miR-155 regulates p27^kip1 expression by directly targeting KPC1. (A) Expression of miR-155 during monocyte differentiation into imDCs and mDCs. (B) Effect of the individual component in the maturation cocktail on miR-155 expression. imDCs at day 6 were treated 10 ng/mL of IL-1β, IL-6, or TNF-α, or 1 μg/mL of PGE₂ for 2 days. Relative expression of miRNA-155 was quantified by real-time PCR and normalized to imDCs without treatment. (C) Mature miR-155 sequence (miR-155WT) and mutated miR-155 sequence (miR-155MUT, 8 mutated nt sequences [in italic]) aligned with human and mouse KPC1 3′-UTRs, including miR-155 binding site (in bold). Mutation of miR-155 binding site sequence is shown below. (D) A lucifearse reporter assay to determine targeting of KPC1 3′-UTR by miR-155. The columns represent normalized relative luciferase activity (RLU) by means with 95% confidence intervals from 3 independent experiments. **P < .01 by 2 sample t test. (E) Overexpression of miR-155 resulted in decreasing KPC1 and increasing p27^kip1 expression. imDCs at day 4 were transfected with miR-155 precursors and negative controls. Twenty-four hours later, cells were analyzed for KPC1 and p27^kip1 expression by Western blot. (F) Reduced KPC1 expression along with up-regulated miR-155 expression during DC maturation. imDCs at day 6 were treated with the maturation cocktail for 2 days (mDC-D2) or 4 days (mDC-D4). Cells were analyzed for miR-155 by real-time PCR and KPC1 expression by Western blot. imDCs at day 6 (imDC) were used as a control. The data shown in each panel are representative of 2 (A,F) or 3 (B,D,E) independent experiments.