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

Abstract

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.

Figure 1.. lncRNA-ROR was highly expressed in myocardial ischemia/reperfusion (I/R) and hypoxia/reoxygenation (H/R). A, Relative lncRNA-ROR expressions in serum of I/R injury patients and normal serum were detected by qRT-qPCR. B, and C, relative lncRNA-ROR expressions in H9c2 cells and human cardiomyocytes (HCM) after H/R treatment were also examined by qRT-PCR. Cells under normoxia served as the control group. Data are reported as means±SE. *P<0.05, **P<0.01 (ANOVA).

Figure 2.. Cells were assigned to normoxia, hypoxia/reoxygenation (H/R), H/R+lncRNA-ROR, and H/R+ASO-lncRNA-ROR groups. A, The release level of lactate dehydrogenase (LDH) and levels of (B) malondialdehyde (MDA), (C) superoxide dismutase (SOD), and (D) glutathione peroxidase (GSH-PX) in H9c2 cells and human cardiomyocytes (HCM) were evaluated by kits according to the manufacturer's instructions. H/R+lncRNA-ROR: H9c2 cells were transfected with lncRNA-ROR after H/R treatment. H/R+si-lncRNA-ROR: H9c2 cells were transfected with si-lncRNA-ROR after H/R treatment. Data are reported as means±SE. *P<0.05, **P<0.01, ***P<0.001 (ANOVA).

Figure 3.. lncRNA-ROR aggravates hypoxia/reoxygenation (H/R)-induced cell injury by regulating cell viability and apoptosis. H9c2 cells and human cardiomyocytes (HCM) were transfected with lncRNA-ROR overexpression vector (lncRNA-ROR) and inhibition vector (si-lncRNA-ROR). After H/R treatment for 24 h, (A) cell viability, (B) apoptosis, and (C) and (D) apoptosis-related factors Bax, Bcl-2, cytochrome C, Smac/Diablo, cleaved-caspase-3, and cleaved-caspase-9 were measured by MTT, flow cytometry, qRT-PCR, and western blot, respectively. GAPDH served as the internal control. Data are reported as means±SE. *P<0.05, **P<0.01, ***P<0.001 (ANOVA).

Figure 4.. lncRNA-ROR mediated myocardial hypoxia/reoxygenation (H/R) by regulating the p38/MAPK pathway. H9c2 cells and human cardiomyocytes (HCM) were transfected with lncRNA-ROR overexpression vector (lncRNA-ROR) and inhibition vector (si-lncRNA-ROR). After H/R treatment for 24 h, (A) the protein levels of ERK and p38 were examined by western blot and (B) the mRNA expressions of ERK and p38 were determined by qRT-PCR. Data are reported as means±SE. *P<0.05, **P<0.01 (ANOVA).

Figure 5.. Inhibitor of p8/MAPK (SB203580) alleviated lncRNA-ROR-induced cell injury. H9c2 cells and human cardiomyocytes (HCM) were transfected with lncRNA-ROR overexpression vector (lncRNA-ROR) and inhibition of p8/MAPK (SB203580, 10 μM). After hypoxia/reoxygenation (H/R) treatment for 24 h, (A) cell viability, (B) apoptosis, and (C) and (D) apoptosis-related factors Bax, Bcl-2, cytochrome C, Smac/Diablo, cleaved-caspase-3, and cleaved-caspase-9 were measured by MTT, flow cytometry, qRT-PCR, and western blot, respectively. GAPDH served as the internal control. Data are reported as means±SE. *P<0.05, **P<0.01, ***P<0.001 (ANOVA).

Figure 6.. lncRNA-ROR promoted reactive oxygen species (ROS) production in H9c2 cells. H9c2 cells were transfected with lncRNA-ROR overexpression vector (lncRNA-ROR) and inhibition vector (si-lncRNA-ROR). After hypoxia/reoxygenation (H/R) treatment for 24 h, (A) intracellular ROS production was analyzed by H2DCF-DA in H9c2 cells; (B) mitochondria ROS production was detected by MitoSOX Red assay; (C) the activity of NADPH oxidase was measured by chemiluminescence assay; (D) the protein level of NOX2 was examined by western blot. Data are reported as means±SE. *P<0.05, **P<0.01 (ANOVA).