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目的:观察转染miR-155后对血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导小鼠主动脉血管平滑肌细胞(vascular smooth muscle cell,VSMC)细胞周期转换的影响并探讨其机制。方法:原代培养小鼠VSMC,用1×10-6 mol/L AngⅡ作用于VSMC48 h后,采用实时荧光定量PCR(quantitative real-time PCR,q-RT-PCR)检测空白对照组及AngⅡ组miR-155表达水平。用q-RTPCR及Western blot检测分别转染miR-155及阴性对照后各组AngⅡ1型受体(angiotensinⅡtype 1 receptor,AT1R)的m RNA及蛋白表达水平;用Western blot检测转染miR-155 mimic及阴性对照后对AT1R下游细胞外信号调节激酶1/2(extracellular signal-regulated kinase 1/2,ERK1/2)、核糖体蛋白S6激酶(P70S6K1)通路的影响。分别检测转染miR-155 mimic及使用血管紧张素受体阻滞剂缬沙坦、m TOR通路抑制剂雷帕霉素、ERK1/2抑制剂U0126对细胞周期素D1(Cyclin D1)的表达影响,并使用流式细胞分析检测细胞周期变化,探讨miR-155对细胞周期的影响及其机制。结果:AngⅡ可显著降低miR-155的表达。miR-155可从m RNA及蛋白水平抑制AT1R表达,并抑制AngⅡ促AT1R表达的作用。转染miR-155 mimic后可显著抑制AngⅡ促进ERK1/2、P70S6K1通路激活的作用。转染miR-155 mimic、使用缬沙坦、雷帕霉素、U0126抑制AngⅡ促Cyclin D1表达的作用,并使细胞周期阻滞在G0/G1期。结论:miR-155可通过抑制AT1R间接抑制AngⅡ促进ERK1/2、P70S6K1通路激活及Cyclin D1表达的作用,抑制AngⅡ促进细胞周期转换的作用。
OBJECTIVE: To investigate the effect of transfection of miR-155 on the cell cycle of mouse aortic vascular smooth muscle cells (VSMC) induced by angiotensin Ⅱ (AngⅡ) and its mechanism. Methods: Primary cultured mouse VSMCs were treated with 1 × 10-6 mol / L Ang Ⅱ for 48 h, then the real-time quantitative PCR (q-RT-PCR) miR-155 expression level. The expression of m RNA and protein of angiotensin Ⅱ type 1 receptor (AT1R) in each group were detected by q-RTPCR and Western blot after transfected with miR-155 and negative control, respectively. Negative control on the expression of extracellular signal-regulated kinase 1/2 (ERK1 / 2) and ribosomal protein S6 kinase (P70S6K1) pathway downstream of AT1R. The effects of miR-155 mimic and angiotensin receptor blocker valsartan, the mTOR pathway inhibitor rapamycin, and the ERK1 / 2 inhibitor U0126 on the expression of cyclin D1 , And the use of flow cytometry to detect cell cycle changes to explore the impact of miR-155 on cell cycle and its mechanism. Results: Ang Ⅱ significantly reduced the expression of miR-155. miR-155 inhibits AT1R expression from m RNA and protein levels and inhibits Ang II-induced AT1R expression. Transfection of miR-155 mimic could significantly inhibit the effect of AngⅡ on ERK1 / 2 and P70S6K1 pathway activation. Transfection of miR-155 mimic with valsartan, rapamycin and U0126 inhibited AngⅡ-induced Cyclin D1 expression and arrested cell cycle in G0 / G1 phase. CONCLUSION: miR-155 can inhibit the activation of ERK1 / 2 and P70S6K1 pathway and the expression of Cyclin D1 by inhibiting AT1R indirectly, and inhibit the effect of AngⅡ on cell cycle transformation.