| MicroRNA(miRNA)is a non-coding sequence composed of about 22 bases.It regulates various biological processes of the body by targeting specific sites in the 3,-UTR region of m RNA to inhibit the translation process of protein-encoding genes.The previous research of this research group showed that miR-126 was differentially expressed in different developmental processes of pig testis.In this study,experimental techniques such as CCK8 /Ed U kit detection,flow apoptosis,flow cycle,ATP detection,Western Blot and q RT-PCR were used to study the effects of miR-126 and its target gene PIK3R2 on the proliferation and apoptosis of porcine immature Sertoli cells through PI3K/AKT signaling pathway,In order to provide a theoretical basis for improving the reproductive performance of boars.The research results are as follows:1.Over-express / inhibit the expression of miR-126 in pig immature support cells,use CCK8 and Ed U kits to detect cell proliferation,flow cytometry to detect cell cycle and apoptosis ratio,and ATP kit to detect intracellular ATP level,real-time fluorescence quantitative PCR technology was used to detect the expression of cell cycle related gene m RNA and Western Blot technology was used to detect the expression of apoptosis related gene protein.The results showed that after overexpression of miR-126,the proportion of pig immature Sertoli cells in G1 phase decreased and that in S phase increased;the m RNA expression of cycle-related genes C-MYC,CCNE1,CCND1 and CDK4 increased;intracellular ATP The level rises.The apoptosis rate decreased,the protein expression levels of the pro-apoptotic genes BAX and Casepase-3 decreased,and the protein expression of the inhibitory gene Bcl-2 increased;Increased expression of PI3 K and AKT phosphorylated proteins in cells.The effect of inhibiting the expression of miR-126 on the immature support cells of pigs is the opposite.The results showed that miR-126 promoted the proliferation of immature porcine support cells and inhibited apoptosis.2.Use Target Scan and Pic Tar and other online software to predict the potential target genes of miR-126 and take the results to intersect,and then use BLAST software to analyze the conservativeness of the binding sites between species and select PIK3R2.Subsequently,miR-126 was overexpressed and inhibited in pig immature Sertoli cells,q RT-PCR and Western Bloting technology were used to detect the expression of PIK3R2 in cells.It can significantly inhibit the expression level of PIK3R2 m RNA and protein(P<0.05),and inhibiting the expression of miR-126 significantly promotes the expression level of PIK3R2 gene m RNA(P <0.01).3.After knocking down the PIK3R2 gene,the proportion of cells in the G1 and S phases decreases;the m RNA expression levels of the cycle-related genes CCNE1,C-MYC,CCND1,and CDK4 increase;the intracellular ATP level increases;The protein expression level of Casepase-3 decreased and the protein expression level of apoptosis-related gene Bcl-2 increased;Increased expression of PI3 K and AKT phosphorylated proteins in cells.The results showed that knocking down the PIK3R2 gene promotes the proliferation of immature porcine support cells and inhibits their apoptosis,which is consistent with the overexpression of miR-126.4.To further clarify that miR-126 regulates the proliferation of porcine immature support cells by knock down the PIK3R2 gene,three sets of co-transfection experiments were designed: miR-126 inhibitor + siRNA PIK3R2,miR-126 inhibitor + siRNA NC and control group inhibitor NC + siRNA NC,and detect cell proliferation and apoptosis.The results showed that transfection of miR-126 inhibitor + siRNA PIK3R2 can promote the proliferation of immature porcine support cells,and the percentage of cell apoptosis decreased and intracellular ATP levels increased.The results of miR-126 inhibitor + siRNA NC group were opposite to those of transfected miR-126 inhibitor + siRNA PIK3R2 group.To sum up,this study preliminarily clarified that miR-126 promotes the proliferation of porcine immature Sertoli cells and inhibits its apoptosis through PI3K/AKT signaling pathway by knock down PIK3R2 gene. |
