miR-3682-3p directly targets FOXO3 and stimulates tumor stemness in hepatocellular carcinoma via a positive feedback loop involving FOXO3/PI3K/AKT/c-Myc

Figure 4 FOXO3 is a direct target of miR-3682-3p. A: Binding sites for miR-3682-3p in the 3′-UTR of FOXO3 were predicted using bioinformatics tools; B-D: The effect of miR-3682-3p on FOXO3 mRNA and protein levels as determined by RT-qPCR and western blot, respectively; E: Dual-luciferase reporter assay was used to detect the interaction between miR-3682-3p and the 3′-UTR of FOXO3; F: Immunohistochemical analysis of FOXO3 expression in xenograft tumors (n = 5) (× 100 magnification scale bar: 200 µm, × 400 magnification scale bar: 50 µm); G: Western blot-based analysis of the effects of FOXO3 on stemness- and PI3K/AKT/c-Myc pathway-related proteins; H: Immunoprecipitation assays for the interaction between endogenous FOXO3 and β-catenin; I: Immunofluorescence staining-based assessment of FOXO3/β-catenin co-localization; J: Immunofluorescence staining of FOXO3 and β-catenin in hepatocellular carcinoma cells (scale bar: 5 μm). ^aP < 0.05, ^cP < 0.001. Experiments were repeated three times. NC: Negative control; wt: Wild-type; mut: Mutant.