Fig. 4

CircANKS1B promotes breast cancer invasion and metastasis by sponge activity of miR-148a-3p and miR-152-3p and up-regulation of USF1.a-b qRT-PCR analysis of ARF4, FGF7, USF1, FZD6, NFAT5 and SOX5 in circANKS1B-overexpressing MCF-7 cells and circANKS1B knockdown MDA-MB-231 cells. c Schematic of luciferase reporter vectors containing wild-type (WT) or mutant (Mut) putative miR-148a/152-3p binding sites of USF1 3′ UTR. d Wild-type or mutant USF1 3′ UTR luciferase reporter vector was co-transfected with miR-148a/152-3p mimics or control mimics into MCF-7 and MDA-MB-231 cells, after 48 h, the luciferase activities were measured. e The relative luciferase activities were analyzed after co-transfection with circANKS1B or control vector, miR-148a/152-3p mimics or control mimics and wild-type (WT) USF1 3′ UTR luciferase reporter vectors. f Immunoblot analysis of USF1 protein expression after co-transfection with circANKS1B or control vector and miR-148a/152-3p mimics or control mimics. GAPDH was used as a loading control. g Transwell invasion assay was performed after co-transfection with circANKS1B or control vector and miR-148a/152-3p mimics or control mimics. h Analysis of USF1 expression in breast cancer patients with or without lymph node metastasis. Using median USF1 H-score value as cutoff. i Representative IHC images of low (patient # 10) and high (patient # 89) USF1 expression in breast cancer tissues. Scale bar = 20 μm. j A strong correlation between circANKS1B and USF1 expression in breast cancer tissues analyzed by Spearman correlation coefficients (r = 0.572, n = 165, p < 0.001). Data were represented as means ± S.D. of at least three independent experiments. **p < 0.01, ***p < 0.001