Smad3 is a direct target of miR-140. Luciferase assays were carried out to address whether Smad3 is directly targeted by miR-140. The wild-type 3′ UTR sequence of Smad3 was cloned downstream of the luciferase gene and this plasmid (WT) was transfected with or without siRNAs mimicking miR-140 or scrambled siRNA. A mutant (MUT) construct was also used that contained mutations in all three predicted target sites of miR-140. Luciferase activities were normalized to transfections without siRNAs. The difference in luciferase activity between wild type and mutant constructs, in the presence of siRNA-140, was statistically significant (P < 0.001 by Students t-test).

Accumulation of miR-140 is transiently suppressed by TGFβ. (A) Time course Northern blot analysis of miR-140 following TGFβ treatment of 10T1/2 cells. TGFβ was applied once (5 ng/mL) and total RNA was extracted 1, 3, 6, 16, 24, and 48 h after the treatment. The membrane was stripped and reprobed with a miR-21 probe to show that the effect is specific for miR-140 or with a U6 probe to demonstrate equal loading. Three independent results are shown for miR-140 accumulation. (B) Accumulation level of miR-140 in 10T1/2 cells is shown without applying TGFβ treatment to demonstrate that the decrease in miR0140 level is due to TGFβ.

Smad3 is regulated at the protein level but not at the RNA level by miR-140. (A) Top panel: Northern blot analysis of Smad3 expression following transfection of cells with LNA-antimiR-449, LNA-antimiR-140, siRNA-96, or siRNA-140. Three independent experiments are shown to demonstrate reproducibility. Membranes were first hybridized with the Smad3-specific probe then following stripping they were rehybridized with a Gapdh-specific probe to show equal loading. Bottom panel: Signal intensities were quantified with a phosphorimager. Smad3 signals were first normalized to the corresponding Gapdh signal then to the corresponding mock sample. There was no statistically significant difference between the samples (student t-test). (B) Top panel: Western blot analysis of Smad3 accumulation in cells transfected with LNA-antimiR-449, LNA-antimiR-140, siRNA-96, or siRNA-140. Equal loading is shown with an Actin probe. Bottom panel: Signal intensities were quantified with Quantity One software (version 4.5.1; Bio-Rad). Smad3 signals were first normalized to the corresponding Actin signal then to the corresponding mock sample. The LNA-antimir-140 treatment gave significantly higher signal than the LNA-antimir-449 or mock (P < 0.05) and the siRNA-140 samples had significantly lower intensity than the siRNA-96 or mock (P < 0.01; both Students t-test).

TGFβ response is suppressed by miR-140. (A) Luciferase assays were carried out to measure the effect of miR-140 on a CAGAC promoter driven Luciferase gene. TGFβ induced a strong response in the presence of a scrambled siRNA (SCR), but the TGFβ mediated induction was suppressed in the presence of miR-140. The difference in luciferase activity between scrambled and miR140-specific siRNA, in the presence of TGFβ was statistically significant (P < 0.001 by Students t-test). (B–D) 10T1/2 cells were transfected with siRNAs mimicking miR-140 or scrambled siRNA. RNA was extracted and cDNA prepared following TGFβ treatment (4 ng/mL). PAI-1 (B), TIMP3 (C), and ADAM12 (D) expression was analyzed using quantitative RT-PCR and expression was normalized to 18S expression. The difference in expression level between scrambled and miR140-specific siRNA, in the presence of TGFβ was statistically significant for all three genes (P < 0.05 by Students t-test).