Biologic effect of increased miR-130a expression. Stable transfection of 32Dcl3 cells with the pEGP-miR-130a, pEGP-miR-223, or pEGP-miR-Null vectors was performed, generating 3 pEGP-miR-130a (Cl 3, Cl 6, and Cl 7) clones, 1 pEGP-miR223 (miR-223) clone, and 1 pEGP-miR-null (null) clone. (A) Relative miR-130a expression in Cl 3, Cl 6, Cl 7, null, and miR-223 was determined by real-time PCR and normalized to Sno234 expression. (B) Relative miR-223 expression in clone miR-223 and null was determined by real-time PCR and normalized to Sno234 expression. (C) Immunoblot showing Smad4 protein expression in the Cl 3, Cl 6, Cl 7, null, and miR-223 clones. The graph shows the relative expression of Smad4 protein relative to the expression of β-actin. The highest value among those being compared was assigned the value 1, and the remaining values were recalculated accordingly. (D) The relative expression level of Smad4 mRNA in the Cl 3, Cl 6, Cl 7, null, and miR-223 clones was measured by real-time PCR and normalized to β-actin expression. Data are shown as the mean ± SD from triplicate measurements. (E) Cell proliferation assays were performed with the clones Cl 3, Cl 6, Cl 7, null, and miR-223 in the presence or absence of human recombinant TGF-β1 (20nM). The graph shows the relative number of cells after 4 days when TGF-β1–stimulated cells are compared with unstimulated cells for each clone. The mean reduction in cell number (Red. in cell no.) for TGF-β1–stimulated cells for each clone was 8% for Cl 3, 31% for Cl 6, 8% for Cl 7, 43% for null, and 52% for miR-223 (the values are based on 3 independent growth experiments). The cell number for the unstimulated cells was assigned the value 1, and the relative number of TGF-β1–stimulated cells was recalculated accordingly. (F) The clones Cl 3, Cl 6, Cl 7, and null were cotransfected with the TGF-β1–inducible firefly luciferase reporter plasmid CAGA₁₂-MLP-Luc, along with a renilla luciferase reporter for normalization of transfection. 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.