miRNA-181a/b通过血清反应因子调控血管平滑肌细胞表型

miRNA- 181a / b REGULATES PHENOTYPES OF VESSEL SMOOTH MUSCLE CELLS THROUGH SERUM RESPONSE FACTOR

目的研究miR-181a/b通过血清反应因子(SRF,serum response factor)对血管平滑肌细胞向合成型表型转化及细胞增殖迁移能力的调控作用。方法将miR-181a/b瞬时转染至人主动脉平滑肌细胞(HAo SMCs)中,用实时定量PCR、CCK-8检测方法和transwell实验分别检测平滑肌细胞的表型标记基因的表达水平、细胞增殖能力和迁移能力的变化。生物信息学分析预测miR-181a/b直接靶向血清反应因子的3’UTR(非编码区),并通过实时定量PCR、western blot及双荧光素酶报告系统分别验证。结果实时定量PCR结果表示在HAo SMCs中过表达miR-181a/b能够抑制收缩表型标记基因的表达,促进合成表型标记基因的表达,CCK-8与transwell实验结果表明,miR-181a/b可增强细胞的增殖和迁移能力。双荧光素酶报告系统与western blot结果表明,miR-181a/b能够直接作用SRF,抑制SRF的蛋白表达。结论 miR-181a/b通过直接作用血清反应因子的3’UTR促进平滑肌细胞由收缩型表型向合成型表型转化。

Objective To investigate the roles of miR - 181 a/b in the smooth muscle cells （ SMC ） phenotypic switch from contractile phenotype to synthetic phenotype through serum response factor （SRF）. Methods miR - 181a/b mimics or inhibitors were transiently transfected into Human Aortic Smooth Muscle Cells （HAoSMCs）, the expression of SMC phenotype marker gene were detected by quantitative real time PCR （qRT -PCR）, the proliferation and migration ability of HAoSMCs were measured by CCK- 8 assay and transwell assay,respectively. In addi- tion, miR - 181 a/b was indicated as directly targeting at 3＇ UTR of SRF based on bioinformatic analysis and identified by qRT - PCR, western blot assay and dual - luciferase assay. Results The results of qRT - PCR showed that miR - 181a/b could down - regulate the expression level of SMC contractile marker genes and up - regulate the expression level of SMC synthetic marker genes. The results of CCK - 8 assay and transwell assay showed that miR - 181 a/b could enhance the proliferation and migration ability of HAoSMCs, respectively. The results of Dual - luciferase assay and western blot assay showed that miR - 181a/b could directly target 3＇ UTR of SRF and inhibit the protein expression level of SRF. Conclusion miR - 181 a/b regulates the shift of SMCs from contractile phenotype to synthetic phenotype through targeting at 3＇ UTR of SRF.