The Mechanism Of Bta-miR-127 Targeting ITGA6 In Regulating Proliferation And Apoptosis Of Bovine Testicular Sertoli Cells

Beef cattle industry is an important part of animal husbandry in China.Efficient breeding has always been a difficult problem for beef cattle industry.In order to solve this problem,many breeders are trying to improve the reproductive rate of cattle,while the basic research content of bull breeding is relatively little.The main factors affecting the reproductive performance of bulls are the number and quality of sperm,and spermatogenesis requires the synergistic action of testicular microenvironment including Sertoli cells(SCs)and Leydig cells,among which SCs provide physical support and hormonal influence for germ cells to promote spermatogenesis.A large number of studies have shown that micro RNAs(miRNAs)play an extremely important role in cell proliferation,apoptosis and secretion,but there are few studies on the effects and molecular mechanisms of miRNAs on immature bovine SCs.Transcriptome sequencing was performed on Angus cattle testis samples at 3 days(before sexual maturity)and 13 months(after sexual maturity),and a large number of differentially expressed non-coding RNAs were screened previously.In this study,the differentially expressed bta-miR-127 was selected as the research object.After differential expression verification,the method of overexpression and interference of bta-miR-127 was adopted.RT-q PCR,Western-Blot,Ed U,flow cytometry,CCK-8 and other experimental techniques were used to explore the effects of bta-miR-127 on the proliferation and apoptosis of bovine SCs.The double luciferase reporter system was used to verify the target genes of bta-miR-127,and the effects of bta-miR-127 on SCs were analyzed by means of interference with target genes.Through the above experiments,the effects of bta-miR-127 on the proliferation and apoptosis of bovine SCs through target genes and the mechanism were analyzed,providing more theoretical basis for molecular breeding of breeding bulls.The main results are as follows:(1)Bta-miR-127 was selected as the research object and the differential expression was verified by RT-q PCR.The results showed that the expression level of bta-miR-127 in bovine testis was significantly higher at birth than at adult stage(P < 0.05),which was consistent with the previous sequencing data.Using bovine primary SCs as a model,by overexpressing and interfering with bta-miR-127,RT-qPCR,Western-Blot,EdU,flow cytometry and CCK-8experiments showed that bta-miR-127 inhibited bovine SCs proliferation,promoted its apoptosis(P < 0.05),and inhibited the expression of secreted factors GDNF,BMP4,CXCL12 and ABP(P < 0.05).(2)Three potential target genes of bta-miR-127,including ITGA6,DGKE and LAS1 L,which may affect the proliferation and apoptosis of bovine SCs,were screened by bioinformatics software Targetscan and transcriptional target prediction combined with literature review.Double luciferase reporter vectors containing wild-type(WT)and mutated(MUT)of the 3’UTR region of these three genes and potential binding sites of bta-miR-127 were constructed.Through RT-q PCR and double luciferase reporter tests,ITGA6 was proved to be the target gene of bta-miR-127.(3)In order to explore the effect of the target gene ITGA6 of bta-miR-127 on the proliferation and apoptosis of bovine SCs and its molecular mechanism,the method of interfering with ITGA6 and the above-mentioned RT-q PCR,Western-Blot and other experimental techniques was adopted.The results proved that after knockdown of ITGA6,the proliferation and the expression of secreted factors GDNF,BMP4 and CXCL12 of bovine SCs were inhibited,and their apoptosis was promoted(P < 0.05).To further reveal the targeting relationship between bta-miR-127 and ITGA6 and its effects on the proliferation and apoptosis of bovine SCs,the bta-miR-127 inhibitor and ITGA6 small interfering RNA were co-transfected into bovine SCs,it was found that ITGA6 gene antagonized the effect of bta-miR-127 on inhibiting the proliferation and the expression of secreted factors GDNF,BMP4 and CXCL12 of bovine SCs and promoting apoptosis(P < 0.05).This study revealed the effects of bta-miR-127 and its target gene ITGA6 on proliferation and apoptosis of bovine SCs,and further verified the targeted silencing effect of bta-miR-127 on ITGA6.These results suggested that ITGA6 can regulate the proliferation and apoptosis of SCs through related genes,thus affecting spermatogenesis and testicular development,which can be used as a marker for breeding of beef cattle.