The Research Of Androgen Receptor Splicing Variants 7 In Castration-Resistant Prostate Cancer

Objective:1.Establish prostate cancer ENZ-R-LNCaP cell model,and verify that the expression of androgen receptor splicing variant 7(AR-V7)in ENZ-R-LNCaP cells.2.Through immunohistochemical method to verify the expression of AR-V7 in low risk,medium risk and high-risk prostate cancer specimens.Methods:1.Culture prostate cancer LNCaP cells in the FBS medium containing activated carbon / glucans,add Enzalutamide(MDV3100)with different concentrations 0.1,0.3,1.0uM for continuous subculture and establish ENZ-R-LNCaP cell model,observe growing status and change of anatomy of LNCaP cells in the absence of androgens under the invert microscope,and use western blot technique to detect expression change of prostate cancer LNCaP cells AR-FL and AR-V7 protein.2.Collect 40 clinical tissue specimens after the prostate cancer operations from the urologic surgery department of the First Affiliated Hospital of Nanchang University in accordance with the standards of 2015 European Association Of Urology,EUA,of which 10 cases should be low-risk,15 cases should be medium,high-risk,use immunohistochemical staining method to detect the expression of AR-V7 proteins in prostate cancer specimens with different risks.Results:1.LNCaP cells,in activated carbon/glucans FBS medium +Enzalutamide,was inhibited clearly in the initial(third generation)growth,it was hard for cells to adherence,a large number of cells died,only a few survived and started to process synapses around.After 9 generations,cells started to gradually process more synapses,and interconnected with each other into a network,cells were autocrine state;when cultured the 12 th generation,the cells were no longer autocrine state,after 4 months of continuous subculture,LNCaP cells started to grow in a spread way,and the state and shape of cells changed,cells became smaller,shaped round or fusiform,synapses became fewer.2.Use western blot to detect AR-FL and AR-V7: compare to the groups without Enzalutamide,the AR-V7 protein of the three experimental groups with 0.1,0.3,1.0uM Enzalutamide(MDV3100)was significantly increased(P<0.05),and the expression were clearly different under different doses(P<0.05).3.The result of mmunohistochemistry showed that vast majority of AR-V7 were in the nucleus,only a small amount were in the cytoplasm;19 of 40 prostate cancer specimens AR-V7 were positive,the total positive rate was 47.5%(19/40),of which,the positive expression low-risk prostate cancer was 20%(2/10),moderate-risk expression of prostate cancer was 40%(6/15)and high-risk expression of prostate cancer was 73.3%(11/15),the difference was statistically significant(P<0.05).Conclusion:1.Adding the androgen receptor antagonist Enzalutamide into the activated carbon/glucans FBS culture medium and continuously subculturing the prostate cancer LNCaP is helpful for the establishment of ENZ-R-LNCaP cell model.2.The expression of AR-V7 may be associated with risk rank of prostate cancer,and may be closely associated with the occurance and progress of castration-resistant prostate cancer.