The Role Of MiR-29b1 In Hypoxia-induced Cardiomyocyte Injury

Background Pathological factors such as acute myocardial infarction,myocardial ischemia-reperfusion and oxidative stress are important inducements of cardiomyocyte apoptosis.The key regulator of mitochondrial apoptotic signaling pathway is Bcl-2 family proteins that regulate the opening and closing of m PTP.The ratio of Bcl-2 / Bax can reflect the actual regulation of apoptosis.mi RNAs is an endogenous non-coding single-stranded small molecule in eukaryotes that recognizes the corresponding 3’ untranslated region(UTR)of the target gene m RNA and regulates gene expression.The mi R-29 family is composed of mi R-29 a,mi R-29 b and mi R-29 c.They are mainly expressed in myocardial fibroblasts and vascular smooth muscle cells.Cx43 is the most important junctional protein in intercellular signal transduction of ventricular working myocytes,which mainly exists in atrial and ventricular muscle tissues,participated in the pathophysiological process of heart development and related diseases.TGF-β is a powerful fibrogenic cytokine and fibroblast chemokine,which can induce fibroblast differentiation in TGF-β / Smad signal transduction,stimulating the synthesis of collagen,fibronectin and proteoglycan.After myocardial infarction,myocardial fibrosis is enhanced by down-regulation of mi R-29 expression.However,the role of mi R-29b1 on cardiomyocytes injury induced by hypoxia is not well known.Thus this research aims to use AC16 and H9C2 cells cultured in vitro under anoxic conditions in a three-air incubator simulated the ischemic and hypoxic state of myocardial cells in vivo,and explored the role of mi R-29b1 in hypoxic cardiomyocyte injury and its potential mechanism involved.And human and rat cardiac myocytes were used to observe whether there was any difference.The purpose of this study was to prepare for further animals experiments in vivo and to provide new targets for the diagnosis and treatment of ischemic heart disease.Methods Chapter Ⅰ Overexpression of mi R-29b1 reduce the apoptosis rate of cardiomyocytes induced by hypoxia We replicated an in vitro AC16 and H9C2 cardiomyocytes ischemia model by hypoxia(1% O2,37℃,24 h and 48h)treatment.After grouping the myocardial cells were transfected into mi R-29b1 mimic and Negative Control,conventional cultured 48 h to ensure that they have already transferred into cells.Cell apoptosis was evaluated by flow cytometry using Annexin V FITC-PI staining assay.Chapter Ⅱ Overexpression of mi R-29b1 in cardiomyocytes up-regulates the ratio of apoptosis-related protein Bcl-2 and Bax,and the expression of Caspase-3 and Caspase-9 proteins.The expression of Bcl-2,Bax,Caspase-3 and Caspase-9 proteins in cardiaomyocytes was detected by Western blot analysis.Chapter Ⅲ The expression of Cx43 protein in cardiomyocytes with overexpression of mi R-29b1 decreased and the expression of TGF-β increased.The expression of Cx43 and TGF-β in cardiomyocytes was detected by Western blot analysis.Results Chapter Ⅰ Overexpression of mi R-29b1 reduce the apoptosis rate of cardiomyocytes induced by hypoxia Flow cytometry showed that both AC16 and H9C2 cells could induce apoptosis due to hypoxia,and the apoptosis rate increased with the prolongation of hypoxia.mi R-29b1 mimic and Negative Control were successfully transfected into AC16 and H9C2 cardiomyocytes.Flow cytometry showed that the apoptosis rate decreased significantly after transfection of mi R-29b1.Chapter Ⅱ Overexpression of mi R-29b1 in cardiomyocytes up-regulates the ratio of apoptosis-related protein Bcl-2 and Bax,and the expression of Caspase-3 and Caspase-9 proteins The Western blot analysis results showed that,compared with those transfected with mi R-NC,the ratio of Bcl-2 and Bax in AC16 cells increased slightly after transfection of mi R-29b1 and the expression of Caspase-3 and Caspase-9 protein increased significantly after transfection of mi R-29b1.The ratio of Bcl-2 and Bax after the overexpression of mi R-29b1 in H9C2 cells in was declined compared with SGOD group and mi R-NC group,the expression of Caspase-3 was not changed compared to mi R-NC group and the expression of Caspase-9 protein was increased.Chapter Ⅲ The expression of Cx43 protein in cardiomyocytes with overexpression of mi R-29b1 decreased and the expression of TGF-β increased.The results of Western blot analysis showed that the expression of Cx43 in AC16 and H9C2 cells in hypoxia environment was lower than that in Control group,and there was no change before and after transfection of mi R-29b1.While the expression of TGF-β in AC16 and H9C2 cells in mi R-29b1 group was lower than that in Control group,the expression of TGF-β was significantly higher than that in SGOD group and mi R-NC group.Conclusions 1.Hypoxia could induce cardiomyocyte apoptosis,and the apoptosis rate increased with the prolongation of hypoxia time.Overexpression of mi R-29b1 reduced markedly the apoptosis rate of hypoxic cardiomyocytes.2.Overexpression of mi R-29b1 inhibits the apoptosis of hypoxic cardiomyocytes through mitochondrial apoptosis pathway.3.The expression of Cx43 protein in hypoxic cardiomyocytes has little change while the expression of TGF-β protein significantly increased after overexpression of mi R-29b1.4.The pathway of apoptosis induced by hypoxia was consistent between human cardiomyocytes and rat cardiomyocytes.