| Background:Androgen is highly correlated with the occurrence of prostate cancer,but the mechanism is not clear.The fluctuation of men's serum testosterone levels increase with age,which may lead to disorder of AR-driven cell growth signaling pathways,which in turn affects the DNA replication process.The DNA mismatch repair(MMR)system mainly guarantees high fidelity of DNA replication process by identifying,excising and repairing mismatched bases during DNA replication.MMR system core gene MLH1 is a risk factor for prostate cancer.The occurrence of prostate cancer is related to MLH1 dysfunction.Therefore,changes in androgen may play a significant role in DNA damage repair,and the underlying mechanism is still unclear.Objective:To detect the effects of dihydrotestosterone(DHT)and its concentration fluctuation on the proliferation of prostate epithelial cells(RWPE-2 cell line)and the expression of mismatch repair gene MutL homologue(MLH1),and to elucidate the underlying mechanism of the change of cell biological behavior.Methods:1.Gradient concentration of DHT(DHT concentration:0,1,10,50,100 nmol/L)was used to treat RWPE-2 cells for different time(0,24,48,72h)to observe the changes of cell growth morphology;Western blot and real-time quantitative PCR were used to detect the changes of MLH1 expression in MMR system,which were used to screen the best matched concentration and intervention time.2.The blank group(0 nmol/L of DHT and the control group added with anhydrous ethanol),the constant group(1,10 nmol/L of DHT)and the fluctuation group(1,10 nmol/L of DHT,fluctuated once every 24 hours)were set up,reference to the expected experimental results.RWPE-2 cells were cultured with different concentrations of DHT for different time(0,24,48,72h)to observe the change of cell state;Cell Counting Kit-8 was used to detect the change of cell proliferation;Western blot and RT-qPCR were used to detect the change of MLH1 expression.3.The related information of androgen,prostate disease and MMR system was predicted by using public database,and the related data of AR and MLH1 gene in prostate disease were obtained and analyzed.The expression levels of target genes in AR signaling pathway(AR,PSA,FKBP5),AKT signaling pathway(P-Akt/Akt,Cyclin D1,BCL-2)and MLH1 were determined by Western blot,RT-qPCR and Immunofluorescence.Results:1.The morphological changes of RWPE-2 cells were concentration-dependent with DHT(1-10 nmol/L)in the constant and fluctuation groups.After DHT stimulated RWPE-2 cells for 72 h,compared with the blank group(6.904 ± 0.143),the relative A values of the constant group(7.073 ± 0.103),(8.508 ± 0.187)and the fluctuation group(8.662 ± 0.327),(9.239 ± 0.167)increased(P<0.01).In addition,the relative A values of fluctuation group was further increased compared with constant group(P <0.05),suggesting that DHT could improve the cell proliferation in concentration-dependent manner and the fluctuation of DHT concentration can further increase this trend.2.The analysis of public data suggested that MLH1 was positively correlated with AR gene(r = 0.3881,P < 0.01).Results of Western blot and RT-qPCR confirmed that the target genes in AR signaling pathway and AKT signaling pathway were increased in constant group and fluctuating group significantly(P<0.05)both in protein levels and mRNA levels,comparing with the group without DHT.The difference in fluctuating group was significant compared with constant DHT concentration group(P<0.05).Whereas,FKBP5 mRNA decreased in fluctuating group comparing with constant DHT concentration group(P<0.05).The distribution of above target genes increased in the nucleus after DHT intervention with more statistical differences in fluctuating group.Conclusion:DHT concentration fluctuation can promote the proliferation of RWPE-2 cells and the expression of mismatch repair gene MLH1.Cell proliferation through the synergistic effect of AR and Akt signaling pathway is one of the possible mechanismsto produce this kind of cell biological behavior change. |
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