Zhang Jingjing, Gao Chuanyu, Zhang Jing, Ye Famin
Coronary Care Unit, Department of Cardiology, People's Hospital of Zhengzhou University, Zhengzhou City, Henan Procince, China.
Department of Cardiology, People's Hospital of Zhengzhou University, Zhengzhou City, Henan Procince, China.
EXCLI J. 2020 Nov 11;19:1520-1532. doi: 10.17179/excli2020-2809. eCollection 2020.
Few studies have addressed the mechanism by which circ_0010729 regulates hypoxia-induced cell injury in cardiovascular diseases. However, its role and its regulatory mechanism in myocardial infarction remain to be explored. Cell viability, cycle, apoptosis, and migration were analyzed using cell counting kit-8 assay, flow cytometry, caspase-3 activity assay kit and transwell assay, respectively. Tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) concentrations were examined by enzyme-linked immunosorbent assay. Glucose metabolism was calculated by detecting ATP production, glucose uptake and lactate production. Levels of circ_0010729, miR-370-3p and TNF Receptor Associated Factor 6 (TRAF6) were detected using quantitative real-time polymerase chain reaction or western blot. The direct interaction between circ_0010729 and TRAF6 or miR-370-3p was verified using dual-luciferase reporter assay and RNA immunoprecipitation assay. Under hypoxia condition, cardiomyocytes suffered from cell viability suppression, cell cycle arrest, cell apoptosis promotion, migration reduction, increase of inflammatory factor IL-6 and TNF-α, as well as glycolysis inhibition. Circ_0010729 expression was up-regulated in the cardiomyocytes at different hypoxia-exposed time points. Circ_0010729 knockdown protected cardiomyocytes against hypoxic dysfunction, while circ_0010729 overexpression showed inverse effects. MiR-370-3p was confirmed to directly bind to circ_0010729 or TRAF6. MiR-370-3p inhibition attenuated the protective effects of circ_0010729 knockdown on hypoxia-modulated cardiomyocyte dysfunction. MiR-370-3p restoration protected cardiomyocytes against hypoxic injury via targeting TRAF6. Besides, circ_0010729 indirectly regulated TRAF6 expression via miR-370-3p. This study demonstrated that circ_0010729 knockdown attenuated hypoxia-induced cardiomyocyte dysfunction via miR-370-3p/TRAF6 axis, indicating a potential therapeutic target for myocardial infarction.
很少有研究探讨circ_0010729调节心血管疾病中缺氧诱导的细胞损伤的机制。然而，其在心肌梗死中的作用及其调节机制仍有待探索。分别使用细胞计数试剂盒-8检测、流式细胞术、caspase-3活性检测试剂盒和transwell检测分析细胞活力、周期、凋亡和迁移。通过酶联免疫吸附测定法检测肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）的浓度。通过检测ATP生成、葡萄糖摄取和乳酸生成来计算葡萄糖代谢。使用定量实时聚合酶链反应或蛋白质印迹法检测circ_0010729、miR-370-3p和肿瘤坏死因子受体相关因子6（TRAF6）的水平。使用双荧光素酶报告基因检测和RNA免疫沉淀检测验证circ_0010729与TRAF6或miR-370-3p之间的直接相互作用。在缺氧条件下，心肌细胞遭受细胞活力抑制、细胞周期停滞、细胞凋亡促进、迁移减少、炎性因子IL-6和TNF-α增加以及糖酵解抑制。在不同缺氧暴露时间点，心肌细胞中circ_0010729表达上调。敲低circ_0010729可保护心肌细胞免受缺氧功能障碍的影响，而过表达circ_0010729则显示相反的效果。证实miR-370-3p直接与circ_0010729或TRAF6结合。抑制miR-370-3p可减弱敲低circ_0010729对缺氧调节的心肌细胞功能障碍的保护作用。恢复miR-370-3p通过靶向TRAF6保护心肌细胞免受缺氧损伤。此外，circ_0010729通过miR-370-3p间接调节TRAF6表达。本研究表明，敲低circ_0010729通过miR-370-3p/TRAF6轴减轻缺氧诱导的心肌细胞功能障碍，提示其可能是心肌梗死的潜在治疗靶点。
