GPR30介导雌激素对三阴性乳腺癌MDA-MB-468细胞系增殖的影响

Effects of estrogen on GPR30-mediated proliferation in triple-negative breast cancer cell line MDA-MB-468

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。

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.