Cell migration assay

A wound-healing assay was performed as described below to test the migration ability of the CRC cells after transfection. The wounds were made in confluent monolayer cells using 10 µL pipette tips, and the culture was changed to a serum-free medium. Wound healing was detected at 0, 12, 24, and 36 hours within the scraped lines, and representative fields were photographed at the different time points. For the Transwell assay, 24-well Transwell plate without Matrigel (8 µm, Corning Incorporated, USA) was used to detect the migration ability of HCT116 cells. After transfection (1×10⁵ cells/well), the HCT116 cells were suspended in 200 µL of serum-free medium and placed in the upper compartment of each chamber, then 800 µL of culture medium with 10% serum was added to the lower chamber as a chemoattractant. Cells were allowed to migrate for 24 hours at 37 °C. Cells on the upper side of the insert filter were completely removed by wiping with a cotton swab, and the filters were fixed with 4% paraformaldehyde. Then, cells were stained with 0.1% crystal violet solution. Cells were observed and imaged using an optical microscope (Biocentury, China).