Upper chamber

LNCaP cells (5 × 10 3 cells/well) were cultured in a 24-well plate in RPMI-1640 culture medium with 10% FBS. The culture plate was incubated overnight at 37°C in a humidified CO2 incubator. Next, the culture medium was removed, and the adherent cell layer was scratched with a sterile 200 μL pipette tip. We washed away the cell fragments with phosphate-buffered saline (PBS). Images of the scratch area at 0 h and 24 h were taken using a built-in camera in the microscope (40x magnification).
Data were evaluated using TScratch imaging software (CSE Lab., ETH, Zurich) to calculate the percent wound area. 9 Transwell assay The Transwell chamber was treated with Matrigel (Solarbio, Beijing, China). After transfection, 2×10 5 LNCaP cells and NC control cells were cultured in the upper chamber (Solarbio, Beijing, China) with serum-free medium. RPMI-1640 medium containing 10% FBS was added to the lower chamber, and the Transwell chamber was removed after continued culture for 24 h and maintained at 37°C in 5% CO2 overnight. Subsequently, cells were fixed with methanol for 10 min, stained with crystal violet (Yuanye, Shanghai, China) and then analyzed by microscopy. The number of cells that had crossed the membrane was calculated, and the experiment was repeated three times.