Liu Yan, Ke Xiao, Guo Wenyu, Wang Xiaoqing, Peng Changnong, Liao Zhiyong, Liu Qiang, Zhou Yingling
Southern Medical University, Guangzhou, China.
Department of Cardiology, The Third People's Hospital of Hui Zhou City, China.
Arch Med Sci. 2019 Aug 7;18(3):732-745. doi: 10.5114/aoms.2019.87205. eCollection 2022.
Myocardial ischemia/reperfusion (I/R) injury is a leading cause of cardiac dysfunction. Circular RNAs (circRNAs) are involved in the pathogenesis of myocardial I/R injury. However, the functions and underlying mechanisms are unclear. The present study determined the role of circ-RHOJ.1 in regulating myocardial cell proliferation and apoptosis after I/R injury.
Myocardial cells isolated from Sprague-Dawley rats were identified with an immunofluorescence assay using cardiac troponin T antibody. Expression of circ-RHOJ.1, miR-124-3p and neuregulin-1 (NRG1) mRNA was assessed with real-time quantitative polymerase chain reaction. NRG1 protein expression was evaluated with western blot and immunofluorescence assays. Dual-luciferase reporter assay was performed to confirm interaction between miR-124-3p and circ-RHOJ.1, and miR-124-3p and NRG1. Effects of circ-RHOJ.1 overexpression or miR-124-3p inhibition on cell proliferation and apoptosis were evaluated using cell counting kit (CCK)-8 assay and flow cytometry. Cytokines levels were analyzed with an enzyme-linked immunosorbent assay.
Myocardial cells were successfully isolated and had down-regulated expression of circ-RHOJ.1 and NRG1, and up-regulated expression of miR-124-3p after I/R injury. circ-RHOJ.1 acted as a sponge for miR-124-3p, and NRG1 served as a target gene of miR-124-3p. circ-RHOJ.1 overexpression or miR-124-3p inhibition increased interleukin (IL)-10 levels and reduced IL-2, IL-6, and tumor necrosis factor-α levels in myocardial cells after I/R injury. Functional assay results illustrated that circ-RHOJ.1 overexpression or miR-124-3p inhibition enhanced proliferation and inhibited apoptosis of myocardial cells after I/R injury.
Circ-RHOJ.1 served as a molecular marker of myocardial I/R injury via regulation of miR-124-3p and NRG1 expression.
心肌缺血/再灌注（I/R）损伤是导致心脏功能障碍的主要原因。环状RNA（circRNA）参与心肌I/R损伤的发病机制。然而，其功能及潜在机制尚不清楚。本研究确定了circ-RHOJ.1在调节I/R损伤后心肌细胞增殖和凋亡中的作用。
用心肌肌钙蛋白T抗体通过免疫荧光法鉴定从Sprague-Dawley大鼠分离的心肌细胞。用实时定量聚合酶链反应评估circ-RHOJ.1、miR-124-3p和神经调节蛋白-1（NRG1）mRNA的表达。用蛋白质免疫印迹法和免疫荧光法评估NRG1蛋白表达。进行双荧光素酶报告基因检测以确认miR-124-3p与circ-RHOJ.1以及miR-124-3p与NRG1之间的相互作用。使用细胞计数试剂盒（CCK）-8法和流式细胞术评估circ-RHOJ.1过表达或miR-124-3p抑制对细胞增殖和凋亡的影响。用酶联免疫吸附测定法分析细胞因子水平。
成功分离出心肌细胞，I/R损伤后circ-RHOJ.1和NRG1表达下调，miR-124-3p表达上调。circ-RHOJ.1作为miR-124-3p的海绵，NRG1作为miR-124-3p的靶基因。circ-RHOJ.1过表达或miR-124-3p抑制可提高I/R损伤后心肌细胞中白细胞介素（IL）-10水平，并降低IL-2、IL-6和肿瘤坏死因子-α水平。功能检测结果表明，circ-RHOJ.1过表达或miR-124-3p抑制可增强I/R损伤后心肌细胞的增殖并抑制其凋亡。
Circ-RHOJ.1通过调节miR-124-3p和NRG1表达，作为心肌I/R损伤的分子标志物。
