Figure 5. MiR-204 inhibits tumor cell migration and invasion by altering AKT/mTOR/Rac1 signaling.

A, miR-204 suppresses activation of AKT and mTOR signaling. HEK-293 cells transfected with miR-204 were grown in serum-free conditions and subjected to western blot analysis using anti-phospho-Ser⁴⁷³-AKT (1∶1000), anti-total-AKT (1∶1000), anti-phospho-Ser^235/236-S6 (1∶1000), anti-total-S6 (1∶1000), anti-phospho-Thr^37/46-4E-BP1 (1∶1000) and anti-total-4E-BP1 (1∶1000). β-actin (1∶10,000) was used as a loading control. Gel photographs are representative of three independent experiments. B, HEK-293 cells transfected with constitutively active AKT T308D/S473D mutant (CA-AKT) in the absence and presence of miR-204 were grown in serum free condition and subjected to western blot analysis as descried in A. C, overexpression of miR-204 abolishes BDNF-induced membrane ruffling and Rac1 translocation. HEK-293 cells transfected with miR-204 or miR-185 were grown in serum-free conditions and treated with or without BDNF (100 ng/mL) for 10 min and stained with Rac1 antibody (red) and FITC-phalloidin (green). Arrows indicate membrane-ruffling regions. D, miR-204 increases the sensitivity of HEK-293 cells to apoptosis as determined by Annexin V/PI staining using the FITC-Annexin V Apoptosis Detection Kit. The percentage cell population shown is the mean±SEM of three independent experiments. E, western blot analysis of HEK-293 cells transfected with miR-204 using anti-caspase-3 (1∶500) antibody and anti-PARP (1∶1000) show increased cleavage of caspase-3 and PARP. β-actin (1∶10,000) was used as a loading control. Gel photograph is representative of three independent experiments.