Fig. 7

The ESRP1/circANKS1B/miR-148a/152-3p/USF1 feedback loop promotes breast cancer invasion and metastasis via inducing TGF-β1/Smad-mediated EMT. a-b Wound healing, transwell migration and invasion assays for circANKS1B-overexpressing MCF-7 cells transfected with si-ESRP1, si-USF1 or miR-148a/152-3p mimics or treated with LY2109761 at a final concentration of 10 μm. Representative images are shown at 0 and 24 h after gap creation. Scale bar = 20 μm. c Transwell migration and invasion assays for circANKS1B silencing MDA-MB-231 cells transfected with ESRP1, USF1 or TGF-β1 vector, or miR-148a/152-3p inhibitors. d Immunoblot analysis of p-Smad2, p-Smad3, Smad2/3, E-cadherin and Vimentin in circANKS1B-overexpressing MCF-7 cells transfected with si-ESRP1, si-USF1 or miR-148a/152-3p mimics or treated with LY2109761 at a final concentration of 10 μM. GAPDH was used as a loading control. e The illustration summarizes our findings. CircANKS1B, as miR-148a-3p and miR-152-3p sponge, increases USF1 expression by eliminating miR-148a/152-3p-mediated repression of USF1, and then, USF1 can respectively transcriptionally up-regulate ESRP1 and TGF-β1 expression via directly binding to the E-box motifs in their promoter regions. Subsequently, ESRP1 promotes circANKS1B generation, and TGF-β1 activates its downstream Smad signaling to induce EMT, thereby enhancing breast cancer invasion and metastasis. Data were represented as means ± S.D. of at least three independent experiments. **p < 0.01 versus control group, ^##p < 0.01 versus circANKS1B overexpression or knockdown group