Fig. 6

EMT-related genes ZEB2 and SMAD4 are direct targets of miR-524-5p. a,b Effects of miR-524-5p overexpression on the expression of the EMT-related genes. A miR-524-5p mimic was transfected to WJ0706 cells for 48 h before the cells were harvested for analysis. The analysis of the expression of ZEB2 and SMAD4 by real-time RT-PCR (a) and Western blot analysis (b). In both experiments, TP53INP1 was included as a control. c,d Experimental validation of miR-524-5p targeting of ZEB2 and SMAD4 in luciferase assays. Based on the predicted miR-524-5p binding sites (red vertical lines) in the 3’-UTRs of the ZEB2 and SMAD4 transcripts, a 3’-UTR luciferase construct of each gene was generated (boxed) (c). The blank pmiRGlo and 3’-UTR luciferase constructs were transfected alone, or in the presence of the miR-524-5p mimic, or a validated negative control (NC) in colon cancer cell line HCT-15 prior to luciferase assays (d). *p < 0.05, relative to the pmiRGlo-only control