| Yak is a seasonal estrous animal with low natural reproduction rate.It is of great significance to study yaks reproductive mechanism.Testis is an important internal reproductive organ in the male mammals,which produces sperms and secretes androgen.Sertoli cells(SCs),located in the seminiferous tubule epithelium,are the only somatic cells in contact with Germ cells(GCs)and are directly involved in the regulation of spermatogenesis.Leydig cells(LCs)are located in the space of seminiferous tubules,androgens from the testis are mainly synthesized and secreted by LCs.Androgen binds to androgen receptors(AR)on the surface of SCs to activate downstream molecules and participate in the regulation of testicular function.The regulation of non-coding RNAs(nc RNAs)in animals has been confirmed,but the gene expression in LCs and SCs are still unclear.Therefore,the purpose of this study was to use next-generation sequencing technology to perform the transcriptome of yak LCs and SCs cultured in vitro,analyze the expression of m RNAs,Lnc RNAs and miRNAs and their regulatory functions.It was found that bta-miR-3431(hereafter referred to as miR-3431)was one of the differentially expressed miRNAs(DEmiRNAs)between the two types of cells.Subsequently,we explored the effects of miR-3431 on LCs and SCs by regulating its expression in cells.The details are as follows :(1)LCs and SCs of yak were isolated and purified by multiple enzyme digestion,Percoll gradient centrifugation,differential adhesion,starvation culture and hypotonic treatment.Then,the expression profiles of m RNAs,lnc RNAs and miRNAs in Leydig cells and Sertoli cells were detected by transcriptome sequencing.After analyzing the sequencing results,there were 84 differentially expressed m RNA(DEm RNAs)(LCs vs.SCs:15 up-regulated and 69 down-regulated),172 DELnc RNAs(LCs vs.SCs: 36 upregulated and 136 down-regulated)and 90 DEmiRNAs(LCs vs.SCs:72 up-regulated and 18 down-regulated).GO and KEGG analysis indicated that the differential expression genes(DEGs)were more enriched in the regulation of actin cytoskeleton,Rap1/MAPK signaling pathway,steroid biosynthesis,focal adhesion and pathways associated with metabolism.miR-3431 was one of the DEmiRNAs between the two types of cells and may regulated cell proliferation,cell jountions and biosynthesis of steroids through SYMPK and CYP11A1.Therefore,the effect of miR-3431 on the related functions of LCs and SCs was mainly studied in the next stage.(2)The regulation of miR-3431/SYMPK on the proliferation and junctions of SCs.Firstly,miR-3431 mimics NC(mimic control),miR-3431 mimics,miR-3431 inhibitor NC(inhibitor control),miR-3431 inhibitor were transfected into SCs,respectively,so that miR-3431 was overexpressed or inhibited.The expression level of SYMPK,the proliferation related genes(PCNA and CCND1)and junction proteins(Occludin、Claudin1、N-cadherin and β-catenin)were detected by RT-q PCR and Western blot,as well as the activity of cell proliferation was detected by CCK-8,and the distribution of junction proteins on SCs were detected by immunofluorescence.The result showed that:when miR-3431 was overexpressed,the expression of SYMPK,PCNA and CCND1 were significantly down-regulated(P < 0.001),the expression of Occludin,N-cadherin and β-catenin was increased significantly(P < 0.05),while the expression of Claudin1 was decreased(P < 0.01),the proliferation activity of SCs was significantly inhibited(P < 0.05),and the membrane localization of Occludin,N-cadherin and β-catenin were promoted,while Claudin1 was inhibited.After miR-3431 was inhibited,the expression trends of SYMPK,PCNA,CCND1,Occludin,N-cadherin,β-catenin and Claudin1 were opposite to the groups of the miR-3431 mimics treated,the proliferation activity of SCs was significantly increased(P < 0.05).These results indicated that miR-3431 could regulate the expression of SYMPK,as well as the proliferation and junctions of SCs.Secondly,we detected the effects of SYMPK on the proliferation and junctions of SCs after SYMPK was silenced.The results showed that the expression of PCNA,CCND1,Occludin,N-cadheri and β-catenin were decreased(P < 0.0001),while the expression of Claudin1 was increased(P < 0.0001);the proliferation activity of SCs was significantly decreased(P < 0.0001);the expression of Occludin、Claudin1、Ncadherin on cell membrane were increased,while no significant localization of Claudin1 on the cell membrane was observed.Finally,the dual-luciferase reporter gene assay showed that there was a effective binding sites between miR-3431 and SYMPK,and SYMPK may be the directly action molecule of miR-3431.(3)The regulation of miR-3431 on the proliferation of LCs.We detected the expression level of SYMPK,PCNA and CCND1 and cell proliferation activity after regulating miR-3431 and SYMPK by cell transfection.The results showed that after overexpression of miR-3431,the m RNA and protein expressions of SYMPK,PCNA and CCND1 were significantly decreased(P < 0.0001),and the cell proliferation activity was significantly decreased compared with the control group(P < 0.05).After miR-3431 was inhibited,their expression was promoted(P < 0.001),and cell proliferation activity was significantly increased(P < 0.05).Similarly,the expression of PCNA and CCND1 were significantly decreased(P < 0.05)and the cell proliferation activity was significantly decreased(P < 0.001)when SYMPK was interfered.These demonstrated that miR-3431/SYMPK also regulated the proliferation of LCs in vitro.The effect of miR-3431 on Luteinizing hormone(LH)-mediated testosterone production in LCs.LCs were treated with different concentrations of LH(0,50,100 and150 ng/m L)for 12,18 and 24 h.By detecting the testosterone content in the cell supernatant and the expression of steroidogenic enzymes,it was finally determined that100 ng/m L LH acted on LCs for 24 h was the best concentration and time for LH to stimulate LCs to secrete testosterone.Then,the expression of miR-3431 was regulated to detected its effect on LH-mediated testosterone production in LCs.The results showed that when miR-3431 was overexpressed,the m RNA(P < 0.05)and protein(P< 0.01)expression of CYP11A1 and CYP17A1 was significantly down-regulated,and the testosterone production level was decreased(P < 0.05).Conversely,when miR-3431 was silenced,the m RNA(P < 0.05)and protein(P < 0.0001)expression of CYP11A1 and CYP17A1 increased significantly,and the testosterone production level increased(P < 0.05),while the change of miR-3431 expression did not affect St AR expression.The results suggested that miR-3431 may be involved in the regulation of LH-mediated testosterone production by regulating the expression of CYP11A1 and CYP17A1.In conclusion,the sequencing results showed that the differential genes between LCs and SCs of yak were mainly involved in the regulation of hormone secretion,cell proliferation and metabolism,and dynamic regulation of cell jounctions.Further studies had shown that miR-3431 can regulate the proliferation of the two types cells in vitro and the expression of junction proteins in SCs through SYMPK,and participated in the regulation of LH-mediated testosterone production in LCs.This study shows that miR-3431 plays an important role in maintaining testicular function,and provides basic research data for the regulation of nc RNAs in yak testicular cells. |
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