Investigation Of The Signal Mechanism Underlying The Regulation Of GREB1 Gene Expression By Estrogen In Malignantly Transformed Mammary Epithelial Cells

ObjectiveTo investigate theeffects of estrogen on the GREB1 gene expression and cell growth and a GPER-CANP2 signaling in human mammary pithelial cells transformed by stimulation with estrogen(E2),aiming at better understanding the signaling mechanismsfor E2-induced biological effects.MethodsMCF-10A mammary epithelial cells were transformed by stimulation with E2 for 13 passages.Morphological changes of cells were observed under microscope and tumorigenesis in nude mice method were used to confirm malignant transformation.Plate cloning assay was used to evaluate cell growth,and qRT-PCR was used to determine the levels of mRNA.MCF-7 breast cancer cells were utilized as in vitro positive control.GPER specific inhibitor G15,CANP specific inhibitor Calpeptin(Calp),and shRNA-CANP2 transfection were appliedto observethe role ofGPER-CANP2 signaling in the E2 actions.Results 1)E2(50nM)inducedmorphological changes in MCF-10A cells:loss of contact inhibition,enlarged cell bodies,and appearance of cloned globules.E2-induced transformed cells were tumorigenic when injected subcutaneouslyinto the mammary fat pad in nude mice(7/10),compared to control(0/10);2)In transformed MCF-10A cells(E2 group),plate colony formation rate was increased by(59.73±7.37)%(P<0.01)compared with control.In MCF-7 cells,E2 increased colony formation rate by(63.72±2.07)%(P<0.01);3)E2(50nM)regulatedthe gene expression of GREB1 in transformed cells by(114.04±7.28)%(P<0.01),compared with control.In MCF-7 cells,E2 increased the levels of GREB1 by(52.02±5.39)%(P<0.05);4)Pretreatment of transformed cells with G15(10?M)significantly inhibited cloning rate by(52.67±4.51)%(P<0.01),with MCF-7 cloning rate decreased by(43.78±5.66)%(p<0.05);5)G15(10?M/L)pretreatment of transformed cells significantly inhibited the E2-induced upregulation of GREB1 by(39.84±6.03)%(P<0.05),with GREB1 expression decreased by(67.74±3.46)%(P<0.01).6)Pretreatment with Calp(10?M)significantly inhibited cell growth by(57.80±8.62)%(P<0.01),while cloning rate in MCF-7 decreased by(83.48±6.07)%(P<0.01);7)Calp(10?M/L)significantly inhibited the E2-induced upregulation of GREB1 in transformed cells by(76.12±7.79)%(P<0.01),with GREB1 level MCF-7 cells decreased by(46.39±7.02)%(P<0.01);8)Silencing of CANP2 with shRNAreduced cell growth in transformed cells by(67.88±3.92)%(P<0.01)and in MCF-7 by(61.38±4.11)%(P<0.01);9)Silencing of CANP2 inhibited E2-induced GREB1 expression in transformed cells by(62.35±6.20)%(P<0.01)and MCF-7 cells by(41.61±3.76)%(P<0.05).ConclusionsE2induces up-regulation of GREB1 gene expression accompanied by increased cell growth in malignantly transformed mammary epithelial cells,and this effect is,at least in part,mediated by GPER-CANP2 signaling,suggesting a novel signaling pathway underlying the biological effects induced by E2.