AZGP1 Is Involved In The Molecular Mechanism Of AR Regulating The Migration, Invasion And Proliferation Of Prostate Cancer Cells

Prostate cancer is not only a common malignancy in the male urogenital system,but also a leading cause of morbidity in the western male population.The occurrence of prostate cancer is closely related to age and the increasingly westernized lifestyle.Androgen Receptor(AR)signal pathway is essential for the growth of normal prostate gland and the progression of prostate cancer,which is closely related to recurrence of prostate cancer and involvement in advanced prostate cancer.However,the molecular mechanisms of this process are not fully elucidated.In order to promote cancer cells growth,faster metabolism is the basic characteristic of tumor cells,including the increase of energy intake and consumption and acceleration of material metabolism.As a multifunctional secretory glycoprotein,AZGP1 involves in controlling fat degradation and energy consumption.More and more evidences suggest that lipid metabolism is closely related to tumor development,indicating that AZGP1 may play a role in promoting process of cancer.Due to the highly homology sequence and structure of MHC I,AZGP1 may differently participate in cancer proliferation and invasion by diverse ways.It can bind to hydrolase to activate the progression of cell apoptosis and inhibition of invasion.For example,AZGP1 inhibits EMT in hepatoma carcinoma cell invasion which is mediated by TGF-β pathway.In addition,early studies reveal that the expression of AZGP1 correlates with lack expression of PTEN,regulating PI3K/AKT pathways.As a result,cascade reactions would be driven in cell proliferation and tumorgenesis.In prostate cancer cells,several researches show that androgen can induce the expression of AZGP1.Nevertheless,the relationship between AZGP1 and AR and effect of AZGP1 on prostate cancer are still unknown.Therefore,in view of problems discussed above,we designed and finished the experiments as follows:Firstly,results from RNA-seq assay of clinic specimen and western blots indicated that the mRNA and protein levels of AZGP1 were significantly higher in prostate cancer specimen and cells by compared with normal prostate specimen and cells.We also found that the expression of AZGP1 was higer in androgen-dependent LNCaP and CWR22Rv1 cells than that in androgen-independent PC-3 and DU 145 cells.And the level of AZGP1 in normal prostate epithelial cell line WPMY-1 was found the lowest.Thus,these results strongly suggested that the high AZGP1 level was significantly related to the initiation and progression of prostate cancer in clinic,suggesting that the expression of AZGP1 might be strongly associated with androgen/androgen receptor(AR)signaling pathway.Secondly,our results showed that Mib treatment can increase AZGP1 mRNA levels and protein levels after hormone-stripped for 3 days in LNCaP and CWR22Rv1 cell lines.According to predicted outcomes from transcription factor binding sites prediction website JASPAR,there were two AR binding sites located within AZGP1 enhancer.Subsequently,by using dual-luciferase reporter assays,we confirmed AZGP1 upregulation by androgen is mediated by activation of AR.Thirdly,LNCaP and CWR22Rv1 cells were transfected with AZGP1 siRNA and hormone-stripped for 48 hours after transfection;and then cells were treated with 10nM Mib for another 24 hours.Results from wound healing assay and transwell invasion assay indicated that Mib-induced migration and invasion of LNCaP and CWR22Rv1 cells were partially impaired by AZGP1 knockdown.Futhermore,flow cytometry analysis in LNCaP and CWR22Rv1 cells also implied that AZGP1 played an important role in G1/S phase transition of cell cycle.Xenograft tumor models in male nude mice further identified that AZGP1 involved in AR-mediated promotion of prostate cancer cell growth and proliferation.In conclusion,AZGP1 was first proved to be a target gene of AR in this study.AZGP1 involving in AR-mediated promotion of proliferation and growth in prostate cancer cell was also illuminated in this study.In addition,other signaling pathways may involve in AZGP1 regulating downstream target genes which lay a solid foundation for further exploration of the functions of AZGP1 in prostate cancer.