MiR-499 InhibitsAnoxia-Reoxygenation-Induced Cardiomyocyte Apoptosis Via Activation Of PI3K/Akt Signaling Pathway

Objectives To investigate the effect of mi R-499 on apoptosis of primary cardiomyocytes induced by anoxia-reoxygenation(A/R),and its potential relationship with PI3K/Akt signaling pathway so as to provide new ways for the clinical treatment of myocardial ischemia reperfusion injury.Methods The primary cultured neonatal rat cardiomyocytes were subjected to hypoxia for 3 hours/ reoxygenation for 4 hours to simulate myocardial ischemia-reperfusion injury.The cardiomyocytes were randomly divided into four groups: normal control group,hypoxia / reoxygenation group,anoxic/reoxygenated mi R-499 agonist agomir preconditioning group,anoxic/reoxygenated mi R-499 agonist agomir PI3 K specific blocker LY294002 group.MTT assay was used to detect the viability of cardiomyocytes in each experimental group.The expression of PI3 K and phosphorylated PI3 K ?caspase-3?Akt and phosphorylated Akt was detected by western blot.The relative expressions of mi R-499 and PI3 K m RNA in each group were detected by fluorescence quantitative RT-PCR.Results 1.The comparison of cell viability in 4 groups :Compared with the Control group(90.24±4.77),the myocardial cell viability of the A/R group decreased significantly(41.27±1.56)(P < 0.05),and that of the Control group was(77.38±2.65)(P < 0.05).2.Relative PI3 K m RNA and mi R-499 expression in cardiomyocytes in 4 groups: The relative expression of PI3 K m RNA and mi R-499 in myocardial cells in A/R group was significantly lower than that in Control group(P < 0.05),and the expression of PI3 K m RNA and mi R-499 in group Agomir was significantly higher than that in group A/R(P < 0.05).3.The comparison of caspase-3 protein expression in cardiomyocytes in 4 groups: Compared with Control group,the expression of caspase-3 in group A/R was significantly higher(P < 0.05),the expression of caspase-3 in group Agomir was significantly lower than that in group A / R(P < 0.05),and the expression of caspase-3 in group Agomir+LY294002 was significantly higher than that in group Agomir(P < 0.05).4.The comparison of PI3 K and Akt phosphorylation levels in 4 groups:As compared with Control group,the protein level of p-PI3 K and p-Akt in A/R group were significantly decreased(P<0.05);compared with A/R group,the protein level of p-PI3 K and p-Akt in Agomir group were significantly increased(P<0.05);compared with Agomir group,the protein level of p-PI3 K and p-Akt in Agomir+LY294002 group were significantly decreased(P<0.05).Conclusions 1.As we compare the Control group,A/R group,Agomir group and Agomir+LY294002 group,we found that compared with Control group,the expression of mi R-499 in A/R group were significantly decreased,but with the upregulation of mi R-499 by preconditioning of mi R-499 agomir,the expression of mi R-499 were significantly increased not only when compared with A/R group,but also higher when compared with Control group,all of these show that in the condition of hypoxia the expression of mi R-499 was decrease and the mi R-499 agomir do improve the expression of mi R-499.2.Comparing of caspase-3 protein expression in cardiomyocytes in 4 groupsthe,The protein level of caspase-3 in A/R group was significantly increased when it compared with Control group,although the Agomir group built the anoxia-reoxygenation modles too,but the protein level of caspase-3 was significant decreased when compared with A/R group,these prove that mi R-499 protected the primary cardiomyocytes from A/R-induced apoptosis.3.The effect of mi R-499 agomir was inhibited by LY294002.which indicate that mi R-499 protected the primary cardiomyocytes from A/R-induced apoptosis,which may be involved in the activation of PI3K/Akt signaling pathway.