Fig. 2

miR-153 downregulates IDO1 expression in colon cancer cells. a miR-153 inhibits reporter expression. At the top is the base pairing between IDO1 3′UTR and miR-153. The seed sequence of miR-153 is underlined. At the bottom is the reduced luciferase activity from reporter carrying the WT or the mutant IDO1 3′UTR under miR-153 overexpression. Y-axis denotes relative luciferase units. Dual luciferase reporter assays were performed three times from 293 T and Hela cells co-transfected with a Renilla luciferase gene (pRL-TK, Promega), a firefly luciferase gene (pGL-3Promoter) upstream of the WT (green) or the mutant (yellow) IDO1 3′UTR, and mirVana® miR-153 mimic (miR-153) and miRNA Mimic Negative Control #1 (NC). b Flow cytometry analyses of IDO1 and PD-L1 expression in DLD-1, HT-29, or HCT-116 cells transfected with miR-153, a negative control (NC), or mock treatment. c Western blotting analyses of IDO1 expression. d Immunofluorescence analyses of IDO1 protein expression (red) in DLD-1 cells expressing miR-153. The plasmids carrying miR-153 or the control had a GFP expression cassette. Nuclei were counterstained with DAPI (blue). e Flow cytometry analyses of IDO1 expression in monocyte-derived dendritic cells with miR-153 overexpression. A bar graph summarized fluorescence density (mean ± SEM) for IDO1 expression from three independent experiments (right). f Simple linear regression analysis showing an inverse relationship between miR-153 and IDO1 expression in 385 colon cancer patients from the TCGA database. *P ≤ 0.05; **P ≤ 0.01