miR-223 targets the Mef2 c-β-catenin pathway during breast cancer cell invasion. (A) miR-223 transfection inhibited Mef2c gene expression. HEK-293T cells were co-transfected with miR-223 and the Mef2 c 3'-UTR-directed luciferase reporter (luciferase-Mef2c; Mef2c 3'-UTR cloned into the pMIR-REPORTER). Luciferase activities were measured as described in Figure 2. Cells transfected with miR-223 and luciferase vector, miR-NC and luciferase-Mef2c, or miR-223 and luciferase-miR-223C (miR-223 complementary sequence cloned in the 3'-UTR of luciferase reporter) were used as controls. Data are averages of triplicates from three independent experiments. * p < 0.05. (B) and (C) miR-223 reduced Mef2c expression and resulted in nuclear accumulation of β-catenin. SKBR3 cells were transfected with miR-223 or miR-NC (control). The expression of Mef2c and localisation of β-catenin in breast cancer cells were then determined by cellular fractionation followed by western blot analysis with anti-Mef2 c, β-catenin and β-actin (B). The localisation of β-catenin in SKBR3 was visualized by indirect fluorescence microscopy. Briefly, cells were incubated with an anti-β-catenin antibody followed by an incubation with a FITC-conjugated anti-mouse secondary antibody. PI was used to visualise the nuclei (C). (scale bar: 20 μm).