Fig. 4

miR-148a inhibits the expression of SMAD2 by downregulating its mRNA. a qRT-PCR analysis of miR-148a overexpression (miR-148a) or knocking down (KD miR-148a) efficacy in the indicated cells. Ctrl, cells transfected with control lentivirus. Data were normalized to U6 and are shown as the fold change to their respective control cells. b, c qRT-PCR (b) and western blot (c) analysis of SMAD2 mRNA and protein level after stably overexpressing or knocking down miR-148a in MHCC-97H and Huh7 or HLF and Hep3B cells respectively. Data were normalized to GAPDH and are shown as the fold change to their respective control cells in (b). GAPDH, as loading control in (c). d Schematic image of binding site between miR-148a and SMAD2–3′-UTR. e The indicated cells were co-transfected with pSicheck2-SMAD2–3’UTR (pSicheck2-SMAD2–3’UTR-mutant) and miR-148a mimic (miR-148a inhibitor). Forty-eight h after transfection, cells were subjected to dual luciferase assay. (f) RIP assays of miR-148a and 3’UTR-SMAD2 enrichments by anti-IgG or anti-Ago2 antibodies in miR-148a overexpressing MHCC-97H cells (left panel) and miR-148a knocking down HLF cells (right panel). Data were normalized to 10% input. Data are shown as Mean ± SEM. *, p < 0.05. **, p < 0.01. n.s., no significance