In vitro migration and invasion assays

An in vitro scratch assay and Transwell assays were used to evaluate the migration and invasion abilities of the tumor cells, respectively. Briefly, cells were grown at monolayer in 12-well plates (2 × 10⁵) for overnight. An artificial scratch was created in cells, and cell debris was removed by washing with 1 × PBS. Cell migration was photographed and the width of the wound was measured. Cell invasion was evaluated by a Transwell system. The polycarbonate filters (8-μm pore size) were pre-coated with Matrigel Matrix (BD Biosciences, Franklin Lakes, NJ, USA), and reconstituted at 37 °C for 30 min. Cells (1 × 10⁵) were suspended in 150 μl of serum-free RPMI 1640 medium and added into the upper chamber, while 600 μl of complete medium was added to the lower chamber. The cells that migrated through the matrigel and adhered onto the lower chamber after 24 h of incubation were fixed with 4% paraformaldehyde for 20 min, and then stained with Mayer’s hematoxylin (Sigma-Aldrich, USA), and counted under microscope (five fields per chamber).