Overexpression of miR-503-5p exacerbates hypoxia/reoxygenation injury in H9C2 cardiomyocytes

Abstract

Acute myocardial infarction (MI) is a leading cause of death worldwide, and the most effective therapy for reducing acute myocardial ischemic injury is myocardial reperfusion, while reperfusion itself can lead to further cardiomyocyte death, called ischemia/reperfusion injury (IRI). A number of microRNAs have been shown to be involved in IRI, however, the role of miR-503-5p, which is implicated in cancers, diabetes mellitus or pulmonary arterial hypertension, remains unknown in myocardial IRI. This study was aimed to investigate the effects of miR-503-5p in myocardial IRI. Cardiac miR-503-5p expression was measured in mice subjected to myocardial IRI achieved by occlusion of left anterior descending artery for 30 minutes followed by reperfusion for 2 hours. H9C2 cardiomyocytes hypoxia/reoxygenation (H/R) was achieved by exposing the cells to glucose deprivation and hypoxia for 10 hours followed by reoxygenation 6 hours. We found that cardiac miR-503 expression level was down-regulated after induction of IRI compared with control, and it was also down-regulated in H9C2 cells after exposure to H/R. In vitro, cell viability was decreased after H/R treatment, and lactate dehydrogenase (LDH) activity was increased. Administration of an agomir designed to overexpress miR503-5p in cells before inducing H/R further reduced cell viability and elevated LDH activity. Moreover, proapoptotic Bax protein showed no significant change after overexpression of miR-503-5p in both normal and H/R states, while anti-apoptotic Bcl protein decreased significantly. We analyzed putative miR-503 targets using the TargetScan software, and found that the phosphoinositide-3-kinase regulatory subunit 1(PI3K p85) was one of the target genes. After overexpression of miR-503-5p, we found that PI3K p85 was decreased in both normal and H/R states. In addition, Akt1 and Akt2 as well as Phospho-Akt were decreased in H/R state, and were further decreased after miR-503-5p overexpressed. Furthermore, we also found that prosurvival protein phosphorylation of signal transducer and activator of transcription (STAT3) was decreased after exposure to H/R, and declined further after overexpression of miR-503-5p. It is concluded that miR-503-5p regulates H9C2 cardiomyocyte injury during H/R, and exacerbates cell injury by inhibiting the activation of prosurvival protein Akt and STAT3.