Evaluating miRNA and KRT80 Functions

The tumor-suppressive functions of miRNAs were evaluated by transient transfection assays using mature miR-139-5p and miR-139-3p. The tumor-promoting functions of KRT80 (loss-of-function assays) were assessed by siRNA transfection assays using siRNAs targeting KRT80. Functional assays (proliferation, migration, and invasion assays) were performed according to procedures of previous studies [51 (link),52 (link)]. Briefly, for proliferation assays, HCT116 or DLD-1 cells were transferred into 96-well plates at 3.0 × 10³ cells/well. Cell proliferation was assessed using XTT assay kit II (Sigma-Aldrich, St. Louis, MO, USA) 72 h after the transfection procedure. For the migration and invasion assay, HCT116 and DLD-1 cells were transfected in 6-well plates at 3.0 × 10⁵ cells/well; 48 h later, transfected HCT116 and DLD-1 cells were added to each chamber at 1.0 × 10⁵ cells/well. Corning BioCoat^TM cell culture chambers (Corning, Corning, NY, USA) were used for the migration assay and Corning BioCoat Matrigel Invasion Chambers were used for the invasion assay. cells on the underside of the chamber membrane were stained and counted for analysis. All experiments were performed in triplicate. The details of the reagents used in these analyses are listed in Table S2.