| Purpose: The aim of this study is to elaborate the mechanism of steroidogenesis of primary progenitor Leydig cells(PLC)inhibiting by alcohol.Method: In this study,PLC was treated with different concentrations of alcohol,then the expression of androgen synthase and androgen production in PLC were detected by molecular biology and Enzyme linked immunosorbent assay(RIA).RT-PCR method was used to analyze the expression of miR-299a-5p in PLC.Next we analyzed the androgen production andthe specific target of regulation in different developmental stages of LC which wastreatedby miR-299a-5p inhibitorby using the method of transcriptome depth sequencing(RNA-Seq).Co-immunoprecipitation(CO-IP)and Informatics methods were used to analyze the signal transduction pathway of miR-299a-5p and its regulatory target NFκB1.Finally,in order to verify the effect of miRNA on the regulation of PLC androgen synthesis,the local injection of long acting miR-299a-5p was performed.Results: RIA determination showed that the alcohol of final concentration of 30μM,120μM and 480μM can make PLCs synthetic progesterone production was 4.62±0.13,3.56±0.41 and 0.91±0.43 ng/106 cells,compared with the control group,were decreased by 26.4%,42.6% and 85%.RT-PCR analysis showed that the alcohol can rapidly inhibit the expression of steroidogenic enzyme gene Hsd3b1 in PLCs.After screening for PLC in micro RNAs,it was found that miR-299a-5p is one of the target genes of alcohol,and is involved in the process of inhibiting the synthesis of PLC.LCs at different developmental stages was taken as the research object,and the miR-299a-5p inhibitor was transfected.The results show that the decreased expression of miR-299a-5p can enhance the expression of Cyclin D,but inhibit the synthesis of androgen production in PLC,ILC and ALC(6.76±1.29,13.84±3.62 和 3.40±0.32 ng/106 cells),compared with the control group,decreased by 88.35%,81.54% and 93.61% respectively.In addition,downregulation of miR-299a-5p could inhibit the expression of HSD3B1.Transcriptome analysis showed that miR-299a-5p was involved in cell and tissue development,stress response,immune system regulation,hormone synthesis and cell proliferation.The results of double luciferase assay showed that the Nfκb1 was the main target gene of miR-299a-5p in LC.The synthesis of androgen production and the expression of Hsd3b1 were inhibited by overexpressied of Nfκb1 in PLC.The analysis of promoter regions of Star、Cyp11a1 and Hsd3b1,which wereandrogen synthesis rate limiting enzymes showed that there was no Nfκb1 binding motif except for Star.The result of Co-immunoprecipitation Assay further confirmed that over expressed of Nfκb1 was not directly related to down-regulation of HSD3B1.Ingenuity Pathway Analysis(IPA)analysis showed that the Nfκb1 mediated differential proteins were mainly concentrated in the androgen pathway and the IL signaling pathway.RT-PCR analysis of cytokine in PLCs showed,the expression of m RNA of IL-1α、IL-1β and IL-6 were significantly increased by 3.54,3.50 and 5.73 times,but the expression of proinflammatory factor TNF-αhas no significant changed.In animal experiments,inhibitied the expression of miR-299a-5p can cause severe inflammatory infiltration in the testis,destruction of the seminiferous tubules and spermatogenesis.Because the IL-1 and IL-6 can downregulate the expression of HSD3B1 and inhibit androgen synthesis in LC has been confirmed,therefore,the mechanism of toxicity of alcohol inhibiting PLCs differentiation and androgen synthesis mainly through downregulatied the expression of miR-299a-5p,and increased Nfκb1 transcripting and activating the expression of IL-6 and IL-1,finally the regulation of PLC differentiation and androgen synthesis by autocrine and paracrine manner.Conclusion: Alcohol can inhibit the expression of PLC in miR-299a-5p,and the target gene of miR-299a-5p in PLC is Nfκb1;Over expression of Nfκb1 could lead to down-regulation of steroid hormone synthesis key enzyme HSD3B1 expression of PLC,thereby affecting the synthesis of PLC and androgen secretion;inhibiting the expression of miR-299a-5p can induce the expression of IL pathway on key proteins IL-1 and IL-6,the animal experiment showed that in the testis of local injection of miR-299a-5p inhibitors can lead to severe inflammation of testis,seminiferous tube structure destroy. |
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