The Role And Regulation Mechanism Of MicroRNA-210 On Cell Apoptosis Induced By H₂O₂ In Endothelial Cells And Cardiomyocytes

Coronary heart disease has become one of the leading causes of human death and disability,with its morbility and mortality rates increasing year by year.Atherosclerosis is the major cause of coronary heart disease.Endothelial apoptosis induced by oxidative stress plays a critical role throughout the whole process of the initiation and development of atherosclerosis.Acute myocardial infarction is the main cause of death and disability in coronary heart disease.Currently,the most effective therapy for acute myocardial infarction is to open the criminal vessel and to restore myocardial reperfusion timely.However,another myocardial injury,named ischemia/reperfusion injury,occurs in this process.Myocardial cell apoptosis induced by oxidative stress plays a critical role in the myocardial ischemia/reperfusion injury.Therefore,it is of great importance to reduce and prevent oxidative stress-induced endothelial cell apoptosis and cardiomyocyte apoptosis for preventing the occurrence of coronary heart disease,improving the prognosis and reducing the mortality and morbidity of coronary heart disease.Micro RNAs?mi RNAs?are a group of small,non-coding RNAs that participate in various cell processes,including cell cycle,DNA repair,cell apoptosis,and cell proliferation,through post-transcriptional downregulation of their target m RNAs.Latest studies have found that micro RNA-210?mi R-210?has an important role in cell function,which has drawn great attention.Mi R-210 was first reported to be linked with hypoxia,and plays an important role in cells reponse to hypoxia.Besides hypoxia,recent studies have found out that mi R-210 also exterts a role in cells under other stimulus,oxidative stress included.The regulation of mi R-210 on cell apoptosis has gradually attracted attention.Our previous study demonstrated that mi R-210 protected H9c2 cardiomyoblasts against H₂O₂ stimulation.However,the role of mi R-210 in HUVECs under oxidative stress and its molecular mechanism still remains unclear.Cell apoptosis is a major mode of cell programmed death.It eliminates individual cells within an organism while preserving the overall structure of surrounding tissue.Apoptosis can be recognized morphologically.The nuclei and cytoplasm,including the mitochondria,shrink in cells undergoing apoptosis,membrane blebbing,chromatin condensation and nuclear fragmentation also occur in apoptosis.Cell contents then become encased in apoptotic bodies surrounded by membrane,which subsequently engulfed by nearby phagocytic cells.Apoptosis participates in the pathlogic process of various cardiovascular diseases,including atherosclerosis,myocardial infarction and reperfusion injury,and heart failure.Thus,it is of great importance to reduce and inhibit apoptosis for the therapy and prevention of cardiovascular diseases.The molecular mechanism of apoptosis is highly complex.The major two pathways of apoptosis are extrinsic apoptosis pathway,also called death receptor pathway,and intrinsic apoptosis pathway,also called mitochondria pathway.The extrinsic apoptosis pathway is mediated by death receptor.After activation,the death receptor recruits multiple apoptotic protein to form death inducing signal complex,caspase-8 and caspase-10 included.The complex ultimately activates apoptosis effector,caspase-3,leading to apoptosis.The intrinsic apoptosis pathway is mediated by mitochondria,and regulated by Bcl-2 family.Pro-apoptotic protein in Bcl-2 family is activated in the intrinsic apoptosis pathway,and forms the mitochondrial membrane permeability transition pore,which leads to the relase of pro-apoptotic protein from mitochondria to cytoplasm,such as cytochrome c.These proteins subsequently activate caspase-9,which in turn activates caspase-3,and leads to apoptosis.CASP8AP2 is an essencial role in extrinsic apoptosis pathway.It activates caspase-8 via interacting procaspase-8.Activated caspase-8 atcivates its downstream caspase,caspase-3 subsequently,thus leading to apoptosis.Some studies found out that under hypoxia,mi R-210 could protect stem cells against apoptosis via downregulation CASP8AP2.Nevertheless,mi R-210 seemed to have no correlatioin with CASP8AP2 in HL-1 cell line.Therefore,the relationship between mi R-210 and CASP8AP2 and its influence on cell apoptosis needs to be explored.BNIP3 is a member of BH3-only family.It regulates intrinsic apoptosis pathway via interacting with anti-apoptotic protein,Bcl-2,thus promoting expression of pro-apoptotic protein.Under hypoxia,mi R-210 could inhibit neuro progenitor cell apoptosis through downregulation of BNIP3.However,the relationship between mi R-210 and BNIP3 in cardiovascular system remains unclear.In this study,we treated HUVECs and H9c2 cells with H₂O₂ to induce oxidative stress.We investigated the role of mi R-210 in HUVECs apoptosis under oxidative stress and the molecular mechanism via detection of CASP8AP2 pathway and BNIP3 pathway related genes expression.Our study provided a new therapy stragedy for coronary heart disease.Methods:1.HUVECs were treated with H₂O₂ to induce oxidative stress,and MTT assay and ATP detection were employed to detect cell viability.Cell apoptosis was detected with flow cytometry.2.Quantitative real-time PCR was performed to detect mi R-210 expression under different concentrantions and duration of H₂O₂ treatment.3.Gain-and-loss-of-function approaches were used via lentivirus infection of HUVECs and H9c2 cells.To investigate the effect of mi R-210 on HUVECs under H₂O₂ treatment,cell viability and cell apoptosis were detected with MTT assay,ATP detection,and flow cytometry,respectively.4.To investigate the relationship between mi R-210 and CASP8AP2 pathway in HUVECs and H9c2 cells under oxidative stress,CASP8AP2 pathway related genes,CASP8AP2,caspase-8,and caspase-3 were detected with quantitative PCR and Western Blot.5.To investigate the relationship between mi R-210 and BNIP3 pathway in HUVECs and H9c2 cells under oxidative stress,BNIP3 pathway related genes,BNIP3,Bax,and Bcl-2 were detected with Western Blot.Results:1.H₂O₂ decreased HUVECs cell viability,and induced cell apoptosis in a dose-dependent manner.2.Mi R-210 expression was increased in HUVECs under H₂O₂ treatment.3.Over-expression of mi R-210 inhibited cell apoptosis in HUVECs induced by H₂O₂,while inhibition of mi R-210 increased cell apoptosis in HUVECs induced by H₂O₂.4.Over-expression of mi R-210 decreased CASP8AP2 expression with or without H₂O₂ treawtment,and decreased cleaved caspase-8 and caspase-3 expression under H₂O₂ treatment in both HUVECs and H9c2 cells5.Over-expression of mi R-210 decreased BNIP3 expression with or without H₂O₂ treawtment,and decreased the ratio of Bax/Bcl-2 under H₂O₂ treatment in both HUVECs and H9c2 cells Conclusion:H₂O₂ induced HUVECs cell apoptosis and mi R-210 expression.Over-expression of mi R-210 inhibited cell apoptosis induced by H₂O₂.The mechanism of the apoptosis inhibition may be via targeting regulation of the expression of apoptotic protein CASP8AP2 and BNIP3,thereby regulating the extrinsic apoptosis pathway and the intrinsic pathway.Our study investigated the anti-apoptotic effect of mi R-210 in HUVECs under H₂O₂ treatment for the first time.We further investigated the relationship between mi R-210 and CASP8AP2 pathway and BNIP3 pathway,and its role in the anti-apoptotic effect of mi R-210 in HUVECs and H9c2 cells.Our study provided a theoretical evidence for the molecular targeted therapy of coronary heart disease.