Long non-coding RNA-ROR aggravates myocardial ischemia/reperfusion injury

Long non-coding RNAs (lncRNAs) play an important role in the pathogenesis of cardiovascular diseases, especially in myocardial infarction and ischemia/reperfusion (I/R). However, the underlying molecular mechanism remains unclear. In this study, we determined the role and the possible underlying molecular mechanism of lncRNA-ROR in myocardial I/R injury. H9c2 cells and human cardiomyocytes (HCM) were subjected to either hypoxia/reoxygenation (H/R), I/R or normal conditions (normoxia). The expression levels of lncRNA-ROR were detected in serum of myocardial I/R injury patients, H9c2 cells, and HCM by qRT-PCR. Then, levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) were measured by kits. Cell viability, apoptosis, apoptosis-associated factors, and p38/MAPK pathway were examined by MTT, flow cytometry, and western blot assays. Furthermore, reactive oxygen species (ROS) production was determined by H2DCF-DA and MitoSOX Red probes with flow cytometry. NADPH oxidase activity and NOX2 protein levels were measured by lucigenin chemiluminescence and western blot. Results showed that lncRNA-ROR expression was increased in I/R patients and in H/R treatment of H9c2 cells and HCM. Moreover, lncRNA-ROR significantly promoted H/R-induced myocardial injury via stimulating release of LDH, MDA, SOD, and GSH-PX. Furthermore, lncRNA-ROR decreased cell viability, increased apoptosis, and regulated expression of apoptosis-associated factors. Additionally, lncRNA-ROR increased phosphorylation of p38 and ERK1/2 expression and inhibition of p38/MAPK, and rescued lncRNA-ROR-induced cell injury in H9c2 cells and HCM. ROS production, NADPH oxidase activity, and NOX2 protein levels were promoted by lncRNA-ROR. These data suggested that lncRNA-ROR acted as a therapeutic agent for the treatment of myocardial I/R injury.

长链非编码RNA（long non-coding RNAs，lncRNAs）在心血管疾病的发病机制中发挥关键作用，尤其与心肌梗死及缺血再灌注（ischemia/reperfusion，I/R）过程密切相关。然而其潜在的分子机制尚未完全阐明。本研究旨在探讨长链非编码RNA-ROR（lncRNA-ROR）在心肌缺血再灌注损伤中的作用及其潜在分子机制。将H9c2细胞与人类心肌细胞（human cardiomyocytes，HCM）分别置于缺氧复氧（hypoxia/reoxygenation，H/R）、缺血再灌注或常氧（normoxia）条件下培养，采用实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction，qRT-PCR）检测心肌缺血再灌注损伤患者血清、H9c2细胞及人类心肌细胞中长链非编码RNA-ROR的表达水平；通过检测试剂盒测定乳酸脱氢酶（lactate dehydrogenase，LDH）、丙二醛（malondialdehyde，MDA）、超氧化物歧化酶（superoxide dismutase，SOD）与谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-PX）的水平；采用MTT法、流式细胞术（flow cytometry）及蛋白质印迹实验（western blot assays）分别检测细胞活力、细胞凋亡、凋亡相关因子以及p38/丝裂原活化蛋白激酶（p38/MAPK）通路的变化情况。此外，采用H2DCF-DA与MitoSOX Red荧光探针结合流式细胞术检测活性氧（reactive oxygen species，ROS）的生成水平；通过光泽精化学发光法（lucigenin chemiluminescence）与蛋白质印迹实验测定NADPH氧化酶（NADPH oxidase）活性及NOX2蛋白的表达水平。实验结果显示，心肌缺血再灌注损伤患者血清以及经缺氧复氧处理的H9c2细胞与人类心肌细胞中，长链非编码RNA-ROR的表达均显著上调。长链非编码RNA-ROR可通过刺激乳酸脱氢酶、丙二醛、超氧化物歧化酶与谷胱甘肽过氧化物酶的释放，显著加重缺氧复氧诱导的心肌损伤。进一步研究发现，长链非编码RNA-ROR可降低细胞活力、促进细胞凋亡，并调控凋亡相关因子的表达水平。此外，长链非编码RNA-ROR可增强p38与细胞外调节蛋白激酶1/2（extracellular signal-regulated kinase 1/2，ERK1/2）的磷酸化水平，而抑制p38/MAPK通路可逆转长链非编码RNA-ROR诱导的H9c2细胞与人类心肌细胞损伤。长链非编码RNA-ROR可促进活性氧生成、增强NADPH氧化酶活性并上调NOX2蛋白的表达水平。上述数据表明，长链非编码RNA-ROR可作为心肌缺血再灌注损伤的治疗制剂。