MiR-139 Regulates Proliferation And Apoptosis In Porcine Immature Sertoli Cells

Spermatogenesis is a relatively complex process in many physiological activities.In addition to the self proliferation and differentiation of spermatogonial stem cells,macrophages,stromal cells and Sertoli cells participate in this process.Sertoli cells exist in the epithelium of seminiferous tubules and form a blood testis barrier through the tight connection between cells,which provides immune and nutritional support for spermatogenic cells.By controlling the number of immature Sertoli cells,it can indirectly affect the physiological activities of spermatogenic cells.Therefore,the study of Sertoli cell proliferation plays an important role in spermatogenesis.miRNAs are a class of small non-coding RNAs with a length of about 22 nt,which can negatively regulate the expression of genes.Our group previously conducted a comprehensive sequencing of testicular tissues from fetal age to sexual maturity in Shaziling boars,identified 353 miRNAs and constructed developmental timing expression profiles,and found that the expression of miR-139 showed significant changes around the estrus stage in Shaziling boars.This thesis intends to investigate the role of miR-139 in immature Sertoli cells of pigs and provide a theoretical basis for further improving the study of male reproduction and solving problems such as male breeding.The main findings of the study are as follows:(1)The high genetic homology of miR-139 in 43 representative animal species,with identical seed sequences across species;In addition,miR-139 has a high degree of variability and complexity in the evolutionary history of different species.(2)25 predicted target genes of miR-139 were obtained through miRDB,miRWalk and Target Scan;KEGG and GO enrichment analysis of the predicted results of Target Scan showed that the KEGG was mainly enriched in prolactin signaling pathway,c AMP signaling pathway,AMPK and other signaling pathways that play important roles in regulating the process of reproduction and growth and development of livestock;GO enrichment results showed that the target genes of miR-139 mainly play binding and pre-transcriptional regulation roles,and most of them function in cells and their exosomes.(3)Overexpression of miR-139 significantly reduced the proportion of cells in the G1phase(P < 0.01)and increased the proportion of cells in the S phase(P < 0.05)of porcine immature Sertoli cells,but there was no significant difference in the number of cells in the G2 phase;whereas inhibition of miR-139 had the opposite result and the number of cells in the G2 phase was highly significantly reduced(P < 0.01).(4)After overexpression or inhibition of miR-139 in porcine immature Sertoli cells,respectively,the proliferation rate of cells was found to increase significantly(P < 0.01)after transfection with miR-139 mimic for 24 h using CCK-8,Ed U,ATP and Annexin V-FITC,and the expression levels of cell proliferation-related genes(BMP4,FGF2,GDNF,PCNA and EGF)were highly significantly increased in the expression levels of BMP4(P < 0.01),FGF2 and GDNF genes(P < 0.05),and the differences in the expression levels of PCNA and EGF genes were not significant;while the cell proliferation rate was highly significantly decreased after 24 h of miR-139 inhibition in the Sertoli cells(P < 0.01),and the expression levels of BMP4,FGF2,GDNF,PCNA and EGF expression levels were all highly significantly decreased(P < 0.01).(5)siRNA-induced EIF4G2 significantly reduced the proportion of cells in G1 phase and increased the proportion of cells in S phase(P < 0.01).Cell proliferation and apoptosis assays showed that inhibition of EIF4G2 significantly inhibited cell proliferation and promoted apoptosis.(6)Three cotransfection experiments were designed to investigate the effects of miR-139 inhibitor + EIF4G2 siRNA,miR-139 inhibitor + siRNA NC and inhibitor NC +siRNA NC on porcine immature Sertoli cells using the above assays.In the EIF4G2 siRNA group,the cell proliferation rate was significantly higher at 24 h,48 h and 72 h after transfection(P < 0.01),and the proportion of cells in the proliferation phase was significantly higher(P < 0.01);in the miR-139 inhibitor + siRNA NC group,the cell proliferation rate was significantly lower at 24 h after transfection(P < 0.05)and at 48 h after transfection(P < 0.05).In the NC group,the cell proliferation rate was significantly reduced after 24 h of transfection(P < 0.05),48 h of transfection(P < 0.01)and 72 h of transfection(P < 0.05),and the proportion of cells in proliferative phase was significantly reduced(P < 0.01).Reducing the expression of EIF4G2 gene inhibited the regulatory effect of miR-139 on the growth of porcine immature Sertoli cells.In conclusion,miR-139 accelerates the cycle of porcine immature Sertoli cells,promotes the proliferation of porcine immature Sertoli cells and inhibits their apoptosis.