中文摘要：

目的探讨雌激素受体GPR30（或称G蛋白偶联雌激素受体G protein-coupled estrogen receptor,GPER）介导雌激素对三阴性乳腺癌细胞系MDA-MB-468增殖的影响。方法免疫荧光法及Western blot法检测MDA-MB-468细胞中受体GPR30的定位及表达量,流式细胞术及CCK-8法检测药物处理后的细胞周期和细胞生长变化,Western blot法检测磷酸化细胞外信号调节激酶（phospho-extracellular regulate kinase,p-ERK）以及周期蛋白CyclinD1的蛋白表达。结果雌激素受体GPR30在MDA-MB-468细胞中高表达,且主要表达于细胞质。17-β雌二醇（E2）、GPR30特异性激动剂（G1）与他莫昔芬（TAM）处理细胞后,均显著促进细胞周期进展和细胞增殖。其中处于DNA合成期（S期）的DNA量分别为空白对照组的（2.81±0.11）、（2.82±0.21）、（2.70±0.20）倍,相对细胞数分别为空白对照组的（1.83±0.18）、（1.94±0.12）、（1.92±0.16）倍,其促生长效应被GPR30特异性拮抗剂（G15）显著抑制（P〈0.05）。E2、G1及TAM处理组中p-ERK及CyclinD1的蛋白相对表达量分别是空白对照组的（2.59±0.21）、（2.43±0.25）、（2.26±0.34）倍以及（1.67±0.06）、（1.51±0.08）、（1.90±0.07）倍,G15可抑制E2、G1及TAM所引发的相应变化（P〈0.05）。结论雌激素活化GPR30刺激下游ERK信号通路上调p-ERK及周期蛋白CyclinD1的表达,促进细胞周期的进展,导致三阴性乳腺癌细胞系MDAMB-468异常增殖。

英文摘要：

Objective To explore the effects of estrogen on the proliferation of triple-negative breast cancer cell line MDA-MB-468 mediated by G protein-coupled estrogen receptor GPR30.Methods Fluorescence immunoassay and Western blotting were performed to examine the localization and expression of GPR30 in MDA-MB-468 cells.Cell cycle and cell proliferation were tested by flow cytometry and CCK-8 assay.The expression of phospho-extracellular regulate kinase（p-ERK） and Cyclin D1 was detected by Western blotting.Results GPR30 was detected with high expression level in the MDA-MB-468 cells and located mostly in the cytoplasm.After treatment with 17-β-estradiol（E2）,GPR30 specific agonist（G1） and tamoxifen（TAM）,the progression of cell cycle and cell proliferation was increased remarkably.The DNA contents in DNA synthesis（S） phase were 2.81 ± 0.11,2.82 ± 0.21,and 2.70 ± 0.20 times higher and the relative cell numbers were 1.83 ± 0.18,1.94 ± 0.12,and 1.92 ± 0.16 times higher than those of the control group,respectively.The above effects induced by E2,G1 and TAM could be blocked by GPR30 specific antagonist G15（P 0.05）.The relative protein level of p-ERK in the E2,G1 and TAM treatment groups was2.59 ±0.21,2.43 ±0.25,and 2.26 ± 0.34 times higher and that of cyclin D1 was 1.67 ± 0.06,1.51 ± 0.08,1.90 ± 0.07 times higher than those of the control group,respectively（P 0.05）.Interestingly,the changes induced by E2,G1 and TAM were inhibited by G15（P 0.05）.Conclusion Estrogen triggers downstream ERK signaling by activating GPR30 to up-regulate the expression of p-ERK and cyclin D1,which accelerates MDA-MB-468 cell cycle progression,leading to abnormal cell proliferation.