FIGURE 3.

Down-regulation of endogenous miR-199a-5p in bladder SMCs decreases cell size and increases proliferation. A, antimiR-199a-5p effectively inhibits miR-199a-5p in the target binding luciferase assay. The perfectly complementary binding site for miR-199a-5p (target) was cloned into pmirGLO luciferase reporter vector and transfected into HEK293 cells stably transduced with lentiviruses expressing miR-199a-5p, antimiR-199a-5p, or scrambled miR control. Renilla luciferase activity expressed from the same vector was used for normalization, and the data were expressed relative (rel.) to the negative control pmirGLO vector. Error bars represent S.E. (*, p < 0.05). B, cultured primary human bladder SMCs (passage 1) were transduced with lentiviruses overexpressing scrambled miRNA control (RFP reporter) or antimiR-199a-5p (GFP reporter) and co-transduced with miR-199a-5p and antimiR-199a-5p. Cells were passaged once and examined live under an LSM microscope. Scale bars, 50 μm. Images of cells collected from random fields (n = 100 cells) were measured. The graph shows the average value in each sample ±S.D. (error bars) (**, p < 0.005). C, 3 × 10⁴ cells were seeded per well, and cell count was analyzed 7 days postplating. Alternatively, cells were labeled with BrdU for 24 h, and a colorimetric proliferation BrdU ELISA was performed. Data are shown as -fold increase related to the initial plating density (left graph) or normalized optical density (right graph) ±S.D. (error bars) (**, p < 0.005).