The Effect And Mechanism Of MicroRNA-214 On Apoptosis Of L6 Skeletal Myoblast In Rats Injured By H₂O₂

Objectives:Skeletal myoblasts transplantation offers hope for patients with myocardial infarction.In myoblast transplantation therapy aftermyoca-rdial infarction,ischemia and anoxic environment of oxidative stress can cause the transplanted cells apoptosis.Low survival rate of transplanted cells seriously affect the effects of treatment.The study intends to establish L6Skeletal myoblast?L6 Skeletal Myobalst,L6SKM?H₂O₂ damage model in vitro,to observe apoptosis under oxidative stress conditions after increasing or inhibiting the expression of miR-214 and discuss its regulatory mechanism.Methods:The cells were recovered and cultured to 3 or 4 generations and then cell suspension was inoculated into 6-holes plates which contain cover glasses or flasks.After 24h,mi R-214 transfection was carried out,and the growth medium was incubated for 24 hours after transfection,followed by 300?mol/L H₂O₂ for 4h.The cells were divided into six groups:normal control group,H₂O₂ group,miR-214 precursor+H₂O₂ group?pre-miR-214+H₂O₂ group?,mi R-214 precursor blank+H₂O₂ group?pre-scramble+H₂O₂ group?,mi R-214inhibitor+H₂O₂ group?anti-miR-214+H₂O₂ group?,miR-214 inhibitor blank+H₂O₂ group?anti-scramble+H₂O₂ group?.The morphological changes of cells were observed under inverted microscope.SP method was to show cyt-c expression;DAPI staining showed the effect of miR-214 on the apoptosis of L6SKM cells injured by H₂O₂;the expression of PTEN was detected by Western blotting.Annexin V-FITC&PI flow cytometry was used to detect the apoptosis rate of L6SKM in above groups and pten inhibitor group.Results:1.Effects of mi R-214 on the growth state and apoptosis of L6SKM cellsUnder microscope,L6SKM in normal control group was flourishing.The dotted brown-yellow granules of cyt-c were found to be scattered in the cytoplasm in the control group and the distribution was even.Cells in H₂O₂treatment group grow slowly.A part of L6SkM shrinks and the cyt-c immunoreactivity was significantly enhanced.Cell heterochromatin distri-buted peripherally or even broken and the number of apoptotic cells increased significantly.The growth status of L6SKM in the pre-miR-214+H₂O₂ group was significantly better than that of the H₂O₂ treatment group.The expression of cyt-c and the apoptosis rate was significantly decreased.While in anti-mi R-214+H₂O₂ group,the number of shrunking cells and dead cells was increased obviously.The expression of cyt-c and the apoptosis rate was significantly increased.2.Effect of miR-214 on expression of PTEN protein in L6SKM injured by H₂O₂.H₂O₂ treatment led to PTEN expression higher than the control group.Compared with H₂O₂ group,the expression of PTEN was reduced signific-antly by miR-214 overexpression and was increased significantly by inhibit-ing miR-214 expression.3.Cell apoptosis rate was detected by flow cytometryThe results showed that the apoptosis rate in H₂O₂ group?39.47±1.89?was significantly higher than that in the control group?7.55±0.42?,P<0.05.Compare with H₂O₂ group?39.47±1.89?,apoptosis rate was obviously lower by miR-214 overexpression?18.57±0.97?,P<0.05,was slightly higher by inhibiting mi R-214 expression?41.16±2.21?but no statistical significance.The apoptosis rate in pre-miR-214+pten inhibitor+H₂O₂ group?11.12±0.95?was obviously lower than that of pre-miR-214+H₂O₂ group?18.57±0.97?,P<0.05.In anti-miR-214+pten inhibitor+H₂O₂ group?17.63±0.73?,apoptosis rate was significantly lower than that in anti-miR-214+H₂O₂ group?41.16±2.21?,P<0.05.Conclusion:1.MiR-214 overexpression can inhibit apoptosis induced by hydrogen peroxide.2.MiR-214 can inhibit myoblasts apoptosis induced by H₂O₂ through decreasing PTEN expression and activating PI3K/Akt signal pathway.