Fig. 3
MiR-219a levels, RhoGDIβ protein expression, and anchorage-independent growth among UROtsa cells treated with BBN for 2 months. A Schematic of miR-219a binding sites and its mutant in RhoGDIβ 3’-UTR luciferase reporter. B WT or miR-219a binding site mutant RhoGDIβ 3’-UTR luciferase reporter and pRL-TK reporter were transiently co-transfected into UROtsaC2mo and UROtsaBBN2mo cells. The luciferase activity of each transfectant was evaluated and results presented as relative RhoGDIβ 3’-UTR activity. The asterisk indicates significant difference from vehicle control (p < 0.05). The club symbol indicates significant difference from WT reporter transfectant (p < 0.05). C miR-219a inhibitor lentivirus was used to infect UROtsaBBN2mo cells, and knockdown efficiency was then determined by using real-time PCR. Results are presented as mean ± SD of triplicate experiments. The asterisk indicates significant decrease vs. control (p < 0.05). D RhoGDIβ protein expression was evaluated using Western blot. GAPDH was used as a protein loading control. E, F Representative images of colony formation by UROtsaBBN2M (Vector) and UROtsaBBN2M (miR-219a inhibitor) cells in the soft-agar assay; colonies were captured and scored under an inverted microscope. Results are presented as colonies/104 cells. Bars represent mean ± SD from three independent experiments. The asterisk indicates significant difference vs. control vector transfectant (p < 0.05)