Figure 6. miR-18a suppresses liver metastasis of colon cancer triggered by direct targeting of Irf2 expressed in Kupffer cells.

(A) Schematic diagram of the putative binding sites of miR-18a in the wide type (WT) IRF2 3′ untranslated regions (UTR). The miR-18a seed matches in the IRF2 3′UTR are mutated at the positions as indicated. CDS, coding sequence. (B) Expression of miR-18a and potential miR-18a targeted genes in macrophages-like RAW264.7 cells was analyzed by real-time PCR. (C) Expression of candidate miRN-18a target gene IRF2 and IFNγ in macrophage RAW264.7 cells assessed by western blotting. (D) IRF2 (red) expression in liver of CT26/OGNVs and CT26/OGNVs/miR-18a treated mice, visualized with a confocal microscopy. Data are representative of three independent experiments (n = 5). (E) Evaluation of IRF2 and IFNγ level in macrophage-like RAW264.7 cells assessed by qPCR, 72 h after transfection of IRF2 siRNA (si-IRF2) or control (Ctrl) siRNA. (F) Expression of IRF2 and IFNγ in aliquots of macrophage-like RAW264.7 cells assessed by western blotting (left), quantification of results (right). (G) Expression of miR-18a and candidate miR-18a target genes in liver F4/80⁺ cells sorted by FACS and assessed by real-time PCR, following intravenous administration of OGNVs/miR-18a mimic and OGNVs/control miRNA. (H) Luciferase activity assays of WT and mutated Irf2 3′UTR luciferase reporters after co-transfection with miR-18a mimic, miRNA mimic control, miR-18a anti-sense RNA (AS-miR-18a), or miRNA anti-sense negative control RNA in RAW264.7 cells. The luciferase activity of each sample was normalized to the Renilla luciferase activity. The normalized luciferase activity of transfected control mimic miRNA was set as relative luciferase activity of 1. Error bars represent S.E.M. Each data point was measured in triplicate.