ESCC cells (5 × 104) were seeded in 6-well plates to reach more than 90% confluence. The samples were then scratched manually using a pipette tip. After scratching with a sterile pipette tip, removal of cell debris by washing 3 times with PBS, the wounded cell samples were then cultured in serum-free medium. Images were acquired at 0 h, 4 h, 8 h and 24 h post scratching by using a microscope. Four wound areas were photographed on each plate and counted under an Olympus inverted fluorescence phase-contrast microscope (Tokyo, Japan). Then the percentage of wound closure was calculated by the following formula: wound closure (%) = (original gap distance-gap distance at the indicated time)/original gap distance × 100%. wound healing assay was performed independently three times, with each group assayed in triplicate.