Figure 2.

Smad4 is a direct target of miR-146a. (A) Overexpression of miR-146a inhibits Smad4 expression and increases vascular endothelial growth factor (VEGF) expression in primary chondrocytes, as assayed by immunoblotting. NC, negative control. (B) Densitometric analysis of immunoblot band intensities for Smad4 normalized by GAPDH. Data are mean ± standard deviation (SD) of three independent experiments. (C) Densitometric analysis of immunoblot band intensities for VEGF normalized by GAPDH. Data are mean ± SD of three independent experiments. (D) On the protein level, knockdown of miR-146a results in the upregulation of Smad4 and the downregulation of VEGF. Relative expression levels of protein shown at the bottom of the bands as normalized by GAPDH level. (E) Densitometric analysis of immunoblot band intensities for Smad4 normalized by GAPDH. Data are mean ± SD of three independent experiments. (F) Densitometric analysis of immunoblot band intensities for VEGF normalized by GAPDH. Data are mean ± SD of three independent experiments. (G) Schematic representation of the Smad4 3' UTR indicating the binding site of miR-146a. WT, wildtype; MT, mutant. (H) miR-146a inhibits Smad4 3' UTR luciferase activity. HEK293T cells were transfected with either pGL3 luciferase vector containing a fragment of Smad4 3' UTR harbouring binding sites for miR-146a, or the corresponding mutant constructs. Ectopic expression of miR-146a led to a remarkable reduction of the luciferase activity of reporter with the wildtype 3' UTR but not that of the mutant reporter. Values are mean ± SD of three independent experiments. *P < 0.05 versus vector. **P < 0.01 versus vector.

Li et al. Arthritis Research & Therapy 2012 14:R75   doi:10.1186/ar3798
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