Transwell plates and cell culture inserts

For in vitro cell motility and invasion assay, Transwell plates and cell culture inserts (BD Biosciences, San Jose, CA) were used. For the coating of invasion assay, Matrigel (BD Biosciences, San Jose, CA) was diluted to 0.3 mg/ml concentration with Coating buffer (0.01 M Tris, 0.7% NaCl, pH 8.0) and 100 μl Matrigel was coated onto upper compartment of cell culture insert. After incubation for 1 h at 37 °C, the cell culture insert was ready for seeding. After transfection of miR-31, SNU-449 and SKHep-1 cells were appropriately (5 × 10⁴ cell/well for motility assay, 1 × 10⁵ cell/well for invasion assay) seeded into the cell culture insert with serum-free media and 5% fetal bovine serum was used as a chemoattractant. After 4 h (motility) or 12 h (invasion) of incubation at 37 °C, migrated or invaded cells were stained using Diff-Quik staining kit (Sysmex, Japan). The images of cells were photographed with Axiovert 200 inverted microscope (Zeiss, Germany) at ×200 magnification and the cell number was counted in three random fields of view.

For in vitro cell motility and invasion assay, Transwell plates and cell culture inserts (BD Biosciences, San Jose, CA) were used. For the coating of invasion assay, Matrigel (Corning Inc, Corning, NY, USA) was diluted to 0.3 mg/ml concentration and coated onto upper compartment of cell culture insert. After transfection of miR-99a and miR-497, HepG2 and Hep3B cells (5 × 10⁴ cell/well for motility assay, 1 × 10⁵ cell/well for invasion assay) were transferred on the top of the cell culture insert with DMEM/F12(Sigma-Aldrich, MO, USA) and 5% fetal bovine serum. After 4 h (motility) or 12 h (invasion) of incubation at 37 °C, migrated or invaded cells were fixed with 1% paraformaldehyde, stained with hematoxylin and photographed with Axiovert 200 inverted microscope at ×200 magnification. The cell number was counted in three random fields of view.