Cell invasion was explored using the Transwell chambers with Matrigel (Millipore, Billerica, MA, USA). Briefly, the upper chambers of Transwell inserts were covered with 1×105 transfected cells resuspended in 200 μL FBS-free DMEM medium. A volume of 500 μL DMEM+10% serum was added to the lower chamber. After 48 h, cells seeded in the upper and lower chambers were obtained using cotton swabs, fixed in 4% PFA and stained with 0.1% crystal violet (Beyotime, China) for 30 min. Stained cells were counted using an upright microscope (Nikon, 200× magnification).

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