Molecular Mechanism Of VDR Regulating HSD3B1 Gene Expression And Its Effect On The Synthesis Of Testosterone In Mice

Vitamin D receptor(VDR)is a nuclear transcription factor that is widely expressed in tissues and regulates gene transcription by binding to the promoter.Our research team found that the reproductive ability of VDR-/-mice was significantly reduced.It is speculated that VDR may regulate hormone synthesis and expression of genes related to reproductive development,and then affect male reproductive performance.Based on proteomic analysis data of WT and VDR-/-mouse testis tissue,this study found that the expression of HSD3B1 was significantly reduced after VDR knockout.HSD3B1 is an enzyme responsible for the steroid production reaction of hormones such as androgens,progestins and glucocorticoids in various tissues.Therefore,revealing the molecular mechanism which VDR regulates HSD3B1 gene expression regulation and hormone synthesis is of great significance for research in the field of male reproduction.In this study,the expression profile of the HSD3B1 gene was analyzed,and the distribution of VDR response elements in the promoter region of the gene was analyzed by bioinformatics tools.According to the VDRE distribution region,constructed luciferase reporter vectors in different segments of the promoter region to identify the activity of different VDREs,and further verified the function of VDRE by site-directed mutations,and systematically explored the molecular mechanism by which VDR regulated HSD3B1 gene expression.Finally,the overexpression of HSD3B1 in TM3 cells was initially explored for its effects on lipid metabolism and hormone synthesis.The results of this study laid the foundation for subsequent studies on VDR and male reproduction.The following experimental results were obtained in this study:1.RT-qPCR was used to map the tissue expression profile of HSD3B1 gene,and it was found to be highly expressed in testis,liver and kidney;testis tissue immunohistochemical experiments showed that HSD3B1 was localized in the testicular stroma,and the expression of HSD3B1 protein was significantly decreased after VDR knockout in mice,at the same time,the gap between the testicular seminiferous tubules and interstitial tissue was significantly widened;VDR positively regulates HSD3B1 in testicular mesenchymal cells.2.Regulatory sequences of 2000 bp before and 180 bp after HSD3B1 transcription initiation site were obtained through the NCBI database.Bioinformatics analysis predicted high score promoters at-1718 —-1668 bp and-300 —-250 bp,but no CpG islands were present.According to the successful construction of the promoter region double luciferase reporter vector,transfection of 293 T cells to identify the activity,the results showed that the F1 fragment promoter activity was higher than the F2 fragment,and VDR had an activating effect on promoter activity.3.Using the transcription factor binding site prediction software to analyze the F1 fragment,we screened two potential VDREs of-225 —-220 bp(TGAACC)and-309 —-304 bp(AGGACA).By constructing different mutation vectors,it was confirmed that-225 —-220 bp(TGAACC)was a VDR-specific regulatory site.4.By constructing the HSD3B1 gene overexpression vector,Elisa experiments demonstrated that both VDR and HSD3B1 promote testosterone synthesis in TM3 cells;HSD3B1 can up-regulate the lipid metabolism pathway genes LPL and Angptl4 to regulate the lipid metabolism pathway in mice,thereby indirectly affecting the male reproductive ability of mice.