Fig. 3

miR-22 post-transcriptionally down-regulated galectin-9 expression by directly targeting its 3’-UTR. a Schematic representation of the predicted binding sites of miR-22 within the 3’-UTR of galectin-9. The lower scheme represents the intact sequence of the wild-type binding site and that of its mutant counterpart within the dual-luciferase reporter vector. b Dual-luciferase assay showing galectin-9 3’-UTR luciferase reporter activity in cultured HepG2 and Hep3B cells transfected with mimic controls (mimic-NCs), inhibitor controls (inhibitor-NCs) and mimics (50 nmol/L) or inhibitors (50 nmol/L) of miR-22-3p, miR-296-3p, miR-455-5p, and miR-491-5p. c Transfection of miR-22 mimics into HepG2 and Hep3B cells resulted in decreased galectin-9 3’-UTR reporter luciferase activity compared to transfection of mimic-NCs. These effects were abolished by a mutation in the putative miR-22-binding site within the galectin-9 3’UTR. d and e, miR-22 negatively regulated galectin-9 expression at the mRNA and protein levels. Endogenous galectin-9 levels in HepG2 and Hep3B cells transfected with miR-22 mimics or NCs or mir-22 inhibitors or NCs were analyzed by qPCR and western blotting at 24 h after transfection (* P < 0.05, **P < 0.01, compared to mimic-NC or inhibitor-NC)