Department of Cardiology, Nanjing Central Hospital, Nanjing 210018, China.
Department of Physical Examination, The Second Clinical College of Jinan University, Shenzhen People's Hospital, Shenzhen 518020, China.
Acta Biochim Biophys Sin (Shanghai). 2018 Mar 1;50(3):288-297. doi: 10.1093/abbs/gmx144.
Recently, microRNAs (miRNAs) have been shown to involve in the process of heart failure. This study aims to investigate the functional role of miR-147b in rat H9c2 cardiomyocytes and explore the underlying molecular mechanisms. Cell viability of H9c2 cells was detected by MTT assay. Cell apoptosis was detected by flow cytometry. Expression of miR-147b and KLF13 mRNA was detected by quantitative real-time PCR. The relationship between miR-147b and KLF13 was verified by dual-luciferase reporter assay. Protein levels were detected by western blot analysis. It was found that H2O2 inhibited cell viability and promoted cell apoptosis of H9c2 cells in a concentration-dependent manner. MiR-147b overexpression suppressed cell viability and increased apoptosis in H9c2 cells, while knock-down of miR-147b increased cell viability and reduced apoptosis in H2O2-treated H9c2 cells. Luciferase reporter assay and in vitro functional assay showed that KLF13 was a downstream target of miR-147b, and KLF13 knock-down suppressed cell viability and induced apoptosis in H9c2 cells. Enforced expression of KLF13 restored the effects of miR-147b overexpression on cell viability and apoptosis in H9c2 cells. MiR-147b modulated the expression levels of apoptosis-related proteins, and the effects of miR-147b overexpression on apoptosis-related proteins levels were prevented by enforced expression of KLF13 in H9c2 cells. The in vivo experiments showed that miR-147b was up-regulated, and KLF13 was down-regulated in the myocardial tissues from rats with chronic heart failure. Collectively, miR-147b inhibits viability and promotes cell apoptosis by targeting KLF13 in H9c2 cells, which may be associated with the pathogenesis of heart failure.
最近，研究表明 microRNAs（miRNAs）参与心力衰竭的过程。本研究旨在探讨 miR-147b 在大鼠 H9c2 心肌细胞中的功能作用，并探讨其潜在的分子机制。MTT 法检测 H9c2 细胞活力。流式细胞术检测细胞凋亡。实时定量 PCR 检测 miR-147b 和 KLF13mRNA 的表达。双荧光素酶报告基因实验验证 miR-147b 与 KLF13 的关系。Western blot 分析检测蛋白水平。结果发现，H2O2 呈浓度依赖性抑制 H9c2 细胞活力并促进细胞凋亡。miR-147b 过表达抑制 H9c2 细胞活力并增加细胞凋亡，而 miR-147b 敲低则增加 H2O2 处理的 H9c2 细胞活力并减少细胞凋亡。荧光素酶报告基因实验和体外功能实验表明，KLF13 是 miR-147b 的下游靶基因，KLF13 敲低抑制 H9c2 细胞活力并诱导细胞凋亡。KLF13 的强制表达恢复了 miR-147b 过表达对 H9c2 细胞活力和凋亡的影响。miR-147b 调节凋亡相关蛋白的表达水平，而 miR-147b 过表达对凋亡相关蛋白水平的影响可被 KLF13 在 H9c2 细胞中的强制表达所阻止。体内实验表明，慢性心力衰竭大鼠心肌组织中 miR-147b 上调，KLF13 下调。总之，miR-147b 通过靶向 KLF13 抑制 H9c2 细胞活力并促进细胞凋亡，这可能与心力衰竭的发病机制有关。
