Research For Protective Effects Of MicroRNA-486 On Hypoxia Injury Of Primary Cultured Myocardial Cells

Objectives 1.We aimed to produce adenovirus vector carrying MiR-486,and artificial synthesize rno-miR-486（Ad-miR-486）and rno-miR-486-antago（Ad-miR-486-antago）.2.Cultivate primary cultured myocardial cells of neonatal rats,and create models of myocardial hypoxia injury via acute ischemia.3.Transfect primary cultured myocardial cells with Ad-miR-486 and Ad-miR-486-antago,and make miR-486 over expression or low expression.Observe biological characteristics of myocardial injury and apoptosis.Illuminate the role of miR-486 in the regulation myocardial ischemic adaptation.And provide new ideas and experimental evidences for therapies of myocardial ischemia.Methods 1.Via miR-486 amplification of recombinant adenovirus with HEK293 cell packaging,produce plenty of adenovirus vector carrying miR-486.Real-time quantitative identification are detected by PCR amplification of adenovirus,adeno-associated virus titer are detected by TCID50 methods.2.Create models of myocardial hypoxia injury via acute ischemia.Using SD neonatal rats 1³ days,under aseptic conditions of separate and predicate myocardial cells in ventricular（at 37 5%CO₂）.72 hours after the transfer experiments carry out in serum-free DMEM,and 12 hours after cultured in normoxic box respectively（37 C,5%CO₂-95%Air）and low oxygen incubator（at 37 1%O₂-94%N2-5%CO₂）supernatant is collected after 48 hours of each cell and cell.In normoxic cultured as control,determination of cell supernatant lactate dehydrogenase（LDH）activity in response to cell damage,myocardial cell activity by CCK-8 PI staining,apoptosis is determined by flow cytometry,and the cells are harvested for total RNA extraction,determined by the expression of hypoxia myocardial cells cultured miR-486 by real-time PCR.3.Experiment of MiR-486 over expression and low expression of the intervention trial.Over expression of miR-486 and low expression of Ad-miR-486 and Ad-miR-486-antago by transfection of primary cultured myocardial cells,experimental groups are as follows:（1）normoxia group（2）;hypoxia;（3）Ad-miR-486 transfected with normoxia group;（4）Ad-miR-486-antago transfected with normoxia group;（5）Ad-miR-486 transfection with hypoxia group;（6）Ad-miR-486-antago transfection with hypoxia group.At the end of the experiment,the cells and supernatant are collected.Results 1 Ad-miR-486 was amplified by HEK293 cells,and the titer of Ad-GFP infection was 2×10¹¹PFU/ml,and the titer of Ad-miR-486 and Ad-miR-486-antago was about 1×10¹¹PFU/ml.2 The models of myocardial injury induced by hypoxia were created.Compared with the cultured myocardial cells,the surface area of myocardial cells was decreased,the intercellular space became larger,the number of dead cells suspended in the culture medium was increased,and the rhythm of cell apoptosis was slowed down after 24 hours of hypoxia.3 Expression of miR-486 in hypoxic cultured myocardial cells changed.After 6h,12 h,24h,36 h and 48 h were cultured,the expression of miR-486 was detected,the expression of miR-486 in 0²4h was significantly up-regulated,and the expression of miR-486 was peaked in 24 h,and the expression of 24h⁴8h decreased gradually.4 In case of hypoxia,LDH activity decreased by 64% in the control miR-486 compared with control group,the cell viability increased 28%,the apoptosis rate decreased by 23%.Antago after transfection of miR-486 cells compared with control group,LDH activity increased by 30%,the cell viability was decreased to 40%,the apoptosis rate increased by 44%.ConclusionIn case of hypoxia,expression of mi R-486 in primary cultured myocardial cells was up-regulated and peaked 24 hours later.Over expression of miR-486 can reduce the apoptosis rate caused by hypoxia,improve cell viability,and reduce cell damage.The low expression of miR-486 can increase cell apoptosis rate caused by hypoxic conditions,decrease cell viability,an increase cell damage.The experimental results show that miR-486 can alleviate the cell injury and inhibit the apoptosis of myocardial cells,and has a certain myocardial protective effect.