G蛋白偶联受体在雌激素诱导人甲状腺未分化癌FRO细胞增殖中的作用及其机制

G-protein coupled receptor improves 17β-Estradiol induced proliferation of human anaplastic thyroid carcinoma FRO cells

目的探讨G蛋白偶联受体(G-protein coupled receptor,GPER)在雌激素促进人甲状腺未分化癌FRO细胞增殖中的作用及其机制。方法不同浓度(0、10-10、10-9、10-8mol/L)的17β-雌二醇(17β-Estradiol,E2)处理FRO细胞不同时间(0、12、24h),MTT法检测细胞增殖率;10-8mol/L的E2处理FRO细胞不同时间(0～24h),流式细胞术检测E2对FRO细胞周期的影响;设计合成针对GPER的小干扰RNA(GPER-siRNA)并转染FRO细胞;Western blot检测FRO细胞中Akt与ERK1/2磷酸化水平与GPER蛋白的表达。结果不同浓度(0、10-10、10-9、10-8mol/L)的E2处理FRO细胞不同时间(0、12、24h),细胞增殖率随浓度和时间的增加而增加,且10-8mol/L的E2处理24h后,细胞增殖率为(16.5±2.1)%;10-8mol/L的E2处理FRO细胞不同时间(0、12、24h),细胞周期S/G2/M期细胞的比例随时间的增加而增加;E2(10-8mol/L)处理FRO细胞不同时间(0、5、10、15、30min),ERK1/2与Akt的磷酸化水平分别在15min与10min达到最大;将GPER-siRNA转染FRO细胞48h,GPER的蛋白表达显著减少;E2作用于分别加入PD98059(30μmol/L)、LY294002(50μmol/L)以及转染GPER-siRNA的FRO细胞15min和10min,与E2处理组相比,ERK1/2和Akt的磷酸化水平降低;E2作用于分别加入PD98059、LY294002及转染GPER-siRNA的FRO细胞24h,与E2处理组相比,增殖率由(16.5±2.1)%降至(11.2±1.3)%、(9.6±1.5)%、(7.2±1.3)%(P<0.05)。结论 E2通过GPER激活ERK1/2、PI3K-Akt途径,促进FRO细胞的增殖。

Objective To investigate the effect of G-protein coupled receptor（GPER） on 17β-Estradiol（E2） induced proliferation of human anaplastic thyroid carcinoma FRO cells.Methods FRO cells were treated with different concentration（0,10-10,10-9 and 10-8 mol/L） of E2 for different time periods（0,12 and 24 h）,and cell growth was evaluated by MTT assay.After the cells were treated by 10-8 mol/L E2 for different time periods（0,12 and 24 h）,cell cycle was analyzed by flow cytometry.RNA interference against GPER（GPER-RNAi） was designed and synthesized,then transfected to FRO cells.The levels of phosphorylated ERK1/2,phosphorylated Akt and GPER were analyzed by Western blot analysis.Results Treatment with different concentration（0,10-10,10-9 and 10-8 mol/L） of E2 for different times（0,12 and 24 h）,the cell growth rates were increased in dose-and time-dependent manners.When FRO cells were treated with 10-8 mol/L E2 for 24 h,the cell growth rate was（16.5±2.1） %.After the cells were treated with 10-8 mol/L E2 for different times（0,12 and 24 h）,the percentages of S/G2/M phase were increased in a time-dependent manner.E2-induced accumulation of phosphorylated ERK1/2 and phosphorylated Akt in FRO cells reached peak at 15 min and 10 min respectively after E2 treatment.The expression of GPER was reduced significantly when GPER-siRNA was transfected to FRO cells for 48 h.Pretreatment with PD98059（30 μmol/L）,LY294002（50 μmol/L） and GPER-siRNA obliterated the inductive effects of E2 on ERK1/2 phosphorylation and Akt phosphorylation at 15 min and 10 min respectively in FRO cells.Meanwhile pretreatment with PD98059,LY294002 and GPER-siRNA obliterated the inductive effects of E2 on cell growth.and the growth rates were reduced to（11.2±1.3）%,（9.6±1.5）% and（7.2±1.3）% respectively compared with those of E2-treated cells [（16.5±2.1）%,P0.05].Conclusion E2 can promote proliferation of FRO cells via ERK1/2 and PI3K-Akt pathway in a GPER-dependent manner.