Supplementary Material for: The microRNA-210/Casp8ap2 Axis Alleviates Hypoxia-Induced Myocardial Injury by Regulating Apoptosis and Autophagy

Coronary heart disease (CHD) is a serious condition comprising atherosclerosis-mediated ischaemic and hypoxic myocardial injury. This study aimed to investigate the mechanism of the miR-210/Casp8ap2 signalling pathway in hypoxic myocardial cells. mRNA and protein expression levels were determined by quantitative real-time PCR and western blotting, respectively. MTT was used to evaluate cell survival, and flow cytometry was used to assess apoptosis and the cell cycle distribution. The interaction between miR-210 and ­Casp8ap2 was detected by dual-luciferase reporter assay. As a result, overexpression of miR-210 significantly inhibited apoptosis and reduced the proportion of cells in G1 phase. Moreover, miR-210 suppressed autophagy by upregulating p62 levels and reducing the LC3-II/I ratio in hypoxic cardiomyocytes. miR-210 regulated apoptosis and autophagy by directly targeting Casp8ap2. Furthermore, the expression levels of Casp8ap2, Cleaved caspase 8, Cleaved caspase 3and Beclin-1 were all decreased in response to miR-210. In short, our results suggest that miR-210 exerts anti-apoptotic and anti-autophagic effects in hypoxic cardiomyocytes, which alleviates myocardial injury in response to hypoxia.

冠心病（Coronary heart disease, CHD）是一类严重的疾病，其病理特征为动脉粥样硬化介导的缺血缺氧性心肌损伤。本研究旨在探讨miR-210/Casp8ap2信号通路在缺氧心肌细胞中的作用机制。研究分别采用实时定量聚合酶链反应（quantitative real-time PCR）与蛋白质免疫印迹（Western Blotting）检测mRNA与蛋白的表达水平；使用噻唑蓝比色法（MTT）评估细胞存活能力，通过流式细胞术（flow cytometry）检测细胞凋亡与细胞周期分布情况。采用双荧光素酶报告基因实验（dual-luciferase reporter assay）验证miR-210与Casp8ap2的靶向结合相互作用。实验结果显示，miR-210过表达可显著抑制缺氧心肌细胞的凋亡，并降低G1期细胞的占比。此外，miR-210可通过上调p62蛋白水平、降低LC3-II/I比值，抑制缺氧心肌细胞的自噬活动。miR-210可通过直接靶向结合Casp8ap2，调控细胞凋亡与自噬过程。进一步检测发现，miR-210过表达可下调Casp8ap2、裂解型半胱氨酸天冬氨酸蛋白酶8（Cleaved caspase 8）、裂解型半胱氨酸天冬氨酸蛋白酶3（Cleaved caspase 3）及Beclin-1的表达水平。综上，本研究结果表明，miR-210可在缺氧心肌细胞中发挥抗凋亡与抗自噬的作用，从而缓解缺氧诱导的心肌损伤。