沉默GPER1表达逆转乳腺癌细胞三苯氧胺耐药的实验研究  

Reversal of tamoxifen resistance in breast cancer cells by silencing GPER1 expression

作　　者：李啸天 薛国军 杨光伦[1] LI Xiaotian;XUE Guojun;YANG Guanglun(Department of Endocrinology and Breast Surgery,First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学附属第一医院内分泌乳腺外科

出　　处：《临床肿瘤学杂志》2019年第9期779-784,共6页Chinese Clinical Oncology

基　　金：国家自然科学基金资助项目(81072149)

摘　　要：目的探讨沉默G蛋白偶联雌激素受体1(GPER1)对乳腺癌MCF-7细胞三苯氧胺(TAM)的逆转作用及可能的作用机制。方法体外培养乳腺癌TAM耐药细胞株MCF-7 TAMR及其亲本非耐药细胞株MCF-7,采用MTT法检测MCF-7 TAMR细胞对TAM的耐药性。shCtrl、shGPER1-1、shGPER1-2和shGPER1-3分别转染MCF-7 TAMR细胞为shCtrl组、shGPER1-1组、shGPER1-2组和shGPER1-3组,另设空白对照组(CTL组),实时荧光定量PCR(QPCR)鉴定转染效果。采用MTT法和Ca 2+荧光检测法检测shGPER1-1组细胞增殖活性和Ca 2+变化。Western blotting检测各组自噬相关蛋白微管相关蛋白1轻链3(LC3-Ⅱ)和Beclin 1表达;采用免疫荧光技术观察细胞自噬泡的形成情况。结果MCF-7和MCF-7 TAMR对TAM的半数抑制浓度(IC 50)分别为1.15 nmol/L和19.47 nmol/L。MCF-7 TAMR的耐药指数(RI)为16.93。MCF-7细胞和MCF-7 TAMR细胞中GPER1的表达水平分别为1.00±0.08和1.27±0.12,差异有统计学意义(P<0.05)。shGPER1-1、shGPER1-2、shGPER1-3组中GPER1的表达水平分别为0.45±0.09、0.66±0.08、0.91±0.07,均较CTL组和shCtrl组明显降低,差异有统计学意义(P<0.05)。shGPER1-1组经TAM作用1~2 min内细胞内Ca 2+浓度迅速升高,而CTL组和shCtrl组变化不明显。0.01、0.1、1、10、100 nmol/L TAM对shGPER1-1组细胞的增殖抑制率分别为(5.44±1.79)%、(17.64±2.34)%、(40.56±3.79)%、(69.51±3.70)%、(76.62±4.15)%,高于CTL组和shCtrl组细胞(P<0.05)。IC 50为2.41 nmol/L。shGPER1-1组对TAM的敏感性较CTL组增加7.08倍。shGPER1-1组Beclin1、LC3-Ⅱ蛋白表达量分别为0.45±0.10、0.33±0.07,低于CTL组和shCtrl组(P<0.05);shGPER1组LC3-Ⅱ荧光斑点的数量明显少于CTL组和shCtrl组。结论沉默GPER1表达可逆转MCF-7 TAMR细胞对TAM的耐药性,其可能的作用机制可能与抑制细胞保护性自噬有关。Objective To investigate the reversal effect of silenced G protein coupled estrogen receptor 1(GPER1)on tamoxifen(TAM)in breast cancer MCF-7 cells and its possible mechanism.Methods The TAM-resistant breast cancer cell line MCF-7 TAMR and its parent Non-drug-resistant cell line MCF-7 were cultured in vitro.The drug resistance of MCF-7 TAMR cells to TAM was detected by MTT method.ShCtrl,shGPER1-1,shGPER1-2 and shGPER1-3 were transfected into MCF-7 TAMR cells as shCtrl group,shGPER1-1 group,shGPER1-2 group and shGPER1-3 group,respectively,with blank control group(CTL group).Real-time fluorescence quantitative PCR(QPCR)was used to identify the transfection effect.MTT assay and Ca 2+fluorescence assay were used to detect the proliferation activity and Ca 2+changes of shGPER1-1 group.The expression of LC3-Ⅱand Beclin-1 in each group was detected by Western blotting,and the formation of autophagic vesicles was observed by immunofluorescence technique.Results The half inhibitory concentration(IC 50)of MCF-7 and MCF-7 TAMR for TAM was 1.15 nmol/L and 19.47 nmol/L,respectively.The resistance index(RI)of MCF-7 TAMR was 16.93.The expression levels of GPER1 in MCF-7 cells and MCF-7 TAMR cells were 1.00±0.08 and 1.27±0.12,respectively,with significant difference(P<0.05).The expression levels of GPER 1 in shGPER1-1,shGPER1-2 and shGPER1-3 groups were 0.45±0.09,0.66±0.08 and 0.91±0.07,respectively,which were significantly lower than those in CTL group and shCtrl group(P<0.05).The intracellular Ca2+concentration in shGPER1-1 group increased rapidly within 1-2 minutes after TAM treatment,but there was no significant change in CTL group and shCtrl group.The inhibitory rates of 0.01,0.1,1,10,100 nmol/L TAM on the proliferation of MCF-7 TAMR cells in shGPER1-1 group were(5.44±1.79)%,(17.64±2.34)%,(40.56±3.79)%,(69.51±3.70)%and(76.62±4.15)%,respectively,higher than those of CTL and shCtrl groups(P<0.05).IC 50 was 2.41 nmol/L.The sensitivity of shGPER1-1 group to TAM was 7.08 times higher than that of CTL group.The express

关 键 词：乳腺癌 GPER1 三苯氧胺 耐药性 自噬 

分 类 号：R737.9[医药卫生—肿瘤]