MiR-23b/

<p>-<b>27b significantly increases E-cadherin expression in castration –resistant cell lines.</b> ALVA31 or PC3-ML cells expressing miR-23b/-27b or transduced with scrambled control and LNCaP cells transfected with antagomiR-23b/-27b or control antagomiR were subjected to western blotting for E-cadherin and actin. Representative blots are shown of a total of 2–6 experiments.</p

MiR-23b/

<p>-<b>27b significantly decreases Rac1 activity but not total Rac1 levels in castration-resistant prostate cancer cell lines.</b> Rac1 activity assays were performed on ALVA31 (A) and PC3-ML (B) cells expressing miR-23b/-27b or transduced with scrambled control. GTP-bound Rac1 was separated from GDP-Rac1 using a pull-down assay as described in Materials and Methods. Complexes containing GTP-Rac1 (active Rac1) were collected, denatured and resolved by SDS-PAGE followed by Western blotting with anti-Rac1 antibodies. Total Rac1 (GDP-and GTP-bound) represents 5% of the original cell lysate. Actin was used as a loading control. Quantification of three independent experiments is shown with error bars representing SEM (*P<0.05), (**P<0.01).</p

MiR-23b/

<p>-<b>27b expression decreases the invasiveness of castration-resistant prostate cancer cells while inhibition of miR-23b/</b>-<b>27b in androgen-dependent prostate cancer cells increases invasiveness.</b> A, Matrigel invasion assays were performed on ALVA31 cells (A) and PC3-ML cells (B) expressing miR-23b/-27b or scrambled control-transduced cells. Upper panels show representative regions of the chamber filters with crystal violet-stained cells. The fold change (±SEM) represents the number of invaded cells per chamber divided by controls from three independent experiments performed in triplicate for A and the means (±SD) of one independent experiment done in triplicate for B (***P<0.001). C, LNCaP cells were transfected with 50 nM control antagomiR or antagomiR-23b/-27b. Matrigel invasion assays were performed 72 hours post transfection as explained above. The means (±SD) of two independent experiments performed in triplicate are shown (***P<0.001).</p

MiR-23b/

<p>-<b>27b expression decreases castration-resistant prostate cancer cell migration while inhibition of miR-23b/</b>-<b>27b increases migration of androgen-dependent prostate cancer cells.</b> Scratch assays were performed on ALVA31 cells expressing miR-23b/-27b or scrambled control (A) and LNCaP cells transfected with 50 nM antagomiR-23b-27b or control antagomiR (B). Images of the cleared zones (representative images shown on the right) were taken before and after a 4 hr incubation and the cleared areas measured using Image J software. The mean percentage of gap closure (±SD) (due to cell migration) is shown for two independent experiments from 7 scratches (A) and 6 scratches (B) (***P<0.001).</p

MiR-23b/

<p>-<b>27b does not regulate prostate cancer cell proliferation.</b> A and B, Proliferation of cells expressing miR-23b/-27b was compared to that of scrambled control-transduced cells. The mean cell number (±SEM) of a total of three independent experiments performed in triplicate is shown for ALVA31 cells and the mean cell number (±SD) of a representative experiment performed in triplicate is shown for PC3-ML cells. C, Proliferation of LNCaP cells transfected with antagomiR-23b/-27b or control antagomiR was assessed. The mean cell number (±SD) from two independent experiments performed in triplicate is shown.</p

MiR-23b/

<p>-<b>27b decreases anchorage independent growth of castration-resistant prostate cancer cell lines.</b> Soft agar assays were performed on ALVA31 and PC3-ML cells expressing miR-23b/-27b or transduced with scrambled control (**P<0.01). Mean colony number (±SEM) per plate from two independent experiments performed in triplicate for each cell line is shown (**P<0.01).</p