Study Of The Regulation Of Apoptosis And Hypertrophy In Cardiac Myocytes By MiR-145

Part 1 Study of the regulation of hydrogen peroxide-mediated apoptosis of cardiac myocytes by miR-145Objective:To investigate the regulatory effects of miR-145 in hydrogen peroxide(H2O2)-mediated apoptosis of cardiac myocytes and the underlying mechanism;to examine the correlation between miR-145 expression and the cardiac ischemia-reperfusion injury.Methods:Quantitative real-time PCR(qPCR)were performed to detect the expression of miR-145 in either ischemia/reperfused mice myocardial tissues or H2O2-treated neonatal rat cardiac myocytes(NRCMs).Adenovirus was employed to over-express miR-145 in NRCMs.DNA ladder,TUNEL staining,DCFH-DA staining and electron transmission microscopy analysis were carried out to evaluate the apoptosis of NRCMs that over-express miR-145 and were challenged with H2O2.Several key signaling proteins in mitochondrial apoptotic pathway were examined by western blot.Moreover,two plasmid constructs were prepared to over-express Bnip3 CDS and Bnip3 CDS+3’UTR in NRCMs,respectively.Finally,a combination of luciferase reporter assay and western blot was utilized to testify if Bnip3 is a direct target of miR-145.Results:qPCR demonstrated that the expression of miR-145 in either ischemia/reperfused mice myocardial tissues or H2O2-treated NRCMs was markedly down-regulated.Over-expression of miR-145 significantly inhibited the H2O2-induced apoptosis,ROS production,mitochondrial structure disruption as well as the activation of several key signaling proteins in mitochondrial apoptotic pathway.These protective effects of miR-145 were abrogated by over-expression of Bnip3 CDS but not Bnip3CDS+3’UTR.Finally,luciferase reporter assay and western blot analysis identified Bnip3 as a direct target of miR-145.Conclusions:miR-145 protects cardiac myocytes from H2O2-mediated apoptosis,through targeting Bnip3,thus inhibiting the mitochondrial apoptotic pathway.The altered expression of miR-145 is associated with myocardial ischemia/reperfusion injury.Part 2 Study of the regulation of isoproterenol-induced hypertrophy of cardiac myocytes by miR-145Objective:To investigate the regulatory effects of miR-145 in isoproterenol(ISO)-mediated hypertrophy of cardiac myocytes and the underlying mechanism;to examine the correlation between miR-145 expression and the increased afterload-induced cardiac hypertrophy.Methods:Quantitative real-time PCR(qPCR)were performed to detect the expression of miR-145 in ISO-treated NRCMs and in the myocardial tissues of the mice undertaking transverse aortic constriction(TAC)procedure.Adenovirus was employed to over-express miR-145 in NRCMs.Fetal gene expression(ANF,BNP andβ-MHC),amino acid uptake rate,and the cell size were analyzed to evaluate the hypertrophy of NRCMs.Several classic hypertrophic signaling pathways were examined by western blot.A plasmid construct was prepared to over-express GATA6 in NRCMs.Luciferase reporter assay and western blot were utilized to testify if GATA6 is a direct target of miR-145.The sub-cellular location of GATA6 was also investigated by confocal fluorescence microscopy,in NRCMs overexpressing miR-145 and NRCMs treated with GSK3β antagonist LiCl or TDZD8.Results:The protective activity of miR-145 was found to be associated with down-regulation of the fetal genes ANF,BNP and P-MHC,a decreased rate of amino acid uptake and inhibited cell growth in NRCMs.These effects were reversed by exogenous over-expression of GATA6.Luciferase reporter assays and western blotting identified GATA6 as a direct target of miR-145.Multiple signaling pathways including MAPK,Akt and mTOR were found to be regulated by miR-145 as well.In addition,two GSK3β antagonists,LiCl and TDZD8,both restored the nuclear accumulation of GATA6 in the presence of miR-145,indicating that miR-145,Akt,GSK3β and GATA6 interact with one another in a regulatory axis.Finally,a dynamic pattern of miR-145 expression was identified in ISO-treated NRCMs and in the hearts of mice that had undergone transverse aortic constriction(TAC).Conclusions:miR-145 regulates both the expression and localization of GATA6,thereby protecting the heart against cardiomyocyte hypertrophy induced by the βAR agonist ISO.