Migration and invasion assay

The migratory ability of DU145 and PC3 cells transfected with si-CCAT2 or si-NC was assessed using a 24-well Transwell chamber (BD Biosciences). For migration assays, PCa cells (1×10⁵ cells) with serum-free DMEM were plated into the upper chambers, and DMEM containing 10% FBS (HyClone; Cytiva) was plated into the lower chambers. Cells were incubated at 37°C with 5% CO₂ for 24 h. Following incubation, the upper surface of the Transwell membrane was wiped to move non-migratory cells. Migratory cells on the lower surface of the Transwell membranes were fixed in 4% paraformaldehyde at 4°C for 20 min, followed by staining with 0.1% crystal violet for 15 min at 25°C. Stained cells were observed in five random fields using a light microscope (magnification, ×20). To assess cell invasion, artificial basement membrane Matrigel^® (BD Biosciences) was pre-coated at 37°C overnight on the bottom of the Transwell culture chamber.