Skip to main content
. 2021 Apr 23;12:2419. doi: 10.1038/s41467-021-22641-y

Fig. 4. IEC-specific deletion of miR-146a promotes CRC development.

Fig. 4

a–c Representative images (a), numbers (b), and sizes (c) of colonic tumors in miR-146afl/fl and IEC-miR-146a−/− (VillinCreMiR-146afl/fl) mice with AOM/DSS-induced CRC (n = 8). d Western blottings of TRAF6, NF-κB subunit phospho-p65 (pp65), and phospho-p38 (pp38) MAPK in CRC tissue from these mice. e In silico target prediction of miR-146a binding the TRAF6 3′-UTR. f Luciferase activity in IEC line (CMT-93) transfected with luciferase construct containing TRAF6 sequence with ctrl or miR-146a mimic (n = 3). g qPCR of TRAF6 from streptavidin pulldown of biotinylated ctrl or miR-146a mimic-transfected CMT-93 IECs (n = 3). h Representative FACS histograms and MFIs of TRAF6 in FACS-sorted IECs from miR-146afl/fl and IEC-miR-146a−/− mice with AOM/DSS-induced CRC. MFI as FC from miR-146afl/fl (n = 5). i Western blottings of TRAF6, pp65, and pp38 in IECs from these CRC mice. j Western blottings of these molecules in miR-146a-sufficient (WT) and miR-146a-deficient (miR-146a−/−) CMT-93 IECs stimulated with IL-17 (25 ng/ml) for 1.5 h. k, l Western blottings of Cox-2 and β-catenin in CRC tissue (k) and IECs (l) from CRC miR-146afl/fl and IEC-miR-146a−/− mice. m Western blottings of Cox-2 and β-catenin in WT and miR-146a−/− CMT-93 IECs stimulated with IL-17a (25 ng/ml). n ELISA of PGE2 in WT and miR-146a−/− CMT-93 IECs stimulated with IL-17a (25 ng/ml) for 48 h (n = 6). o, p qPCR of PTGES2 in CRC tissue (o) and IECs (p) from CRC miR-146afl/fl and IEC-miR-146a−/− mice. qPCR data as FC from miR-146afl/fl (n = 8). q, r Western blotting of PTGES2 in CRC tissue (q) and IECs (r) from CRC miR-146afl/fl and IEC-miR-146a−/− mice. s Western blotting of PTGES2 in WT and miR-146a−/− CMT-93 IECs stimulated with IL-17a (25 ng/ml). t In silico target prediction of miR-146a binding the PTGES2 3′-UTR. u Luciferase activity in CMT-93 IECs transfected with luciferase construct containing PTGES2 sequence with ctrl or miR-146a mimic (n = 4). v qPCR of PTGES2 from streptavidin pulldown of biotinylated ctrl or miR-146a mimic-transfected CMT-93 IECS (n = 3). w Representative colonic histopathology (scale bar = 100 µm) with Ki67 staining in WT and miR-146a−/− mice with AOM/DSS-induced CRC. x, y Representative images (x) and numbers (y) of colonic tumors in miR-146afl/fl and IEC-miR-146a−/− mice treated with anti-IL-17a (500 μg/ml) twice a week throughout AOM/DSS CRC induction (n = 5). z Model depicting IEC-intrinsic miR-146a in limiting tumorigenic IL-17 signaling in IECs. Data are representative of ≥2 independent experiments. n = biologically independent replicates per group (b, c, h, o, p, y) or replicates pooled from independent experiments (f, g, n, u, v). Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, by two-way ANOVA with Tukey’s adjustment (y) or two-tailed Student’s t-test (b, c, f–h, n–p, u, v). Source data are provided as a Source data file.