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

LI Xiaotian; XUE Guojun; YANG Guanglun

doi:10.3969/j.issn.1009-0460.2019.09.003

Abstract：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?7TAMR and its parent Non?drug?resistant cell line MCF?7 were cultured in vitro.The drug resistance of MCF?7TAMR cells to TAM was detected by MTT method. ShCtrl, shGPER1?1, shGPER1?2 and shGPER1?3 were transfected into MCF?7TAMR cells as shCtrl group, shGPER1?1 group, shGPER1?2 group and shGPER1?3 group, respectively, with blank control group (CTL group). Real?time fluores?cence quantitative PCR (QPCR) was used to identify the transfection effect.MTT assay and Ca2+fluorescence assay were used to detect the proliferation activity and Ca2+changes of shGPER1?1 group. The expression of LC3?Ⅱ and 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(IC50)of MCF?7 and MCF?7TAMR for TAM was 1.15 nmol／L and 19.47 nmol／L, respectively.The resistance index (RI) of MCF?7TAMR was 16.93. The expression levels of GPER1 in MCF?7 cells and MCF?7TAMR cells were 1.00±0.08 and 1.27±0.12, respec?tively, 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 pro?liferation of MCF?7TAMR 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). IC50 was 2.41 nmol／L. The sensitivity of shG?PER1?1 group to TAM was 7.08 times higher than that of CTL group. The expression levels of Beclin 1 and LC3?Ⅱ in shGPER1?1 group were 0.45±0.10 and 0.33±0.07, respectively, which were lower than those in CTL group and shCtrl group (P＜0.05). The num?ber of LC3?Ⅱfluorescent spots in shGPER1 group was significantly less than that in CTL group and shCtrl group. Conclusion Silen?cing GPER1 expression can reverse the resistance of MCF?7TAMR cells to TAM, and its possible mechanism may be related to the inhibi?tion of protective autophagy.