miR-26a mimic was significantly attenuated by treatment of the cells with AR234960(P<0.05).The result of luciferase reporter gene assay confirmed the binding of miR-26a to CTGF.Conclusion miRNA-26a can effectively alleviate vascular calcification by lowering the level of CTGF,reducing ALP activity and the expressions of OPN,BMP-2 and collagen II,and increasing the expression of OPG.