Department of Cardiology, The First College of Clinical Medical Science, China Three Gorges University, Yichang, Hubei, China.
Institute of Cardiovascular Diseases, China Three Gorges University, Yichang, Hubei, China.
J Cell Physiol. 2020 Apr;235(4):3753-3767. doi: 10.1002/jcp.29270. Epub 2019 Oct 6.
Apoptosis is the major cause of cardiomyocyte death in myocardial ischemia/reperfusion injury (MI/RI). Increasing evidence suggests that microRNAs (miRNAs) can contribute to the regulation of cardiomyocytes apoptosis by posttranscriptional modulation of gene expression networks. However, the effects of miR-327 in regulating MI/RI-induced cardiomyocytes apoptosis have not been extensively investigated. This study was performed to test whether miR-327 participate in cardiomyocytes apoptosis both in vitro and in vivo, and reveal the potential molecular mechanism of miR-327 regulated MI/RI through targeting apoptosis repressor with caspase recruitment domain (ARC). Sprague-Dawley (SD) rats were subjected to MI/RI by left anterior descending coronary artery occlusion for 30 min and reperfusion for 3 hr. H9c2 cells were exposed to hypoxia for 4 hr and reoxygenation for 12 hr to mimic I/R injury. miRNA-327 recombinant adenovirus vectors were transfected into H9c2 cells for 48 hr and rats for 72 hr before H/R and MI/RI treatment, respectively. The apoptosis rate, downstream molecules of apoptotic pathway, and the target reaction between miRNA-327 and ARC were evaluated. Our results showed that miR-327 was upregulated and ARC was downregulated in the myocardial tissues of MI/RI rats and in H9c2 cells with H/R treatment. Inhibition of miR-327 decreased the expression levels of proapoptotic proteins Fas, FasL, caspase-8, Bax, cleaved caspase-9, cleaved caspase-3, and the release of cytochrome-C, as well as increasing the expression levels of antiapoptotic protein Bcl-2 via negative regulation of ARC both in vivo or vitro. In contrast, overexpression miR-327 showed the reverse effect. Moreover, the results of luciferase reporter assay indicated miR-327 targets ARC directly at the posttranscriptional level. Taken together, inhibition of miR-327 could attenuate cardiomyocyte apoptosis and alleviate I/R-induced myocardial injury via targeting ARC, which offers a new therapeutic strategy for MI/RI.
细胞凋亡是心肌缺血/再灌注损伤（MI/RI）中心肌细胞死亡的主要原因。越来越多的证据表明，microRNAs（miRNAs）可以通过转录后调节基因表达网络来调节心肌细胞凋亡。然而，miR-327 调节 MI/RI 诱导的心肌细胞凋亡的作用尚未得到广泛研究。本研究旨在检测 miR-327 是否在体外和体内参与心肌细胞凋亡，并通过靶向凋亡抑制因子 with caspase recruitment domain（ARC）揭示 miR-327 调节 MI/RI 的潜在分子机制。通过左前降支冠状动脉闭塞 30min 再灌注 3h 建立 SD 大鼠 MI/RI 模型。H9c2 细胞在缺氧 4h 复氧 12h 条件下模拟 I/R 损伤。miR-327 重组腺病毒载体转染 H9c2 细胞 48h 和大鼠 72h 后，分别进行 H/R 和 MI/RI 处理。评估细胞凋亡率、凋亡途径下游分子以及 miRNA-327 和 ARC 之间的靶反应。结果显示，MI/RI 大鼠心肌组织和 H/R 处理的 H9c2 细胞中 miR-327 上调，ARC 下调。抑制 miR-327 通过负调控 ARC，减少 Fas、FasL、caspase-8、Bax、cleaved caspase-9、cleaved caspase-3 的表达水平和细胞色素 C 的释放，同时增加抗凋亡蛋白 Bcl-2 的表达水平，在体内和体外均有此作用。相反，过表达 miR-327 则表现出相反的效果。此外，荧光素酶报告基因实验表明 miR-327 可直接在转录后水平靶向 ARC。综上所述，抑制 miR-327 通过靶向 ARC 可减轻心肌细胞凋亡，缓解 I/R 诱导的心肌损伤，为 MI/RI 提供了一种新的治疗策略。
