Role And Mechanism Of E3 Ubiquitin Ligase AREL1 In Prostate Cancer

BackgroundProstate cancer is a cancer closely related to androgens,and its growth requires androgens to maintain.After castration treatment,prostate cancer patients often relapse into castration-resistant prostate cancer(CRPC)within one to two years.Some studies have reported that tumor tissues of CRPC patients have the ability to synthesize androgen de novo,and the expression of a series of androgen synthase genes in tumors is up-regulated.Some drugs targeting androgen synthase have also been successfully marketed.CYP11A1 is a key rate-limiting enzyme in the androgen synthesis pathway.At present,there is no research report on CYP11A1 as a target to develop prostate cancer treatment drugs.Purpose:Construct androgen-independent LNCaP-AI cell subline model;study the effect of AREL1 on prostate cancer cell LNCaP;luciferase reporter gene experiment to verify the transcription factor interacting with AREL1 promoter.Method:The expression of AREL1 protein in benign prostatic hyperplasia,well differentiated prostate cancer and well differentiated prostate cancer was detected by immunohistochemistry.LNCaP cells were cultured in 1640 medium containing 10%CSS for 3 months to construct androgen-independent LNCaP-AI cell subline model;qRT-PCR was used to detect AREL1,AR,StAR,CYP11A1 and HSD3β1 gene expression changes in LNCaP and LNCaP-AI cells;pCMV-N-Flag-AREL1 was transiently transfected into LNCaP cells with Lipo2000 transfection reagent,and the protein levels of AREL1 and CYP11A1 in LNCaP cells were detected by Western Blot after AREL1 overexpression,and testosterone levels in cell LNCaP supernatants was detected by radioimmunoassay after AREL1 overexpression,PSA(KLK3)gene expression changes in LNCaP cells were detected by qRT-PCR after AREL1 overexpression,LNCaP proliferation ability were detected by MTT after AREL1 overexpression,and LNCaP migration ability were detected by scratch assay after AREL1 overexpression,Transwell Detect the LNCaP invasion ability after AREL1overexpression;RNAi Max transfection reagent was used to transiently transfect si RNA-AREL1 into LNCaP cells,and the above method was used to reversely verify the role of AREL1 in LNCaP cells;ALGGEN-PROMO predicted AREL1 transcription factors,recombinant plasmids such as pCMV-N-Flag-C/EBPβ,pCMV-N-Flag-GR,pCMV-N-Flag-YY1 and pGL-Basic-AREL1 Promoter were constructed,Using Lipo2000 transfection reagent,pCMV-N-Flag-C/EBPβ,pCMV-N-Flag-GR and pCMV-N-Flag-YY1 were transiently co-transfected with pGL3-Basic-AREL1 Promoter and p RL-TK,respectively,in 293 T cells.Fluorescence intensity was detected by the enzyme reporter gene assay,to verify whether these transcription factors interact with the AREL1 promoter;pCMV-N-Flag-C/EBPβ,pCMV-N-Flag-GR and pCMV-N-Flag-YY1 were transfected with Lipo2000 transfection reagent LNCaP cells were transiently transfected,and the effects of these transcription factors on AREL1 gene expression were detected by qRT-PCR.Result:1.The expression of AREL1 was negative in benign prostatic hyperplasia,but decreased with the increase of malignant degree of prostate cancer.2.After long-term culture in 1640 medium containing 10% CSS,the expression of AREL1 gene in LNCaP cells was down-regulated,and the expression of AR,StAR,CYP11A1 and HSD3β1 and other genes were up-regulated.3.After overexpression of AREL1,the expression of CYP11A1 protein in LNCaP cells was down-regulated,the level of testosterone in the cell supernatant was decreased,and the expression of PSA gene was down-regulated,and the proliferation,migration and invasion of LNCaP cells were inhibited.4.After AREL1 interference,the expression of CYP11A1 protein in LNCaP cells was up-regulated,the level of progesterone in the cell supernatant was increased,the expression of PSA gene was up-regulated,and the expression of CYP11A1 in LNCaP cells was up-regulated.Proliferation,migration and invasion are all promoted.5.ALGGEN-PROMO predicts that AREL1 transcription factors are: C/EBPβ,GR and YY1,Ualcan prediction results show that C/EBPβ and GR,gene expression is down-regulated in prostate tumor tissue;dual luciferase reporter gene experiments verify C/EBPβ,GR are transcription factors of AREL1,qRT-PCR results showed that AREL1 gene expression was up-regulated in LNCaP cells after overexpression of C/EBPβ and GR.Conclusion:(1)Long-term deprivation of androgen in LNCaP cell culture medium leads to decreased AREL1 expression in LNCaP cells.(2)The expression of AREL1 was decreased with the increase of malignant degree of prostate cancer.(3)Overexpression of AREL1 can reduce testosterone synthesis in LNCaP cells through ubiquitination and degradation of CYP11A1,down-regulate PSA gene expression,thereby inhibiting the Its proliferation,migration and invasion,knockdown of AREL1 results in the opposite.(4)C/EBPβ and GR are AREL1 transcription factors,C/EBPβ and GR are down-regulated in the prostate cancer database.