miR-509-5p and miR-1243 suppress cell motility and invasion through an MET phenotype alteration and directly target VIM, HMGA2, SMAD2 and SMAD4. (a) The number of viable cells 24–96 hours after transfection with 5 nmol/L of miR-NC, miR-509-5p and miR-1243 in Panc1 cells was assessed by the WST-8 assay. Each data point represents the mean of triplicate experiments (bars, SD). (b,c) Transwell migration and invasion assays were performed in 24-well modified Boyden chambers without and with Matrigel, respectively. Panc1 cells (4 × 104 cells per well [migration and invasion assay]) that were transfected with each miRNA were transferred into the upper chamber, and the migrated or invaded cells on the lower surface of the filters were fixed, stained and counted after 24 hours of incubation. Experiments were performed in triplicate, and each data point represents the mean (bars, SD). (d) Western blot analysis of SMAD2, SMAD4 and HMGA2 in Panc1 cells 72 hours after transfection with 10 nmol/L of miR-NC, miR-509-5p and miR-1243. (e) Results of the luciferase reporter assays in Panc1 cells after co-transfection of the pmirGLO Dual-Luciferase vectors containing wild-type (WT) of SMAD2, SMAD4, VIM and HMGA2 or mutant variants of these genes and miR-NC, miR-509-5p and miR-1243. Student’s t-test was used for statistical analysis, and asterisks represent P < 0.05 versus miR-NC transfectants.