MiRNA-34c And MiRNA-449b Regulate The Proliferation And Apoptosis Of Sertoli Cells By Targeting AXL Gene

Spermatogenesis is a complex biological process,which requires the synergy of spermatogenic cells and testicular somatic cells.There are many kinds of cells in the seminiferous tubules of the testis.As a somatic cell with important functions for the testis,supporting cells can form a blood-testis barrier,secrete a variety of secretory factors,and can also provide biological support for germ cells.Abnormal regulation of gene expression in Sertoli cells may lead to impaired spermatogenesis,leading to male sterility.Studies have shown that microRNAs(miRNAs)have a certain regulatory effect on the proliferation,secretion and apoptosis of supporting cells.The preliminary transcriptome data analysis of our laboratory found that miRNA-34 c and miRNA-449 b are differentially expressed in the testicular tissues of newborn bulls and adult bulls.We speculate that miRNA-34 c and miRNA-449 b may have a regulatory role in the spermatogenesis of bulls.,But its role and mechanism are still unclear.In this study,the interference and overexpression vectors of miRNA-34 c and miRNA-449 b were constructed.EdU and qRT-PCR were used to identify the effects of miRNA-34 c and miRNA-449 b on the proliferation and secretion functions of supporting cells.Flow cytometry and TUNEL,tested the effects of miRNA-34 c and miRNA-449 b on the apoptosis of bovine Sertoli cells.The results showed that after miRNA-34 c was overexpressed,the expression level of secretory factors synthesized by Sertoli cells decreased,the proliferation of Sertoli cells was inhibited,and the apoptosis of Sertoli cells was promoted.When miRNA-34 c was inhibited,the result was the opposite;when miRNA When-449 b is overexpressed,the expression level of secretory factors synthesized by Sertoli cells also decreases,and the level of apoptosis increases.Based on the prediction of the target relationship in Targetscan and our laboratory's transcriptome data,the potential target gene receptor tyrosine kinase(AXL)was screened.A wild-type(WT)and mutant(mut)dual fluorescein vector containing potential binding sites for miRNA-34 c and miRNA-449 b in the 3'UTR region of the AXL gene was constructed.The results of dual luciferase report experiment,qRT-PCR and western blot showed that the expression level of AXL gene was significantly reduced when miRNA-34 c and miRNA-449 b were overexpressed(p<0.05),when miRNA-34 c and miRNA-449 b were inhibited The expression of AXL increased(p<0.05);the ratio of luciferase activity in bovine Sertoli testis cells transfected with wild-type luciferin vector was significantly lower than that in bovine Sertoli testis cells transfected with mutant luciferin vector.It shows that AXL gene is the direct target of miRNA-34 c and miRNA-449 b.In addition,an overexpression vector of the AXL gene was constructed,EdU and qRT-PCR were used to verify the regulation of the AXL gene on the proliferation and secretion function of supporting cells,and the apoptotic cells were detected by flow cytometry and qRT-PCR was used to detect the effects of AXL gene on apoptosis.The PCR-array method detects the changes in the expression level of 80 reproduction-related genes.The results show that miRNA-34 c regulates the proliferation,secretion and apoptosis of bovine Sertoli cells by directly targeting the AXL gene,and miRNA-449 b also regulates the secretion and apoptosis of Sertoli cells by targeting the AXL gene,but has an effect on the proliferation of Sertoli cells Not obvious.This study reveals the targeting relationship between miRNA-34 c and miRNA-449 b and AXL gene and their influence on the proliferation,secretion and apoptosis of bovine Sertoli cells,and the regulation of AXL gene on the expression of reproduction-related genes,suggesting that AXL gene may be supported by influence The expression of cell-related genes thus indirectly affects the development of the testis and spermatogenesis.