双氢睾酮浓度波动对前列腺上皮细胞中错配修复基因表达的影响及其机制

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

目的探讨双氢睾酮(DHT)浓度波动对前列腺上皮细胞(RWPE-2细胞系)中错配修复基因MutL同源物(MLH1)表达的影响及其机制。方法将RWPE-2细胞分为空白组,恒定组(DHT浓度为1、10 nmol/L)以及波动组(DHT浓度为1、10 nmol/L,每24 h波动1次),以不同浓度的DHT干预不同的时间(0、24、48、72 h),观察细胞的形态、生长状态及细胞计数试剂盒(CCK-8)检测细胞增殖能力;应用蛋白质印迹法(Western blot)、实时定量反转录聚合酶链反应(RT-qPCR)技术检测雄激素受体(AR)信号通路靶基因[前列腺特异抗原(PSA)、FK506结合蛋白(FKBP5)]、苏氨酸蛋白激酶(Akt)信号通路靶基因[细胞周期蛋白D1(Cyclin D1)、B淋巴细胞瘤-2(bcl-2)]和MLH1基因表达的改变;免疫荧光法检测基因分布位置的变化。应用Graph Pad 7.0统计软件分析,组间差异采用t检验分析。结果恒定组与波动组中RWPE-2细胞形态学变化与DHT作用具有浓度(1~10 nmol/L)依赖性;CCK-8结果提示,72 h后,与空白组(6.904±0.143)比较,恒定组(7.073±0.103)、(8.508±0.187)与波动组(8.662±0.327)、(9.239±0.167)相对吸光度(A)值均增加,提示DHT干预增强细胞增殖,呈时间浓度依赖性(1~10 nmol/L),差异有统计学意义(t=6.805、4.922、10.6,P<0.01);Western blot结果提示,与空白组比较,波动组与恒定组中各靶基因的mRNA及蛋白表达水平均上调,波动组较恒定组中上述基因表达进一步上调,波动组FKBP5基因较恒定组表达下调,差异有统计学意义(t=5.488、15.730、7.976、3.695、3.952、2.830,P<0.05);免疫荧光结果显示,与空白组比较,波动组和恒定组中各靶基因核内分布增多,且波动组上述基因分布差异更显著。结论DHT能上调RWPE-2细胞中错配修复基因MLH1的表达,而DHT波动会使这种趋势增加,通过AR和Akt信号通路介导的细胞增殖是其可能机制之一。

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.