c-Fos expression is regulated by miR155. (A) Expression of c-Fos mRNA was analyzed by real-time RT-PCR in unstimulated and LPS-stimulated BM-DCs from WT or miR155^−/− mice (left) and in DC²¹¹⁴ cells transduced with empty vector or the BIC expression vector (right). Results are represented as relative c-Fos mRNA expression. The means and SDs derived from 3 independent experiments are shown; *P < .05. (B) Expression of c-Fos protein was analyzed by Western blotting in BM-DCs prepared from WT and miR155^−/− mice. Actin was used as internal control. A gel representative of 3 independent experiments is shown (left). c-Fos signals were quantified and normalized relative to actin (right). The results represent the mean fold increase derived from 2 independent experiments. (C) Schematic representation of mouse c-Fos mRNA. The sizes in nucleotides of the 5′UTR, open reading frame (ORF), and 3′UTR are indicated. The 3′UTR contains 2 predicted binding sites (black boxes) for miR155. The sequence of mouse miR155 is shown aligned with its predicted target sites in the 3′UTR of c-Fos mRNAs from the indicated species. A-U and G-C base pairs are represented by solid lines; G-U base pairs are represented by dotted lines. The miR155 seed region and its complementary sequences in c-Fos mRNAs are enclosed by boxes. Sequences of the mutated (Mut) 3′UTRs of human (Hs) and mouse (Mm) c-Fos mRNA are indicated. (D) Luciferase reporter constructs containing the WT or mutated 3′UTRs of human or mouse c-Fos mRNA were transfected into 293T cells. A reporter construct containing the 3′UTR of BACH1 mRNA, a known target of miR155, was used as positive control. The constructs were transfected together with the indicated amounts of human or mouse miR155. Luciferase activity was measured 24 hours after transfection, normalized with respect to the activity obtained with a control reporter vector, and is expressed as relative luciferase activity. The means and SDs derived from 3 independent experiments are shown.