Fig. 6

miR-1246 suppressed the expression of SPRED2 by binding to its 3’UTR. (a) Relative level of SPRED2 between normal and tumor tissues (TCGA data, 50 normal cases and 378 tumor cases) (left panel); The expression of SPRED2 was assessed by qRT-PCR between tumor and adjacent normal tissues in our patient samples (right panel) (n = 30). (b) The expression of SPRED2 and miR-1246 were analyzed in 30 cases of clinical CRC tissues (Pearson’s correlation coefficient, r = 0.5003, *P < 0.01, n = 30). (c) The binding sites of miR-1246 in the 3’UTR of SPRED2 mRNA and the wide type or mutant type of binding site were shown. (d) The effect of miR-mimics on the pGL3-SPRED2-WT and pGL3-SPRED2-Mut reporters in DLD-1 and HCT116 cell lines was measured by dual luciferase reporter gene. (e) RIP assay confirmed the binding between SPRED2 and miR-1246 in DLD-1 and HCT116 cell lines. (f) Relative level of SPRED2 when treated with miR-1246 mimics or miR-1246 inhibitors. (g, h) Relative level of SPRED2 was detected by qRT-PCR and western blot treated with miR-mimics or miR-inhibitors in the presence of stable knocked-down and overexpressed METTL3 cell lines. (i) The phosphorylation status of RAF/MEK/ERK pathway when co-transfected with oeSPRED2 plasmids and miR-1246 mimics or siSPRED2 and miR-1246 inhibitors. Data are presented as means ± standard deviation (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)