The Mechanism Of Bta-miR-375 Affecting Bovine Sertoli Cell Proliferation And Apoptosis Through Targeting STARD6 Gene

Sertoli cells in the seminiferous tubules play an important role in spermatogenesis and testicular development in male mammals.Fetal Sertoli cell proliferation is the basis of testicular cord formation,and testicular cord affects the normal development and function of testis in adulthood.The development degree of Sertoli cell population before puberty determines the spermatogenic ability in adulthood.The damage during Sertoli cell development or proliferation may lead to the decline of sperm quantity and semen quality.Previous studies have shown that micro RNAs(miRNAs)are involved in the regulation of spermatogenesis and testicular development in testis,but the mechanism of action of most miRNAs in bovine testicular Sertoli cells remains unclear.The transcriptome sequencing results of Angus cattle testis showed that the expression of bta-miR-375 was significantly different in testis before and after bovine sexual maturation.Therefore,this research explored the mechanism of bta-miR-375 regulating the proliferation,secretion and apoptosis of immature bovine Sertoli cells.RT-q PCR,Western blot,flow cytometry,CCK-8 and Ed U methods were used to detect the proliferation and apoptosis of bovine Sertoli cells.The results are as follows:(1)RT-qPCR showed that bta-miR-375 was highly expressed in bovine testis at sexual maturity(P < 0.01).Overexpression of miR-375 can inhibit the proliferation and secretion of bovine testicular Sertoli cells and promote their apoptosis.Inhibiting the expression of miR-375 can promote the proliferation and secretion of Sertoli cells and inhibit their apoptosis.(2)Combined with previous RNA-seq analysis data and Targetscan prediction results,in this study,three potential target genes of miR-375(STARD6,CSTF2 and NFE2L2)were screened.The results of double luciferase reporter assay and RT-q PCR assay showed that STARD6 was the target gene of bta-miR-375.(3)Interfering with the expression of STARD6 can inhibit the proliferation and secretory factor expression of bovine Sertoli cells and promote the apoptosis of Sertoli cells.Meanwhile,silencing STARD6 inhibited the promoting effect of miR-375 inhibitor on proliferation and secretion of immature Sertoli cells in cattle.Silencing STARD6 attenuated the inhibitory effect of miR-375 inhibitor on Sertoli cell apoptosis.These results suggested that miR-375 can inhibit the proliferation and expression of secretory factors of bovine Sertoli cells and the promote apoptosis of Sertoli cells by targeting STARD6.