Effect Of BAP18 On Transcriptional Regulation Of Androgen Receptor-mediated Genes And Its Role In Polycystic Ovary Syndrome

Objective: BAP18 is an important androgen receptor cofactor that upregulates AR-mediated gene transcription and has been reported to play an important role in the development of prostate cancer.However,the function of BAP18 in non-neoplastic diseases such as polycystic ovary syndrome(PCOS),which hyperandrogenemia is the main pathological feature,needs to be further explored.The objectives of this study were: 1.To clarify the expression of BAP18 in granulosa cells of patients with PCOS and its relationship to the disease;2.To elucidate whether BAP18 influences the development of PCOS by affecting androgen receptor-mediated gene transcription;3.To explore the mechanism by which BAP18 influences AR-mediated gene transcription;4.To explore the link between BAP18 and the expression of AR itself and its causes;5.To explore the biological function of BAP18 in granulosa cells.Methods: 1.To explore the association between BAP18 expression and PCOS case indicators by collecting granulosa cells from women with PCOS and normal non-PCOS women in both groups.2.Use protein immunoprecipitation assays(co-IP)to clarify whether BAP18 interacts with AR in granulosa cells,use immunofluorescence assays to verify the localization of BAP18 to AR in granulosa cells,and then use double luciferase reporter gene assays to verify the effect of BAP18 on AR-mediated gene transcriptional activity.3.An attempt was made to delve into the molecular mechanisms by which BAP18 affects androgen signaling pathways.Looking for androgen receptor downstream target genes that BAP18 may affect in granulosa cells,and then use chromatin immunoprecipitation assays(ChIP)to validate the changes in recruitment and histone modification levels in the transcriptionally active regions of AR target genes by knockdown of BAP18.4.Use protein immunoblotting(western blotting)and q-PCR experiments to verify whether BAP18 can up-regulate the transcription of the AR itself,followed by Ch IP experiments to verify whether BAP18 is recruited to the promoter region of the AR gene and affects the transcription of the AR itself,and finally using co-IP experiments to clarify whether BAP18 interacts with transcription factors that affect transcriptional activation of the AR gene.5.Knockdown BAP18 gene in granulosa cells by using lentiviral or interfering RNA transfection and detect androgen and estrogen levels in the culture medium of granulosa cells by ELISA,and test whether the cell cycle of granulosa cells is affected.Results: 1.The expression of BAP18 was significantly lower in the PCOS group than in the control group,and the level of BAP18 expression was highly correlated with clinical indicators of PCOS.The data showed that BAP18 expression was negatively correlated with LH/FSH,T and AMH expression.2.There is co-localization and interaction between BAP18 and the androgen receptor in granulosa cells,and BAP18 can upregulate AR-mediated gene transcription.3.Silencing of BAP18 can downregulate the expression of AR target genes CYP19A1 and FSHR and can significantly affect the recruitment of the AR to its target genes CYP19A1 and FSHR androgen response components and reduce the level of modification of histone H3K4me3.4.Silencing of BAP18 can significantly downregulate the expression of the AR mRNA,and overexpression of BAP18 can significantly upregulate the expression of the AR.We found that BAP18 can interact with the most important driver of AR,Sp1,and that BAP18 can recruit with Sp1 to the binding site of the AR promoter region and affect the level of modification of histones H3K4me3,H3 ac and H4 ac.5.Knockdown of BAP18 caused granulosa cells to stagnate in the G1 phase.Moreover,knockdown of BAP18 significantly affected CYP19A1 expression,which in turn affected androgen to estrogen conversion.Conclusion: 1.BAP18 is low expressed in polycystic ovary syndrome and highly correlated with clinical indications;2.BAP18 can interact with AR and up-regulate AR-mediated gene transcription;3.BAP18 can affect the expression of AR target genes CYP19A1 and FSHR by affecting AR recruitment and histone modification levels;4.BAP18 can significantly affect the expression of the AR gene by binding to the transcription factor Sp1 and affecting histone modifications in its DNA-binding region;5.BAP18 can affect the cell cycle of granulosa cells and affect androgen-estrogen conversion.