Effect of Overexpression of miRNA-424-5p Targeting AKT3 on Apoptosis of Endometrial Epithelial Cells in Dairy Cows
[Objective]In this study,overexpression of miRNA-424-5p was performed to clarify the mechanism of the effect of miRNA-424-5p and its target gene AKT3 on the apoptosis of cow endometrial epithelial cells,and provide experimental basis and theoretical basis for elucidating the mechanism of miRNA-424-5p in the process of cow placental separation.[Method]The experiment was divided into three groups:Blank control group(Control),negative control group(miRNA-424-5p mimics NC)and overexpressed group(miRNA-424-5p mimics).The expression of miRNA-424-5p in cow endometrial epithelial cells after miRNA-424-5p mimics was detected by Real-time quantitative PCR using stem loop method.Western blotting and Real-time quantitative PCR were used to detect the expression changes of AKT3 and apoptosis-related factors after overexpression,and Annexin V-FITC/PI was used to detect the apoptosis of cow endometrial epithelial cells.[Result]Compared with blank control and negative control groups,the expression of miRNA-424-5p mRNA in endometrial epithelial cells was extremely significantly increased(P＜0.01),the mRNA expression of Bax,Caspase3 and Caspase9 was extremely significantly increased(P＜0.01),and the mRNA expression of AKT3 and Bcl-2 was extremely significantly reduced(P＜0.01)after transfection with miRNA-424-5p mimics.The relative expression of Bax and Caspase3 proteins was extremely significantly increased(P＜0.01),Caspase9 protein was significantly increased(P＜0.05),and AKT3 and Bcl-2 proteins were extremely significantly reduced(P＜0.01).Apoptosis rate of cells was extremely significantly increased(P＜0.01).[Conclusion]Overexpression of miRNA-424-5p could increase the expression of apoptotic factors Bax,Caspase3 and Caspase9,and decrease the expression of Bcl-2 in endometrial epithelial cells of dairy cows through targeted regulation of AKT3,thus leading to increased apoptosis.
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