MicroRNA-503 mediates myocardial Ischemia/Reperfusion injury via inhibiting PI3K/Akt and STAT3 dependent pro-survival signaling pathways

Abstract

Acute myocardial infarction is a leading cause of death worldwide. Restoration of blood flow to previously ischemic myocardium may paradoxically lead to Ischemia/Reperfusion (I/R) injury. microRNAs (miRs) are noncoding small RNAs, which modulates gene expression by mRNA degradation or translational repression. Accumulated evidence has shown that miRs are implicated in cardiovascular disease, however, the potential role of microRNA-503 (miR-503) in myocardial I/R injury is largely unknown. Consequently, this study is aimed to investigate whether and how miR-503 affects myocardial I/R injury. In vivo, mice were randomly divided into I/R and Sham group; The mouse model of myocardial I/R injury was established by ligation of left anterior descending coronary artery for 30min followed by 2h of reperfusion (I/R). Creatine Kinase MB (CK-MB) in serum and myocardial infarct size were examined as myocardial injury markers. In vitro, H9c2 cells were exposed to glucose deprivation and hypoxia for 10h followed by 6h of reoxygenation (H/R). Before H/R stimulation, cells were separately pretreated with N-acetylcysteine (NAC, 1mM or 2 mM), or transfected with miR-503 agomir (Agomir-503), Antagomir-503, and their corresponding negative control (NC). miR-503 expression in mice hearts or cells was determined by RT qPCR. Cell viability was analyzed by MTT assay, lactate dehydrogenase (LDH) activity was detected in cell culture supernatant. Cell apoptosis and protein expression were examined by TUNEL assay, and Western blotting separately. Results showed that mice exhibited myocardial infarction following I/R, and serum CK-MB concentration was increased markedly. miR-503 expression level was down-regulated in vivo and in cells upon 10h of hypoxia or H/R stimulation. In vitro, cell viability was reduced but LDH activity increased significantly in H/R+NC group compared with Control (Con)+NC group, Agomir-503 treatment further reduced cell viability, increased LDH activity and cell apoptosis. In contrast, the injurious effects were reversed by Antagomir-503 treatment. NAC (1mM) pretreatment improved cell viability and reduced LDH activity, it decreased miR-503 expression level upon H/R stimulation. Mechanistically, both BCL2 and phosphoinositide-3-kinase regulatory subunit 1 (PI3K p85) are target genes of miR-503. In vivo, protein PI3K p85 and its downstream protein phospho-Akt (p-Akt, T450 and T308), p-GSK-3β(S9) in mice hearts were down-regulated in I/R group, but phosphorylated signal transducer and activator of transcription 3 (p-Stat3, Y705) was increased significantly. In vitro, both PI3K p85 and Bcl-2 were decreased significantly in H/R+NC group compared with Con+NC group, Agomir-503 treatment decreased both proteins under normoxia or H/R condition; Protein Akt1, Akt2, p-Akt (T450), p-Akt (T308), Stat3 and p-Stat3 (Y705) were decreased remarkably in H/R+NC group, and further decreased by Agomir-503 treatment except p-Akt (T308); p-GSK-3β (S9) was decreased significantly by Agomir-503 treatment under H/R condition. In contrast, Antagomir-503 treatment increased protein levels of PI3K p85 and Bcl-2 under normoxia or H/R condition, it also enhanced p-Akt (T450) and p-Stat3 protein levels. It is concluded that miR-503 is implicated in the development of myocardial I/R injury and may mediate I/R injury by inactivation of PI3K/Akt and STAT3 pathways. Thus, miR-503 may act as a therapy target in myocardial I/R injury.