Figure 5.

miR-5188 Augments Breast Cancer Stemness, Metastasis, Proliferation, and Chemoresistance through FOXO1-Mediated β-Catenin Ubiquitination and Translocation

(A) Western blot analysis of stemness, metastasis, proliferation, chemoresistance, and Wnt/β-catenin signaling-associated proteins expression in FOXO1-overexpressed MCF-7 cells, miR-5188-overexpressed MCF-7 cells, miR-5188-overexpressed MCF-7 cells with FOXO1 overexpression, FOXO1-silenced MDA-MB-231 cells, miR-5188-silenced MDA-MB-231 cells, miR-5188-silenced MDA-MB-231 cells with FOXO1 knockdown, and their control cells. (B) Endogenous coimmunoprecipitation analysis validated the interaction between FOXO1 and β-catenin in MCF-7 cells. (C) Immunofluorescence costaining of FOXO1 and β-catenin was performed in MCF-7 cells. The fluorescence intensities along the dark arrow crossing the cytoplasm were calculated to show the colocalization of FOXO1 and β-catenin. (D) Western blot and quantification analysis of the effects of miR-5188 and FOXO1 on β-catenin stability in MCF-7 and MDA-MB-231 cells treated with cycloheximide at different time points. (E) Immunofluorescence costaining of FOXO1 and β-catenin was performed to detect their expression and subcellular localization in MCF-7 and MDA-MB-231 cells (scale bars, 10 μm). (F) Nucleic and cytoplasmic proteins were extracted for β-catenin detection by western blot. (G) Coimmunoprecipitation analysis of the effects of miR-5188 and FOXO1 on the interaction between β-catenin and ubiquitin in MCF-7 cells treated with MG132. (H) Western blot analysis of total β-catenin and active β-catenin expression in MCF-7 and MDA-MB-231 cells treated with FOXO1 plasmid, FOXO1 siRNA, or lithium (LiCl). (I) Immunohistochemistry analysis of Sox2, Oct4, Nanog, E-ca, N-ca, and Vimentin expression in xenograft tumors derived from MCF-7 cells after stable miR-5188 overexpression, MDA-MB-231 cells after stable miR-5188 knockdown, and their controls (scale bars, 40 μm) (n = 5). *p < 0.05; **p < 0.01; ***p < 0.001.