Fig. 4

c-Myc promotes invasion, EMT and metastasis of CRC cell by repressing miR-200b-3p. a The luciferase activity of PGL3-promoter-miR-200b-3p constructs of wild type and mutant types after transfection of pCDA3.1-c-Myc constructs in SW480 cells. b ChIP assay in LoVo and SW480 cells. Three regions containing putative c-Myc binding sites in the R1, R2 and R3 of miR-200b-3p promoter were amplified by PCR with specific primers. IgG was used to be negative controls, while input positive controls. c The effect of overexpression of c-Myc and co-expression of c-Myc and miR-200b-3p on the invasion of SW480 cells by Boyden chamber. Scale bars represent 20 μm. d Key EMT-related markers were detected by western blot in SW480 cells after overexpression of c-Myc and co-expression of c-Myc and miR-200b-3p. The grey value of the protein was normalized to that of the corresponding GAPDH. e Intestinal and hepatic metastatic nodules after subcutaneous tumors derived from SW480/Mock, SW480/c-Myc and sw480/c-Myc+miR cells were transplanted in the mesentery at the distal end of cecum in mice (n = 5) for 6 weeks. Red arrows point at potential metastatic nodules in intestines. Scale bars represent 50 μm. f, g The number of hepatic metastatic nodules (f) or intestinal metastatic nodules (g) of mice with tumors derived from SW480/Mock, SW480/c-Myc and sw480/c-Myc + miR cells. The number of hepatic metastatic nodules per mouse were counted under the microscope, with five HPF observation (***p < 0.001)