MicroRNA-124 Regulates Cardiomyocyte Apoptosis And Myocardial Infarction Through Targeting Dhcr24

Background and Aim:During myocardial ischemia,various pathological stimuli induced cardiomyocytes apoptosis is the basic pathological process of myocardial infarction and post-MI ventricular remodeling.Our group has recently found that mir-124 is involved in vessel smooth muscle cell(VSMC)proliferation,migration,phenotypic switching and vascular injury-induced neointima formation.However,the role of mir-124 in cardiomyocytes apoptosis is still unknown.In the present study,we try to investigate the functional role of miR-124 in cardiomyocytes apoptosis,as well as its potential mechanism.Methods:Primary cardiomyocytes isolated from neonatal mice birth within 3 days and H9C2 cell line were cultured in vitro,miR-124 were evaluated during H2O2 and hypoxic stilulation.Using gain-/loss-of function assay,the role of miR-124 on cardiomyocytes apoptosis were assessed.The functional target of miR-124 in cardiomyocytes was predicted by RNA sequence,various bioinformatics softeare and Dual-Luciferase Reporter Assay.Using overexpressing target gene,the potential mechanism of target gene was evaluated.Meanwhile,miR-124 inhibitor and its negative contral were intra-myocardially injected at the ischemic area to investigate the effect of miR－124 on cardiomyocytes apoptosis in the myocardial infarction model.Finally,serum of patients with normal coronary arteries or acute myocardial infarction(AMI)were collected to detect the expression of miR-124.Results:After serum-free starvation,the expression of miR-124 in cardiomyocytes and H9C2 was significantly increased during H2O2 and hypoxia stimuli.CCK-8 and flow cytomertry assay found that overexpression of miR-124 significantly reduced cell viability and increased cardiomyocytes apoptosis,whereas inhibition of miR-124 improved cell viability and decrease cardiomyocytes apoptosis.Dhcr24 was predicted as the functional target gene of miR-124 by using RNA sequence and several computational prediction tools.Dhcr24 was down-regulated by miR-124 on both mRNA and protein levels.Dual-Luciferase Reporter Assay indicated that luciferase activity was significantly repressed by miR-124 over－expression in vector harboring wild-type Dhcr24 3’UTR,while it did not change in mutant Dhcr24 3’UTR5 confirmed Dhcr24 is the functional target of miR-124.Co-transfection miR-124 mimic and Dhcr24 over-expression plasmids showed that miR-124 over-expression or Dhcr24 activation alone in cardiomyocytes significantly decreased or increased cell viability,respectively,re-activation of Dhcr24 abolished the effects of miR-124 on the cells.Meanwhile.Dhcr24 protected cardiomyocytes via mediating p38MAPK/ERK and JNK signaling.miR-124 inhibitor significantly increased Dhcr24 expression levels,reduced Tunel positive cell,fibrosis area and cardiac dysfunction in myocardial infarction heart.Finally and importantly,mir－124 were significantly higher in AMI patients,and positive relationships between miR-124 and myocardial injury markers(Tnl and CK-MB)were observed in the AMI patient,and miR-124 and LVEF was inversely correlated.Conclusion:Our finding demonstrated that miR-124 had a crucial role in hypoxia induced cardiac myocytes injury and myocardial infarction induced cardiac dysfunction,and suggested its potential therapeutic application for ischemic heart disease.