MicroRNA-145 Targeting Down-Regulation Of PDCD4 To Protect Myocardial Injury After Myocardial Ischemia

Objectives 1.To investigate the expression profile of miR-145 in the myocardial infarction(MI)rat model.2.To investigate the beneficial role of miR-145 overexpression on the cardiac function and myocardial injury of MI rat.3.To determine the protective effects of miR-145 overexpression on the hypoxia/ reoxygenation(H/R)-injured cardiomycocytes.4.To identify the downstream target of miR-145 and further determine its role on MI by dual luciferase reporter assay.Methods: 1.The sequences of miR-145 and sham control were subcloned into plasmid vevtor(LVmiR-145 and LV-miR-NC)and then contransfected into HEK-293 T cells by lentivector packaging system.2.Forty SD rats were randomized into four experiments groups: sham group,MI group,MI rats injected with lentivirus containing miR-145 sequences(LV-miR-145 group)and MI rats injected with lentivirus containing negative control sequences(LV-NC group).3.24 hours after MI,rats were anesthetized and echocardiography was studied.LVAWD,LVPWD,LVEF and LVEFS were evaluated by a Vevo-770 high-frequency ultrasound system.Rats were sacrificed after two weeks.BW,HW and LVW were weighted.Moreover,the infarct area were indentified and expressed by a percentage of the total area of LV.4.The expression level of miR-145 in heart tissues was dertermined by RT-qPCR and the protein expression levels of Bcl-2,Bax and cleaved caspase-3 in heart tissues were evaluated by Western blot.5.The cardiomyocytes isolated from the 1-3-day-old SD ratswere transfected with miR-145 or scrambled control miRNA and subjected to H/R treatment.After that,the cardiomyocytes apoptosis was evaluated by flow cytometry.The cationic dye JC-1 was used to detect mitochondrial membrane potential(MMP)in different cardiomyocyte groups.The dual luciferase reporter assay was performed to explore the potential direct target of miR-145.Results: 1.The BW in different groups was no significant.The expression level of miR-145 was significantly reduced in the infract areas.2.24 hours after anterior descending artery(LAD),the LVEF and LVFS in MI group were decreased,while the LVAWd and LVPWd were increased compared to sham group.The reduction of LVEF and LVFS,and the elevation of LVAWd and LVPWd in LV-mi R-145-treated rats were attenuated.Moreover,the LVW-to-BW ratio and HWto-BW ratio were increased in MI group.However,the LVW-to-BW ratio and HW-toBW ratio were reduced in MI rats transfected with LV-miR-145.3.The structural disorder of myofibril,extensive necrotic lesions and muscle fiber swelling were observed in MI group,and these pathological changes were attenuatedin MI rats transfected with LV-miR-145.Moreover,the infarcted area and cardiomyocyte apoptosis were decreased in MI rats treated with LV-miR-145 compared to MI rats treated with LV-NC.The expression levels of pro-apoptotic proteins cleaved caspase-3 and Bax were decreased and anti-apoptotic protein Bcl-2 was increased in LV-miR-145 group.4.The expression of miR-145 in H/R-treated cardiomyocytes was reduced.The cell apoptosis in H/R treated-cardiomyocytes transfected with miR-145 was significantly decreased.By dual luciferase reporter assay,the PDCD4 was identified as the potential target gene of miR-145.Moreover,the western blot analysis indicated that miR-145 overexpression could reduce the expression of PDCD4 protein.PDCD4 overexpression restored cardiomyocytes apoptosis inhibited by miR-145 after H/R injury.5.The MMP was decreased in H/R-treated cardiomyocytes and this effect was inhibited by miR-145.Moreover,the release level of cytochrome c from mitochondrial was suppressed by miR-145.Conclusion: 1.The reduced expression of miR-145 in MI-injured myocardial tissues suggestted that miR-145 may be implicated in the develpment and progression of MI.2.MiR-145 overexpression attenuates the apoptosis in MI-injured myocardial tissues and improves cardiac function,indicating that miR-145 might be act as a potential therapeutic target for MI.3.MiR-145 overexpression could stabilize the MMP and inhibit cytochrome c release in H/R-treated cardiomyocytes,thus leading to the inhibition of cell apoptosis.4.MiR-145 exerts its protective role partly through regulating its potential direct target PDCD4 in MI.