Fig. 3

CircANKS1B functions as a sponge for miR-148a-3p and miR-152-3p in breast cancer. a RIP analysis of circANKS1B, linear ANKS1B and CDR1as using anti-AGO2 antibody in MCF-7 and MDA-MB-231 cells. CDR1as, which has been validated to bind to AGO2 protein, was used as a positive control. b Biotinylated-circANKS1B pull-down assays were performed to test the specificity of circANKS1B probe. c-d The top ten candidate miRNAs predicted by CircNet database were showed and their expression levels were measured by qRT-PCR after the biotinylated-circANKS1B pull-down assays in MCF-7 and MDA-MB-231 cells. e-f Wild-type (WT) or mutant (Mut) biotinylated-miR-148a/152-3p mimics were transfected into circANKS1B-overexpressing MCF-7 and MDA-MB-231 cells, after streptavidin capture, circANKS1B expression levels were determined by qRT-PCR. g Schematic of luciferase reporter vectors containing wild-type (WT) or mutant (Mut) putative miR-148a/152-3p binding sites of circANKS1B. h Wild-type (WT) or mutant (Mut) circANKS1B 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 assessed. i FISH assays were conducted to determine the co-localization between circANKS1B and miR-148a-3p or miR-152-3p in breast cancer cells. Scale bar = 10 μm. Data were represented as means ± S.D. of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001