Mechanisms Of MiR-499 Regulates Immature Porcine Sertoli Cell Growth By Targeting PTEN Gene

The size of testes in male animals can affect the amount of ejaculation and the orderly spermatogenesis to a certain extent,and play an important role in the reproductive ability of male animals.MicroRNA(miRNA)are a class of endogenous non-coding RNAs that regulate post-transcriptional gene expression and play important regulatory roles in testicular development and spermatogenesis.In the early stage,we used RNA-seq technology to identify 374 miRNA in porcine testis tissues at different developmental stages.It was found that miR-499 was differentially expressed in testis tissue before and after sexual maturity in Shaziling.In this study,we used a variety of molecular experiments to study the effects of miR-499 and its target gene PTEN on the proliferation and apoptosis of porcine immature Sertoli cells through PI3K/AKT signaling pathway at the cellular level,in order to improve the reproductive capacity of male animals and theoretical basis.The research results are as follows:(1)The expression of PTEN gene by miR-499 targeting regulation in porcine immature Sertoli CellsBased on miR-499 target gene prediction information,pmirGLO-PTEN-3 ?UTR-WT / MUT vector was constructed and co-transfected with porcine immature Sertoli cells with miR-499 mimic and mimic NC to detect luciferase activity.The results showed that the fluorescence activity of miR-499 mimic + pmirGLO-PTEN-3 ?UTR-WT co-transfection group was significantly lower than the other three groups(P<0.01),indicating that miR-499 can target PTEN 3 ?UTR region prediction.Binding site.Subsequently,the expression and expression of miR-499 in porcine immature Sertoli cells were overexpressed and inhibited.Real-time quantitative PCR and Western Blot were used to detect the expression of PTEN gene in each group.The results showed that PTEN gene was overexpressed after miR-499.The expression level was significantly decreased(P<0.05),while the expression level of PTEN gene was significantly increased after inhibition of expression of miR-499(P<0.05).(2)Effects of miR-499 and PTEN genes on the growth of porcine immature Sertoli cellsIn order to clarify the functions of miR-499 and PTEN genes,overexpress or inhibit the expression of miR-499 and PTEN genes in porcine immature Sertoli cells,CCK8 and EdU kits were used to detect cell proliferation,and cell loss and cell cycle were detected by drain cells.The mortality rate,ATP kit was used to detect the ATP level of cells,and the expression of cell cycle,apoptosis and PI3K/AKT signaling pathway related genes were detected by realtime fluorescent quantitative PCR and Western Blot.The results showed that after overexpression of miR-499,the proliferation of porcine immature Sertoli cells was promoted;the proportion of cells in G1 phase of porcine immature Sertoli cells decreased,the proportion of cells in S phase increased,and The expression of cycle-related genes C-MYC,CCNE1,CCND1 and CDK4 is increased;the percentage of apoptosis in porcine immature Sertoli cells is decreased,the mRNA and protein expression of apoptosis-inducing genes Bax and Casepase-3 are decreased,the mRNA and protein expression of apoptosis-inhibiting gene Bcl-2 and intracellular ATP levels were elevated;porcine immature Sertoli increased expression of PI3 K and AKT phosphorylated proteins in cells.The results of interference with the PTEN gene were consistent with the results of overexpression of miR-499,whereas the inhibition of expression of miR-499 was reversed.(3)Effect of miR-499 targeting PTEN gene on the growth of porcine immature Sertoli cellsTo further clarify that miR-499 regulates porcine immature Sertoli cells growth by targeting PTEN gene,three co-transfection experiments were designed in this study: miR-499 inhibitor + siRNA PTEN,miR-499 inhibitor + siRNA NC and inhibitor NC + siRNA NC,which was tested for its function on porcine immature Sertoli cells using various assays described above.The results showed that the proliferation of porcine immature Sertoli cells was promoted in the transfected miR-499 inhibitor + siRNA PTEN group;the proportion of cells in the G1 phase of porcine immature Sertoli cells was decreased,the proportion of cells in S phase was increased,and The expression of cycle-related genes C-MYC,CCNE1,CCND1 and CDK4 is increased;the percentage of apoptosis in porcine immature Sertoli cells is decreased,the mRNA and protein expression of apoptosis-inducing genes Bax and Casepase-3 are decreased,and the mRNA and protein of apoptosis-related gene Bcl-2 are inhibited.Increased expression;increased expression of PI3 K and AKT phosphorylated proteins in porcine immature Sertoli cells.Transfection of the miR-499 inhibitor + siRNA NC group was the reverse of the transfection of the miR-499 inhibitor + siRNA PTEN group.In summary,miR-499 can promote the proliferation of porcine immature Sertoli cells and inhibit its apoptosis through PI3K/AKT signaling pathway by targeting PTEN gene.