Transwell invasion assay

Cells at the logarithmic growth phase were prepared for transfection experiments. After transfection with miR-212-3p mimics or antagomir for 24 hours, cell migratory and invasive capacities were analyzed using transwell assay (BD Sciences) as described previously.25 Cell suspensions (5×10⁴ cells/mL) were prepared using a serum-free medium. Into each well of the upper chamber, 100 μL of the cell suspension was added; the lower chamber contained 600 μL of the culture medium supplemented with 10% FBS. For transwell invasion assay, 100μL Matrigel (1mg/mL; BD Sciences) was added into the upper chamber and incubated at 37°C for 30 min. The cells were washed, seeded, and incubated at 37°C with 5% CO₂ for 24 hours and the uninvaded cells swabbed. The cells in the lower chamber were fixed with 75% ethanol for 10 min and stained using 0.5% crystal violet (Solarbio) at room temperature for 20 minutes and observed under an optical microscope (Leica, Mannheim, Germany). Each experiment was repeated three times.