Fig. 3: FOXO3a controlled miRNAs to influence IRS1 expression and the negative feedback suppression of the IGF-1R/Akt/mTOR signaling.

a SKBR3 or BT474 cells were transfected with miR-128-3p or/and miR-30a-5p inhibitor (upper) or transfected with miR-128-3p or/and miR-30a-5p mimics (bottom). The expression of IRS1 and β-actin was examined by western blot assays. b SKBR3 or BT474 cells were transfected with miR-128-3p or/and miR-30a-5p inhibitor followed by rhIGF-2 treatment at indicated concentration for 24 h. The expression of IRS1 and β-actin was examined by western blot assays. c SKBR3 or BT474 cells were transfected with FOXO3a shRNA followed by rhIGF-2 treatment at indicated concentration for 24 h. The expression levels of miR-128-3p and miR-30a-5p were measured by qRT-PCR, ****p < 0.0001. d A schematic representation of FOXO3a binding sites within the 2 kb putative promoters of miR-128-3p and miR-30a-5p. The first base of the precursors of miR-128-3p and miR-30a-5p is defined as ‘+1’. e SKBR3 or BT474 cells were treated with rhIGF-2 at indicated concentrations for 24 h. The enrichment of FOXO3a at miR-128 or miR-30a promoter was evaluated by ChIP-qPCR. The chromatin was precipitated with an anti-FOXO3a antibody. The precipitated chromatin was then analyzed by qRT-PCR with primers specific for the putative FOXO3a binding sites, ****p < 0.0001. n = 3 biological independent samples (c, e). Data are presented as mean values ± SEM (c, e). Statistical significance was determined by a two-tailed Student’s t-test (c, e). Data show a representative of three independent experiments (a, b). All data are provided in the Source Data file.