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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 role of G-protein coupled receptor (GPER) in estrogen-induced proliferation of human thyroid carcinoma cell line FRO and its mechanism. Methods FRO cells were treated with 17β-estradiol (E2) at different concentrations (0,10-10,10-9,10-8 mol / L) for different time (0,12,24 h) Proliferation rate; FRO cells treated with 10-8 mol / L E2 for different time (0-24 h), the effect of E2 on FRO cell cycle was detected by flow cytometry; GPER-siRNA was designed and synthesized and transfected FRO cells. The phosphorylation of Akt and ERK1 / 2 and the expression of GPER protein in FRO cells were detected by Western blot. Results FRO cells treated with E2 at different concentrations (0, 10-10, 10-9, 10-8 mol / L) for different times (0,12,24h) increased the cell proliferation rate with increasing concentration and time, The cell proliferation rate was (16.5 ± 2.1)% after treatment with 8 mol / L of E2 for 24 h, while the cells treated with E2 at 10-8 mol / L for different periods (0,12,24 h), cells in S / G2 / M phase The proportion of ERK1 / 2 and Akt phosphorylation reached the maximum at 15 min and 10 min after treatment with E2 (10-8 mol / L) for different times (0, 5, 10, 15 and 30 min) GPER-siRNA transfected FRO cells 48h, GPER protein expression was significantly reduced; E2 treated with PD98059 (30μmol / L), LY294002 (50μmol / L) and GPER-siRNA transfected FRO cells 15min and 10min, The phosphorylation level of ERK1 / 2 and Akt decreased compared with the control group. E2 increased the proliferation rate of FRO cells treated with PD98059, LY294002 and GPER-siRNA for 24 h, respectively. The proliferation rate was increased from (16.5 ± 2.1) % To (11.2 ± 1.3)%, (9.6 ± 1.5)%, (7.2 ± 1.3)%, respectively (P <0.05). Conclusion E2 can activate ERK1 / 2 and PI3K-Akt pathway through GPER and promote the proliferation of FRO cells.