Figure 5

Expression of miR-299 decreased cell migration and promoted an epithelial phenotype. Migration and expression of EMT markers were compared between induced and uninduced C4-2B cells stably expressing miR-299-3p, and between induced PC-3 cells transiently expressing miR-299-3p or scr RNA. (A,B) Bright field images taken at 0 hours and 24 hours (C4-2B) or at 0 hours and 14 hours (PC-3) after making the scratch showing the relative rates of migration of C4-2B-299 induced cells compared to the uninduced cells and induced PC-3-299 cells compared to the induced scr RNA expressing cells. Two scratches were made in each dish and the experiments conducted in triplicate. Scale bar: 200 μm. (C) Relative rates of cell migration. Data presented as the ratio of the distance migrated by the uninduced or induced scr RNA control cells compared to induced cells expressing miR-299. Data show the mean ± SD of minimum three independent assays. (D–G) Expression of miR-299 promoted conversion of EMT to epithelial phenotype. (D,E) Representative images of the immunoblot analysis of Slug, vimentin (PC-3 cells) and E-cadherin in induced and uninduced C4-2B-299 cells and induced PC-3-299 or scr RNA control PC-3 cells. Antibodies against α-tubulin and GAPDH were used as the loading controls. (F,G) Densitometric analysis showing reduced expression of Slug and vimentin, and increased expression of E-cadherin in induced cells compared to the uninduced or induced scrRNA control cells. Values were normalized to internal controls. Data represent mean ± SD of at least 3 independent assays.