Effect of miR-130a on Smad4 expression. (A) HEK293 cells were transfected with miR-130a and a scrambled pre-miR (miR-neg), respectively. The immunoblot shows down-regulation of Smad4 protein in cells transfected with pre-miR-130a compared with scramble pre-miR at 48 hours. The graph displays the relative expression of Smad4, normalized to β-actin expression. The experiment was repeated 3 times with similar results. (B) The relative expression of Smad4 mRNA in HEK293 cells transfected with pre–miR-130a and miR-neg as in panel A. The Smad4 mRNA level was measured by real-time PCR and normalized to β-actin expression. Data are shown as the mean ± SD from triplicate measurements. (C) Western blot analysis showing the expression level of Smad4 protein in A549 cells transfected with siRNA-Smad4, pre–miR-130a, miR-neg, or siRNA-neg at 48 hours after transfection. The graph shows the relative expression of Smad4 normalized to β-actin expression. (D) The relative expression level of Smad4 mRNA in A549 cells transfected with siRNA-Smad4, pre–miR-130a, miR-neg, or siRNA-neg at 48 hours after transfection. Smad4 mRNA expression was measured by real-time PCR and normalized to β-actin expression. Data are shown as the mean ± SD from triplicate measurements. (E) A549 cells were cotransfected with a TGF-β inducible firefly luciferase reporter plasmid (CAGA₁₂-MLP-Luc) that carries the Smad-binding element of the plasminogen activator inhibitor-1 gene, a renilla luciferase reporter for normalization of transfection, and with siRNA-Smad4, pre–miR-130a, miR-neg, or siRNA-neg, respectively. The cells were cultured in standard medium for 24 hours and then incubated with or without human recombinant TGF-β1 (20nM) for an additional 6 hours before determination of luciferase activity by use of the Dual-Luciferase Reporter Assay System. The experiment was repeated 3 times with similar results. The highest value in the transfection series was assigned the value 1, and the relative expression of the other samples was recalculated accordingly.