Figure 3

Downregulation of let-7 and miR-200 levels initiates and maintains OSM-induced EMT phenotype. (a, b) MCF-7 cells were treated with 10 ng/ml of OSM and the expression levels of the let-7 (a) and miR-200 (b) families were analyzed by real-time RT–PCR at the indicated time points. (c) MCF-7 cells were transfected with let-7b/d/g and miR-200b/c. After transfection for 24 h, the cells were treated with 10 ng/ml of OSM for 5 days. The expression of E-cadherin, fibronectin, ZEB1 and Snail was analyzed by western blot. (d) MDA-231 cells were transfected with let-7b/d/g and miR-200b/c for 48 h. The expression levels of E-cadherin, fibronectin, vimentin and ZEB1 were analyzed by western blot. (e, f) MCF-7 and MDA-231 cells were transfected with let-7b/d/g and miR-200b/c. The transfected MCF-7 cells were treated with 10 ng/ml of OSM for 5 days. The invasive activities of the cells were determined by Matrigel invasion assays after 10 h (MDA-231 cells) or 48 h (MCF-7 cells) of culture. The invading cells were stained (e) and quantified (f). (g) MCF-7 cells were co-transfected with let-7 inhibitors and miR-200 mimics for 24 h. Then the cells were treated with OSM. The expression levels of E-cadherin, fibronectin and ZEB1 were analyzed by western blot after OSM induction for 4 days. Error bars indicate s.e. (N⩾3). NC, nonspecific miRNA mimics; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.