miR-155抑制AngⅡ诱导的主动脉血管平滑肌细胞周期转换
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国家自然科学基金面上项目(81170250)


Inhibition of miR-155 on AngⅡ-stimulated cell cycle progression of vascular smooth muscle cells
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    摘要:

    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VSMC 48 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-RT-PCR及Western blot检测分别转染miR-155及阴性对照后各组AngⅡ 1型受体(angiotensin Ⅱ type 1 receptor,AT1R)的mRNA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、mTOR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mRNA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。

    Abstract:

    Objective: To investigate the effect of miR-155 on cell cycle progression of mice vascular smooth muscle cells under treatment of AngⅡ, and explore the detailed mechanism. Methods: The vascular smooth muscle cells(VSMCs) derived from C57 mice were cultured by the adherent method (1×10-6 mol/L AngⅡ for 48 h). Quantitative real-time PCR (q-RT-PCR) was performed to detect the miR-155 expression levels of the blank control group and the Ang Ⅱ group. q-RT-PCR and Western blot assay were performed to detect the mRNA and protein levels of angiotensin Ⅱ type 1 receptor(AT1R) in the transfected miR-155 group and the negative control group; Western blot assay was performed to detect the effect of transfected miR-155 mimics and negative control group on extracellular signal-regulated kinase 1/2 (ERK1/2) of AT1R downstream and ribosomal protein S6 kinase (P70S6K1) signaling pathway. The expression of cyclin D1(Cyclin D1) was examined by transfection of miR-155 mimics,angiotensin receptor blocker valsartan,mTOR pathway inhibitor rapamycin,and ERK1/2 inhibitor U0126. In the end,we used flow cytometer to analyze the change of cell cycle to investigate the effect of miR-155 on cell cycle and its mechanism. Results: AngⅡ significantly decreased the expression of miR-155. miR-155 mimics remarkably attenuated AT1R expression from mRNA and protein levels,inhibited the AngⅡ-activated ERK1/2 and P70S6K signaling pathway,and decreased the AngⅡ-enforced expression of Cyclin D1. Transfection of miR-155,valsartan,rapamycin and U0126 suppressed the AngⅡ-induced G1-to S-phase progression of VSMC. Conclusion: MiR-155 can inhibit the expression of AngⅡ-induced ERK1/2,activation of P70S6K1 signaling pathway,and Cyclin D1 by blocking AT1R,and thereby inhibit the function of Ang Ⅱ-induced cell cycle transition.

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张紫微,杨丽霞,郭瑞威,吕晋琳,陆霓虹,王先梅,石燕昆. miR-155抑制AngⅡ诱导的主动脉血管平滑肌细胞周期转换[J].南京医科大学学报(自然科学版),2017,(3):281-286,334

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  • 收稿日期:2016-07-11
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  • 在线发布日期: 2017-04-07
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