Invasion assay

To detect the invasive ability of MDA-MB-231 invasion assay was performed. After 24 h of transient transfection, 2.5 × 10⁴ cells were harvested and seeded in the 8 μm pore size upper Transwell chamber (Corning, USA) coated with Matrigel (CAS:356234, Corning, USA), which cultured in basic medium. The optimal coating concentration ratio was DMEM serum-free medium: Matrigel = 8:1 (V/V), and 2 h incubation at 37 °C. 900 μL complete medium containing 5% FBS was added into the lower chamber as a chemoattractant. After 24 h incubation cells were fixed with 4% paraformaldehyde and stained with 1% crystal violate. Migratory cells in lower chamber were counted from each well and photographed at 20× (Olympus, Japan). The experiment was repeated three times.