Effect of dihydrotestosterone fluctuation on mismatch repair gene mutL homolog 1 in prostate epithelial cells and its mechanism

Abstract

Objective To investigate the relationship between the fluctuation of dihydrotestosterone concentration and the expression of mutL homolog 1 (MLH1) in mismatch repair system in prostate epithelial cells (RWPE-2 cell line) and its mechanism. Methods RWPE-2 cells were randomly divided into blank group [anhydrous ethanol solvent and dihydrotestosterone (DHT) concentration of 0 nmol/L], constant concentration group (concentration of DHT: 1, 10 nmol/L) and fluctuating concentration group (concentration of DHT: 1, 10 nmol/L, fluctuating every 24 h). After different time (0, 24, 48 and 72 h), cell counting kit-8 was use to examine cell proliferation and the target genes in androgen receptor (AR) signaling pathway [prostate specific antigen (PSA), FK506 binding protein 51 (FKBP5)], protein kinase B (Akt) signaling pathway [Cyclin D1, B cell lymphoma/leukemia-2 (bcl-2)] and MLH1 was detected by Western blotting, real-time quantitative polymerase chain reaction (Real-time PCR) and immunofluorescence. Graph Pad 7.0 statistical software was used for analysis and differences between groups were analyzed by t test. Results The morphological changes of RWPE-2 cells had a certain concentration-dependent relationship with DHT: constant group and fluctuating group had more cells and better growth than blank group. After RWPE-2 cells were stimulated with DHT for 72 h, as compared with the blank group (6.904±0.143), the relative A values of the constant group [(7.073±0.103) and (8.508±0.187)] and the fluctuation group [(8.662±0.327) and (9.239±0.167)] increased statistically (t=6.805, 4.922, 10.6, P<0.01). Western blotting and RT-qPCR results confirmed that the mRNA and protein levels of every target gene were increased significantly in constant group and fluctuating group (P<0.05) as compared with blank group. Meanwhile, the difference in fluctuating group was more significant than constant group. Whereas, FKBP5 mRNA decreased in fluctuating group as compared with constant group (t=7.101, 10.760, 8.289, 8.088, 8.519, 9.157, 11.330, P<0.05). The distribution of above genes increased in the nucleus after DHT intervention with more significant difference in fluctuating group. Conclusion DHT can up-regulate the expression of the mismatch repair protein MLH1 in RWPE-2 cells, meanwhile, DHT fluctuations can increase this trend and AR and Akt signaling pathways-mediated proliferation is one of the mechanisms. Key words: Dihydrotestosterone fluctuation; Cell proliferation; Mismatch repair; Androgen receptor signal pathway; Protein kinase B signal pathway