Objective: To investigate the effect of miR-133b on cardiomyocyte apoptosis induced by myocardial ischemia-reperfusion (I/R) and explore the mechanism.
Methods: Thirty-six adult SD rats were randomized into sham-operated group, I/R group, AdmiR-NC group and AdmiR-133b group, and rat models of myocardial I/R were established in the latter 3 groups with myocardial injections of saline or recombinant adenoviruses in the left ventricle. The expression of MiR-133b was detected using RT-qPCR, and cardiac function of the rats was determined using FDP 1 HRV and BRS analysis system. Serum CK-MB and cTnI levels were determined by ELISA, myocardial injury was evaluated with HE staining, cardiomocyte apoptosis was detected by flow cytometry, and ROS content was determined using a DCFH-DA probe. In the in vitro experiment, H9C2 myocardial cells with hypoxia/reoxygenation (H/R) treatment were transfected with Mir-NC or MiR-133b mimic, and the cellular expression of MiR-133b, cell apoptosis, and ROS content were determined. Dual luciferase reporter assay was performed to verify the targeting relationship between miR-133b and YES1. The effects of pc-YES1 or miR-133b mimic transfection on YES1 expression, apoptosis, and ROS content in H9C2 cells were evaluated.
Results: Compared with those in I/R group, miR-133b expression was obviously up-regulated, LVEDP, cTnI and CK-MB levels were significantly decreased, and LVSP, +dp/dt, -dp/dt, HR and CF levels were increased in admiR-133b group (P < 0.01). The rats in admiR-133b group showed obviously reduced pathological damage, cell apoptosis and ROS content compared with those in I/ R group (P < 0.01). In H9C2 cells exposed to H/R, transfection with miR-133b mimic significantly up-regulated miR-133b expression and decreased cell apoptosis and ROS content (P < 0.01). The results of dual luciferase reporter assay suggested a direct targeting relationship between miR-133b and YES1, and MiR-133b mimic transfection significantly down-regulated YES1 protein expression in cells with H/R exposure (P < 0.01). Co-transfection with pc-YES1 reversed the effect of miR-133b overexpression on myocardial cell apoptosis and ROS accumulation.
Conclusions: miR-133b can inhibit I/R-induced myocardial cell apoptosis and ROS accumulation by targeting YES1 to reduce myocardial I/R injury in rats.
目的: 探究miR-133b对心肌缺血再灌注（I/R）诱导心肌细胞凋亡的作用及机制。
方法: 体内实验，将大鼠随机分为4组：假手术组、模型组、转染腺病毒对照组、转染miR-133b腺病毒组，每组9只。在左心室心肌的6个随机点注射转染复合物后，利用左侧冠状动脉前降支打活结方法诱导心肌I/R。通过RT-qPCR检测miR-133b的表达水平，FDP-1 HRV和BRS分析系统测定心功能，酶联免疫吸附法测定血清肌酸激酶同工酶（CK-MB）和心肌肌钙蛋白（I CTnI）水平，HE染色观察心肌损伤，流式细胞术检测细胞凋亡，2, 7-二氯荧光素双乙酸酯（DCFH-DA）探针测定ROS含量。体外实验，心肌H9C2细胞经缺氧/复氧（H/R）处理后，转染miR-NC或miR-133b mimic，RT-qPCR检测miR-133b的表达水平，流式细胞术检测细胞凋亡，DCFH-DA探针测定ROS含量；双荧光素酶报告实验验证靶向关系；转染pc-YES1或miR-133b mimic后，Western blot检测YES1的表达水平，流式细胞术检测细胞凋亡，DCFH-DA探针测定ROS含量。
结果: 与I/R组相比，AdmiR-133b组大鼠心肌中miR-133b的表达显著上调，LVEDP、cTnI和CK-MB水平显著降低，LVSP、+dp/dt、-dp/dt、HR和CF水平显著升高，病理损伤显著减轻，心肌细胞凋亡和ROS积累显著减少（P < 0.01）。与H/R组相比，miR-133b mimic组H9C2细胞的miR-133b表达显著上调，细胞凋亡和ROS积累显著减少（P < 0.01）。miR-133b与YES1在3’UTR区存在靶向结合位点，共转染YES1 WT和miR-133b mimic可以显著降低荧光素酶活性（P < 0.01）。miR-133b mimic组与H/R组相比H9C2细胞中YES1蛋白的表达显著下调（P < 0.01）。共转染pc-YES1逆转miR-133b过表达对心肌细胞凋亡和ROS积累的作用。
结论: 无论是在体内还是体外，miR-133b靶向YES1抑制I/R引起的心肌细胞凋亡和ROS的积累，从而减轻心肌I/R损伤。
Keywords: YES1; apoptosis; miR-133b; myocardial ischemia-reperfusion; reactive oxygen species.