The Role Of MicroRNA 146a In Myocardial Ischemia Reperfusion Injury In Mice

Objectives Myocardial ischemia-reperfusion injury is still a major stumbling block in the therapeutic effect of myocardial ischemia-reperfusion.Many microRNAs have been found to participate in the pathogenesis.This study aimed to explore the expression,role and mechanism of MicroRNA 146 a in myocardial ischemia-reperfusion injury.Methods1.Models of myocardial ischemia-reperfusion injury in wild-type mice were established to explore the changes of MicroRNA 146 a expression in myocardial tissues at different time after reperfusion;2.To detect the effect of endogenous MicroRNA 146 a on myocardial ischemia-reperfusion injury: a.In vivo models of myocardial ischemia-reperfusion injury were established in MicroRNA 146 a knockout(MicroRNA 146a-/-,KO)mice and wide type(WT)mice;b.On the 1th day,3 th day and 7th day,the changes of cardial function in mice were detected by echocardiography;c.Evans Blue / TTC staining of myocardium was performed at 4 h after myocardial ischemia reperfusion to detect the change of ischemic and infarct area in myocardium;d.LDH level in plasma was detected with ELISA at 2h after myocardial ischemia-reperfusion;e.Apoptotic cells were detected by Tunel at 2h after myocardial ischemia-reperfusion;3.Target genes of MicroRNA 146 a were screened and identified using targetcan,RT-PCR and dual luciferase reporter;4.Myocardial tissue was extracted at 2h after myocardial ischemia-reperfusion to detect the expression of target gene and the possible downstream apoptosis signaling protein in myocardial ischemia-reperfusion by Western Blot;5.Rescue study was performed to verify the role of the target gene in myocardial ischemia-reperfusion.Results1.MicroRNA 146 a increased compensatoryly at 2h after myocardial ischemia-reperfusion;2.Cardiac echocardiography showed that compared with WT,the cardial function of KO mice decreased obviously on the 7th day after reperfusion;Evans Blue / TTC Staining showed that the area of infarct myocardium in KO mice was significantly increased compared with that in WT mice.ELISA showed that LDH was increased in KO mice compared with that in WT mice.Tunel indicated that the in situ apoptosis of cardiomyocytes in KO group was significantly increased compared with that in WT group.3 Gene microarrays indicated that there were 136 possible apoptosis-related MicroRNA 146 a target genes,in which nineteen of them could combine with MicroRNA 146 a.The results of RT-PCR showed that Med1,Dapk3,Lspl and Aldh2 were significantly up-regulated in the ischemic myocardium of KO mice.Among them,Med1 increased most obviously.The dual luciferase reporter result suggested that Med1 was one target gene of MicroRNA 146 a.The result of Western Blot showed that the expression of Med1 in myocardial tissues of KO group was significantly increased after myocardial ischemia and reperfusion,accompanied with the up-regulation of Bax / Bcl2 ratio and cleaved-Caspase3.5.Rescue study showed that cell apoptosis was alleviated with the inversion of Med1 expression in H9C2 which experienced hypoxia-reoxygenation.Conclusion MicroRNA 146 a can decrease the apoptosis of cardiocytes by partially inhibiting the target gene Med1,thus to exert its cardioprotective effect.