Study On The Role And Mechanism Of MiR-322 In H/R Myocardial Injury

Objective:Explore the role and mechanism of miR-322 in hypoxia/reoxygenation myocardial injury.In this study,stem cells were differentiated into cardiomyocytes in a targeted manner.The myocardial cell H/R experiment was used to simulate myocardial ischemia reperfusion in the context of myocardial infarction.As the research follow on,we hope to provide new ideas for clinical prevention and treatment of myocardial injury.Methods:1.Culture of mouse embryonic fibroblasts?MEF?and embryonic stem cells?m ESC-D3?.2.Preparation and transfection of lentivirus.The lentivirus with the target gene was constructed,miR-322 overexpression,miR-322 knockdown,celf1 overexpression,celf1 knockdown,miR-322/celf1-co-overexpression,and then transfected into mESC-D3 cells,and screened the expression target gene cells.3.Differentiation of mESC-D3 cells directed into cardiomyocytes.The cells were divided into 8 groups:no-H/R group?Normal?;wild cell group+H/R?Wild?;empty virus cell+H/R?Empty?;miR322-up+H/R?322-up?;miR322-down+H/R?322-down?;Celf1-LV+H/R?Celf1-LV?;Celf1-RNAi+H/R?Celf1-RNAi?;miR322-up+Celf1-LV+H/R?322-up+Celf-LV?.The differentiation of embryonic stem cells was induced by classical hanging drops method for 4.5d.The embryoid body was culturedin the differentiation medium in 6cm petri dish.4.Hypoxia/reoxygenation of myocardial cells.After successful differentiation,the myocardial cells were treated with hypoxia/reoxygenation when mature?the 8th day after differentiation?.Hypoxia:5%C0₂,95%N₂ 3h;reoxygenation:5%C0₂,95%O₂ 2h.5.Test indicators.The cells were observed in various groups after the hypoxia/reoxygenation;The spontaneous beating frequency of myocardial cells in each treatment group was observed by fluorescence microscope;The cell survival rate was detected by MTT colorimetry before and after hypoxia/reoxygenation;The activity of lactate dehydrogenase in cell culture medium and the ATP content were detected;Real-time quantitative RT-PCR,GAPDH was used as the standard control to determine the changes of myocardial cell genes?OCT-4,Nkx-2.5,MLC-2V,Sox-2,?-MHC?in each group;The expression levels of Nkx-2.5 and cTnT were detected by western blot.6.Statistical analysis.Statistical analysis applied t test to evaluate the differences between the two groups,P<0.05.Result:1.MESC-D3 cells successfully induced differentiation into cardiomyocytes that could generate spontaneous beating;2.MiR322-overexpression and Celf1-knockdown can protect myocardial cells differentiated by stem cells from the H/R injury.Compared with the normal group,the survival rate and beating frequency of myocardial cells in the wild group decreased significantly,the ATP level in the cells of the wild group decreased significantly,while the LDH activity in the culture medium increased,indicating that H/R was successful.Compared with the control group,the survival rate and beating frequency of myocardial cellsincreased significantly,the ATP level increased significantly too,the LDH activity was evidently reduced in the treatment groups of miR-322 overexpression?Celf1 knockdown.The PCR results showed that pluripote-ncy correlation genes?OCT-4,Sox-2?and myocardial cell genes?Nkx-2.5,MLC–2V,?-MHC?genes are raised,WB results showed that the myocardial cell protein?Nkx-2.5 and cTnT?are raised.3.MiR-322 can protect myocardial cells differentiated by stem cells from the H/R injury by inhibiting the expression of Celf1.Compared with control group,the survival rate and beating frequency of myocardial cellsincreased significantly,the ATP level increased significantly too,the LDH activity was evidently reduced in the322-up+Celf1-LV group,but the changes of each index were not as significant as that of 322-up group.The results of PCR showed that these genes Oct-4,Sox-2,Nkx-2.5,MLC-2 v,?-MHC were up-regulated,and the results of WB showed that the myocardial cell protein Nkx-2.5 and cTnT were all up-regulated.Celf1 might be the downstream protein for miR-322,miR-322 can regulate the downstream genes and proteins expression through regulating Celf1,so as to give play to the role of protect myocardial cells,however,miR-322 do not completely inhibit Celf1-overexpression,so there are many complex mechanisms,intermediate molecules need to be researched.Conclusion:1.MiR-322-overexpression has protective effect on hypoxia/reoxygenation myocardial injury;2.Celf1-knockdown has protective effect on H/R myocardial injury;3.MiR-322 have protective effect on H/R myocardial injury by inhibiting the expression of Celf1;...