MiR-155对血管紧张素Ⅱ诱导小鼠主动脉血管平滑肌细胞表型转换的影响及机制研究

摘要

目的:观察miR-155对血管紧张素Ⅱ(AngⅡ)诱导小鼠主动脉血管平滑肌细胞(VSMC)表型转换的影响并研究其可能的机制.方法:原代培养小鼠VSMC,用不同浓度、时间AngⅡ作用于VSMC,然后用实时荧光定量聚合酶链式反应(qRT-PCR)检测不同处理组miR-155表达的变化情况.用qRT-PCR检测过表达及抑制表达miR-155后空白对照组、miR-155 mimics组、mimics阴性对照组、miR-155 inhibitors组、inhibitors阴性对照组的miR-155水平变化情况,检测转染效率.用免疫蛋白印迹(Western blot)法检测过表达及抑制表达miR-155后空白对照组、AngⅡ组、miR-155 mimics组、AngⅡ+miR-155 mimics组、miR-155 inhibitors组、AngⅡ+miR-155 inhibitors组对AngⅡ激活哺乳动物雷帕霉素靶蛋白(mTOR)及细胞外信号调节激酶1/2(ERK1/2)通路的影响.Western blot检测转染miR-155、使用雷帕霉素、细胞外信号调节激酶1/2抑制剂(U0126)后空白对照、AngⅡ组、miR-155 mimics组、AngⅡ+miR-155 mimics组、AngⅡ+U0126组、AngⅡ+雷帕霉组AngⅡ促进VSMC表型转换的影响,检测两个收缩表型蛋白标志物即平滑肌22α(SM22α)与 α-平滑肌肌动蛋白(α-SM-actin),及一个合成表型蛋白标志物骨桥蛋白(OPN).结果:与作用0 h或0 mol/L AngⅡ相比,AngⅡ作用于VSMC后AngⅡ以浓度、时间依赖方式降低miR-155的表达.转染miR-155后可显著减少基础及AngⅡ促进mTOR与ERK1/2通路激活作用,而转染miR-155抑制剂可进一步增加基础及AngⅡ促进mTOR与ERK1/2通路激活作用.雷帕霉素、U0126及转染miR-155均可抑制AngⅡ减少收缩表型蛋白标志物SM22α 与 α-SM-actin的表达,同时抑制AngⅡ促进合成表型蛋白标志物OPN的表达.结论:AngⅡ通过抑制miR-155表达促进mTOR与ERK1/2激活促进VSMC表型转换.%Objective: To observe the effect of miR-155 on angiotensin Ⅱ (AngⅡ)-induced mice vascular smooth muscle cell (VSMC) phenotype switching with its possible mechanism. Methods: Primary cultured mice VSMCs were treated by AngⅡ at different concentrations and time periods, relevant expressions of miR-155 were examined by RT-PCR. qRT-PCR was conducted to determine miR-155 changes in Blank control group, miR-155 mimics group, miR-155 mimics negative control (NC) group, miR-155 inhibitor group and miR-155 inhibitor NC group. Western blot analysis was performed to measure the effect of miR-155 on AngⅡ-enforced ERK1/2 and mTOR signaling pathway in Blank control group, AngⅡ group, miR-155 mimics group, AngⅡ+miR-155 mimics group, miR-155 inhibitor group and AngⅡ+miR-155 inhibitor group; to detect the impact of miR-155, rapamycin (Rap) and U0126 on AngⅡ promoted VSMC phenotype switching in Blank control group, AngⅡ group, miR-155 mimics group, AngⅡ+miR-155 mimics group, AngⅡ+U0126 group and AngⅡ+Rap group, and to detect protein expressions of SM22α, α-SM-actin (contractile phenotype marker protein) and OPN (synthetic phenotype marker). Results: AngⅡ decreasing miR-155 expression was in a dose- and time-dependent manner. miR-155 could reduce the basal and AngⅡ-promoted ERK1/2, mTOR signaling pathway, while miR-155 inhibitor could elevate the above effect. Rap, U0126 and miR-155 could inhibit AngⅡ-attenuated expressions of SM22α, α-SM-actin and meanwhile inhibit AngⅡ-enforced expression of OPN. Conclusion: miR-155 could inhibit mice AngⅡ-promoted VSMC phenotype switching which might be via inhibiting the activations of mTOR and ERK1/2.