miR-19b通过靶向ARC促进心肌缺血再灌注损伤

miR-19b promotes myocardial ischemia reperfusion injury by regulating activity-regulated cytoskeleton associated protein

目的 探索miR-19b通过靶向调节活性调节细胞骨架相关蛋白(activity-regulated cytoskeleton-associated protein, ARC)促进心肌缺血再灌注损伤(myocardial ischemia reperfusion injury, I-RI)的作用及其机制。方法 选择健康雄性C57B/L6小鼠20只按照体重随机分为2组,正常对照组(假手术组)和心肌缺血再灌注损伤组(n=10)。离体水平,利用过氧化氢(HO)诱导心肌细胞损伤,分为正常对照组、过氧化氢处理组、过氧化氢+miR-19b组和过氧化氢+阴性对照组。利用Real-time PCR检测小鼠心肌和心肌细胞中miR-19b的表达水平;蛋白免疫印迹技术检测ARC蛋白表达水平;利用MTT检测细胞活力,采用试剂盒检测乳酸脱氢酶(LDH)的活性,分析心肌细胞损伤情况。构建miR-19b拟似物(miR-19b mimics)、miR-19b抑制物(AMO-miR-19b)、miR-19b拟似物的阴性对照(NC-miR-19b mimics)和miR-19b抑制物的阴性对照(NC-AMO-miR-19b),利用瞬时转染技术,将其转染至心肌细胞中,进行心肌细胞中的miR-19b的过表达和干扰,进而分析miR-19b对ARC蛋白表达的影响。结果 (1)miR-19b在缺血再灌住损伤小鼠心脏和HO诱导损伤的心肌细胞中表达上调(P<0.05);(2)miR-19b过表达加重HO诱导心肌细胞损伤(P<0.05);(3)ARC在心肌缺血再灌住损伤小鼠心脏表达下调(P<0.05);(4)miR-19b负性调控了ARC的表达(P<0.05)。结论 miR-19b能够通过负性调控ARC的表达从而促进心肌缺血再灌注损伤。

Objective To explore the effect of miR-19 b on aggravation of myocardial ischemia reperfusion injury through regulating the activity-regulated cytoskeleton associated protein(ARC) and its mechanism. Methods Twenty male SPF grade C57 B/L6 mice were selected and randomly divided into 2 groups, normal control group(10 mice) and myocardial ischemia reperfusion injury group(10 mice). In vitro, cardiomyocytes injury by using hydrogen peroxide was induced. The expression level of miR-19 b was detected by Real-time PCR. The protein expression level was measured by Western blot. Cell vitality and LDH activity were used to inflect the injury degree of cardiomyocytes. MiR-19 b mimics, AMO-miR-19 b, NC-miR-19 b mimics and NC-AMO-miR-19 b was constructed, then which were transfected into cardiomyocytes to over-express or interfere the expression of miR-19 b in cells. Results(1)miR-19 b was up-regulated in the mice heart of myocardial ischemia reperfusion injury and injury cardiomyocytes induced by HO(P<0.05).(2)Over-expression of miR-19 b alleviated the cardiomyocytes injury based on HOtreatment(P<0.05).(3)The protein expression of ARC was down-regulated in the mice heart of myocardial ischemia reperfusion injury(P<0.05).(4)miR-19 b negatively regulated the protein expression of ARC(P<0.05). Conclusion Over-expression of miR-19 b may contribute to myocardial ischemia injury by negatively regulating ARC expression.