Effect And Mechanism Of MicroRNA-486 On P53/Bbc3/BCL-2 Pathway In Prevention And Treatment Of Myocardial Injury Induced By Coronary Microembolization

Background: Coronary microembolization(CME)is a coronary artery microcirculation embolization and myocardial micro-infarction caused by spontaneous rupture of coronary atherosclerotic plaque or coronary intervention(PCI).It's considered to be a strong predictor of long-term major adverse cardiovascular events.CME activates multiple apoptotic pathways,leading to significant cardiomyocytes apoptosis in infarct-related areas and cardiac dysfunction,which is defined as myocardial injury.The regulatory mechanism of apoptotic pathway is not entirely clear in CME.P53 is an important apoptosis-related gene which can regulate the death receptor pathway,Bax / BCL-2,NF receptor and Fas protein pathway by Bbc3,and induce apoptosis.The expression of p53 was up-regulated in myocardial ischemia-reperfusion.When the mitochondrial apoptosis pathway was inhibited,the apoptosis of myocardium was decreased and the expression of p53 was down-regulated.However,whether p53 activates mitochondrial apoptotic pathway in CME,and whether p53 can regulate CME-induced myocardial apoptosis is not clear.Micro RNAs(miR,miRNAs)act as a negative regulator of m RNA expression by promoting m RNA hydrolysis or inhibiting translation.Mi RNAs are also involved in apoptosis.However,whether miRNAs can interfere with p53-mediated mitochondrial apoptotic pathway in cardiomyocytes is not clear.Therefore,the aim of this study was to observe the expression of miR-486 and p53 in CME rats and isolated cardiomyocytes,and to investigate the relationship between miR-486 / p53 and BCL-2-related mitochondrial apoptotic pathway.We also manipulated the miR-486 and p53 expression levels to reveal the roles of them in CME-induced cardiomyocyte apoptosis and cardiac dysfunction.Part I : Dynamic changes of miR-486 and p53 expression during coronary microembolization and their association with myocardial injury.Objective: Establishing the rats coronary microembolization(CME)model to observe the dynamic changes of miR-486 and p53 expression levels in CME,and to analyze their relationship with myocardial apoptosis and cardiac dysfunction.Methods: 12 SD rats were randomly divided into CME and sham groups.The CME group was established by injecting polyethylene microspheres into the left ventricle.The sham group was administered normal saline.The cardiac function was measured by ultrasonography after 6 hours.And then took the left ventricular myocardium specimens.TUNEL staining was used to evaluate the apoptotic index.RT-PCR and western blot were used to detect the expressions of miR-486,p53 and Bbc3Results: The CME group compared to sham group,the expressions of p53 and Bbc3 were increased(P <0.05),the expression of miR-486 was decreased(P<0.05),the apoptotic index was increased(P <0.05),left ventricular ejection fraction(LVEF)were significantly lower,and the left ventricular short axis shortening(FS)and cardiac output(CO)were decreased(P<0.05).Spearman linear correlation analysis showed that the expression of miR-486 was negatively correlated with the cardiomyocyte apoptotic index(P),negatively correlated with LVEF(P);p53 expression was positively correlated with cardiomyocyte apoptosis index(P)and LVEF(P).The expression of Bbc3 was positively correlated with the apoptotic index of myocardial cells(P),which was positively correlated with LVEF(P).Mi R-486 was negatively correlated with p53expression(P),and p53 was positively correlated with Bbc3 expression(P).Conclusion: Both miR-486 and p53 are involved in CME-induced myocardial injury and have a significant correlation,which may be involved in this process by regulating the expression of myocardial Bbc3.Part II: Role of p53 / Bbc3 / BCL-2 pathway in myocardial injury induced by coronary microembolization in RatsObjective: Cardiomyocyte apoptosis is a primary cause for coronary microembolization(CME)-induced cardiac dysfunction.p53 induces cell growth retardation and apoptosis through stress pathway;however,its role in CME is yet unclear.The present study investigated the mechanism of p53-induced myocardial apoptosis and cardiac dysfunction by activating the mitochondrion apoptotic pathway following CME.Methods: 40 SD rats were equally divided into microembolization(CME),sham operation(sham),CME+si RNA-p53,and CME+control-p53 groups.The CME rat model was established by injecting microembolization spheres via the left ventricle.The CME and sham groups were administered normal saline,while the other two groups were injected with adeno-associated virus injections,carrying p53-si RNA and control-si RNA sequences,respectively,before CME.Cardiac ultrasound,TUNEL,fluorescence quantitative PCR,and Western blot assessed the cardiac function indicators,cardiomyocyte apoptosis,and the expressions of m RNA and protein in myocardial tissues,respectively.Results: Echocardiography revealed a significantly reduced cardiac function of the CME group than the sham group(P<0.05)while the CME-induced cardiac dysfunction was improved in the CME+si RNA-p53 group(P<0.05).The CME group's myocardial apoptosis indicators increased significantly than the sham group(P<0.05);those of the CME+si RNA-p53 group decreased significantly than the CME group(P<0.05).Fluorescence quantitative PCR and Western blot demonstrated that p53,Bbc3,and cleaved caspase-3 expressions were significantly increased(P<0.05),and BCL-2 expression was declined(P<0.05)in myocardial tissues of the CME group compared with the sham group.A contrasting result was observed in the CME+si RNA-p53 group compared with the CME group.Conclusion: P53 is involved in the CME-induced cardiac dysfunction,which may up-regulate Bbc3 to activate BCL-2/caspase3 mitochondrial apoptotic pathway and induce myocardial apoptosis.Inhibiting the p53 expression can effectively suppress this pathway,thereby reducing cardiomyocyte apoptosis and cardiac dysfunction.Part III: Mi R-486 regulates cardiomyocyte apoptosis by p53-mediated BCL-2 associated mitochondrial apoptotic pathwayObjective: Cardiomyocyte apoptosis is a common pathological manifestation that occurs in several heart diseases.This study aimed to explore the mechanism of micro RNA-486(miR-486)in cardiomyocyte apoptosis by interfering with the p53-activated BCL-2 associated mitochondrial pathway.Methods: miR-486 mimics and inhibitors were transfected into the primary cardiomyocytes of suckling Sprague-Dawley rat pups,and H2O2 was used to induce apoptosis.Flow cytometry and TUNEL were both used to detect cardiomyocyte apoptosis,while the relative m RNA transcript and protein levels of miR-486,p53,Bbc3,BCL-2,and cleaved caspase-3 were detected using RT-PCR and western blot analysis,respectively.Results: miR-486 overexpression significantly decreased the expressions of p53,Bbc3 and cleaved caspase-3(P<0.05),and BCL-2 expression was significantly increased(P<0.05),which in turn caused a significant decrease in the rate of cardiomyocyte apoptosis(P<0.05).In contrast,miR-486 silencing resulted in an elevated rate of cardiomyocyte apoptosis(P<0.05).Conclusion: miR-486 may regulate cardiomyocyte apoptosis via p53-mediated BCL-2 associated mitochondrial apoptotic pathway.Therefore, up-regulating miR-486 expression in cardiomyocytes can effectively reduce the activation of the BCL-2 associated mitochondrial apoptotic pathway,consequently protecting cardiomyocytes.Part IV: Effect of micro RNA-486 on the regulation of p53 / Bbc3-mitochondrial apoptosis pathway in the reatment of coronary microembolizationObjective : Micro-RNAs play regulatory roles in coronary artery microvascular(CME)-induced myocardial injury.This study was designed to investigate the role of micro RNA-486(miR-486)in the prevention and treatment of myocardial injury by interfering with p53/Bbc3-activated mitochondrial pathway.Methods: 40 SD rats were randomly divided into CME,sham,CME +miR-486 up,and CME + miR-486 control groups.CME + miR-486 up group and CME + miR-486 control group were injected with adeno-associated virus carrying miR-486 sequence and control sequence from the tail vein,and 14 days later,CME model and sham operation model were constructed.The cardiac function was measured by ultrasonography.TUNE was used to detect cardiomyocyte apoptosis.The expression of p53,Bbc3,BCL-2 and cleaved caspase-3 in myocardium were detected by quantitative PCR and western blot.Results: Compared with sham group,the cardiac function of CME group was significantly decreased(P <0.05).There was no significant difference in cardiac function between CME group and CME + miR-486 control group(P>0.05),but the CME + miR-486 up group was significantly improved compared with CME group(P <0.05).The apoptotic index of myocardial cells in CME +si RNA-p53 group was significantly lower than that in CME group(P <0.05).Compared with sham group,the expression of p53,Bbc3 and cleaved caspase-3in CME group were significantly increased(P <0.05),but the expression of BCL-2 was decreased(P <0.05).The expression of p53,Bbc3 and cleaved caspase-3 in CME + miR-486 up group was significantly lower than that in CME group,while the expression of BCL-2 was increased.Conclusion: Mi R-486 could regulate CME-induced myocardial injury by interfering with p53 / Bbc3 / BCL-2 / caspase-3-related mitochondrial apoptotic pathway.Upregulation of miR-486 expression can effectively reduce myocardial apoptosis and cardiac dysfunction.