Overexpression Of MicroRNA-29c Suppresses Proliferation,and Promotes Apoptosis And Differentiation In P19 Cells By Inhibiting Akt3

Research background:Congenital heart disease is the most common birth defect disease,its pathogenesis has not been elucidated.miR-29 c is a miRNA highly expressed in the myocardial tissue of the ventricular septal defect using microchip technology.Previous studies have found that miR-29 c expression changes significantly affect P19 cell proliferation.The bioinformatics analysis and luciferase report showed that Akt3 was the target gene involved in cell proliferation related signaling pathway.The expression of Akt3 in myocardial tissue was significantly inhibited,suggesting that the mechanism of mir-29 c induced fetal heart malformation may be related to the regulation of PI3K-Akt3 signaling pathway.In this study,the effects of miR-29 c overexpression and silencing on apoptosis and differentiation of myocardial cells were observed at the cellular level.Furthermore,the molecular mechanism of mir-29 c induced fetal heart development was further studied with the PI3K-Akt3 signaling pathway as the starting point.The study of miR-29 c induced fetal heart malformation and its mechanism has not been reported in the literature.This study is innovative and can provide new clues and potential intervention targets for early prevention andtreatment of congenital heart disease.Methods:1.After transfection of P19 cells with miR-29 c mimic,CCK8 experiment,flow cytometry,Hoechst staining experiment,real-time quantitative PCR and western blot were used to detect the effects of miR-29 c overexpression on proliferation,cycle and apoptosis of P19 cells.2.After miR-29 c mimic transfection with P19 cells,bioinformatics analysis combined with luciferase reporter experiment and western blot experiment to determine the target gene of miR-29 c.3.After transfection of P19 cells with si-Akt3,CCK8 experiment,flow cytometry,Hoechst staining,real-time quantitative PCR and western blot experimental were used to detect the effects of Akt3 silence on proliferation,cycle and apoptosis of P19 cells.4.After transfection of P19 cells with miR-29 c mimic and si-Akt3,dimethyl sulfoxide(DMSO)induced P19 cell differentiation.Real-time quantitative PCR was used to detect the expression of myocardial specificity marker genes myosin heavy chain(alpha myosin heavy chain,αMHC),GATA4 transcription factor,myocardial enhancement factor 2c(Myocyte enhancer factor 2c,Mef2c)and cardiac troponin T(cardiac troponin T and cTNT),to clear the effect of miR-29 c overexpression andAkt3 silence on the differentiation of P19 cell.Results:1.After transfection of P19 cells with miR-29 c mimic,the proliferation ability of cells was inhibited,and cell cycle was blocked in G0/G1 phase,the expression level of Bax increased significantly,and the apoptosis rate increased.2.The fluorescease report and the protein stamp experiment confirmed Akt3 is a target gene of miR-29 c.3.The proliferation ability of P19 cell lines treated by si-akt3 was inhibited,and cell cycle was blocked in G0/G1 phase,while the mRNA expression level of Bax increased significantly,while the protein expression level of bcl-2 was significantly decreased,and the apoptosis rate increased.4.After treatment with miR-29 c mimic and si-Akt3,P19 cells formed a more homogeneous and stable embryonic form,and more pulsation myocardial cells were formed in the later stage.The expression level of the myocardial marker genes αMHC,Mef2 c,GATA4 and cTNT was significantly higher than that of negative control.Conclusions:1.miR-29 c overexpression can inhibit the proliferation of P19 cells,promote apoptosis,block cell cycle,and promote differentiation of myocardial cells.2.Akt3 is a downstream target gene of miR-29 c,and overexpression of miR-29 c affects its biological effect on P19 cells by inhibiting the expression of Akt3 target gene.