Role Of G Protein Coupled Estrogen Receptor(GPER) Signaling In The Estrogen-induced Transformation Of Mammary Epithelial Cells

Objective: To investigate 17β-estradiol(E2)-induced transformation and coincident altered expression of focal adhesion kinase(FAK) in mammary epithelial cells line MCF-10 A and the role of G protein coupled receptor(GPER) in the E2 action. Methods: MCF-10 A cell were treated with DMSO(control), E2, G1, G15, E2 +G15, G1+G15 for 11 repeated treatment cycles. Western blotting was used to assess protein expression, soft-agar colony formation assay was employed to observe anchorage-independent growth, wound healing assay was applied to observe cell migration, cell growth curve was employed to observe cell growth, si RNA was used to knockdown FAK gene expression and reduce the activity of GPER, q RT-PCR was employed to evaluate levels of m RNA of target genes. Results: 1)After treatment of wild type MCF-10 A cell with E2 and G1 for 11 generations, the cells displayed morphological changes, increased proliferation, and migration( the E2-treated group and the G1-group cell mobility rate increased 100.02% and 100.78%, as compared with control(**P < 0.01)(Fig. 3A). Colony formation(E2 group and G1 clone formation rate increased 173.08% and 153.85%, as compared with control(**P < 0.01, Fig. 3B), indicating a transformed phenotype; 2) E2 and G1 treatment caused upregulation of GPER expression at both m RNA and protein levels in mammary epithelial cells MCF-10 A,the E2-treated group and the G1-group cell GPER expression increased 109.58%(**P < 0.01) and 60.55%(*P < 0.05), as compared with control(**P < 0.01); 3) GPER antagonist G15 obviously inhibited the E2 and G1 induced transformation, the cells displayed morphological, decreased proliferation, and migration,colony formation; 4) E2 and G1 stimulated FAK were profoundly suppressed by G15.si RNA knockdown FAK can make the E2-treated cell migration decrease 22.61%(**P < 0.01). Conclusion: E2 and G1 induce transformation of MCF-10 A mammary epithelial cells;E2 and G1 upregulate the expression of GPER m RNA and protein; E2 evoked MCF-10 A cell transformation may be associated with the GPER signaling,and FAK may be play a role in the downstream of GPER...