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- 2018
miRNA-21通过TPM1参与高糖诱导的人主动脉平滑肌细胞表型转化与增殖
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Abstract:
摘要：目的 探讨miRNA-21在高糖诱导的人主动脉平滑肌细胞表型转化与增殖中的作用，以及对靶基因原肌球蛋白1(tropomyosin 1, TPM1)表达的影响。方法? ?体外培养人主动脉平滑肌原代细胞，利用脂质体转染miRNA-21 mimic和inhibitor至细胞中，BrdU法检测高糖刺激miRNA-21过表达和缺失情况下人主动脉平滑肌细胞的增殖情况，荧光定量PCR检测转染前后高糖刺激时细胞miRNA-21和TPM1的表达情况，Western blot检测人主动脉平滑肌细胞中TPM1、骨桥蛋白(osteopontin, OPN)、平滑肌蛋白22α(smooth muscle 22 alpha, SM22α)的表达情况。结果? ?10、20、25mmol/L D-葡萄糖刺激时人主动脉平滑肌细胞较正常对照组(0.613±0.022)显著增殖(0.725±0.026、0.913±0.024、1.513±0.082)，差异有统计学意义(P<0.01)；10、20、25mmol/L D-葡萄糖刺激时miRNA-21表达较对照组升高1.1、2.0、3.1倍(P<0.01)；正常组细胞转染miRNA-21 mimic后细胞增殖能力增强1.6倍，转染miRNA-21 inhibitor后高糖组细胞增殖能力减弱(P<0.01)；转染miRNA-21 mimic后TPM1表达减弱，SM22α表达下调，OPN表达增加(P<0.01)，转染miRNA-21 inhibitor后TPM1表达上调，SM22α表达上调，OPN表达减少(P<0.01)。结论? ?miRNA-21可下调细胞TPM1的表达，从而促进人主动脉平滑肌细胞表型转化与增殖。
ABSTRACT: Objective? ?To investigate the effect of miRNA-21 on human aortic smooth muscle cells (HASMC) phenotype switching and proliferation induced by high glucose and expression of its target tropomyosin 1 (TPM1). Methods?? miRNA-21 mimic and inhibitor were transfected into HASMC by lipofectamine 2000 in vitro. BrdU method was employed to test the proliferation of HASMC under high glucose and overexpression and absence of miRNA-21; qRT-PCR was used to detect the expressions of miRNA-21 and TPM1 under different conditions. Western blot was used to detect the expressions of TPM1, osteopontin (OPN), and smooth muscle 22 alpha (SM22α). Results?? HASMC significantly proliferated (0.725±0.026, 0.913±0.024, 1.513±0.082) under high glucose (10, 20 and 25mmol/L) compared with control group (0.613±0.022, P<0.01). The expression of miRNA-21 was higher than that in the control group under high glucose (P<0.01). Cell proliferation ability was enhanced 1.6 times in miRNA-21 mimic group than in normal group and decreased in the group of miRNA-21 inhibitor (P<0.01). After transfected with miRNA-21 mimic, the expressions of TPM1 and SM22α reduced and that of OPN increased (P<0.01). After transfection with miRNA-21mimic,TPM1 was upregulated, the expression of SM22α increased while that of OPN decreased (P<0.01). Conclusion?? miRNA-21 promotes the switching and proliferation of HASMC phenotype by down-regulating the expression of TPM1
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