Figure 4.

MiR-5188 directly targets FOXO1 to augment β-catenin-mediated tumor stemness, metastasis, proliferation, and chemoresistance in HCC. (A) Western blot analysis was utilized to examine stemness, metastasis, proliferation, chemoresistance, and Wnt/β-catenin signaling-associated protein expression levels in HCC cells. (B) Bioinformatics analysis was used to predict miR-5188-binding sequences within the FOXO1 3'-UTR. (C) QPCR and Western blot analysis were used to examine FOXO1 mRNA and protein levels in miR-5188-overexpressing HCC cells, miR-5188-silenced HCC cells, and the corresponding control cells (n=3 independent experiments, Student's t-test). (D) Immunohistochemical analysis detected FOXO1 protein expression in xenograft tumors derived from HCCLM3 cells treated with antagomir, Huh7 cells stably overexpressing miR-5188 and the corresponding control cells (scale bar: 10 μm) (n=5). (E) Luciferase reporter assays were conducted to validate the interaction between miR-5188 and the 3'UTR of FOXO1 (n=3 independent experiments, one-way ANOVA). (F) RIP was conducted to validate the interaction between AGO2-bound miR-5188 and FOXO1 mRNA (n=3 independent experiments, Student's t-test). (G) Western blot analysis of stemness, metastasis, proliferation, chemoresistance, and Wnt/β-catenin signaling-associated protein expression levels in HCC cells. (H) Endogenous coimmunoprecipitation was used to test the interaction between FOXO1 and β-catenin. (I) Immunofluorescence costaining was utilized to detect the colocalization of FOXO1 and β-catenin. Fluorescence intensities along the red arrow crossing the cytoplasm were calculated to show the colocalization of FOXO1 and β-catenin. (J) Immunofluorescence costaining was carried out to detect the expression and localization of FOXO1 and β-catenin (scale bar: 5 μm). (K) Nucleic and cytoplasmic β-catenin protein expression was detected by Western blotting. All data are presented as the mean ± SD. Experiments were repeated three times.