Figure 4. miR-155 and miR-146b levels control LTB₄-mediated SOCS-1 inhibition and enhanced MyD88 levels.

(A) Thioglycollate-elicited macrophages from WT were transfected with 30 nM of miR-155, miR-146b, or miR-125b antagomir for 24 h, then stimulated with 100 nM LTB₄ for another 24, followed by determination of (A) MyD88 or (B) SOCS-1 mRNA levels or protein (C). (D and E) WT and 5-LO−/− macrophages were transfected with 30 nM of the mimics miR-155, miR-146b, or miR-125b for 48 h followed by determination of MyD88 (D) and SOCS-1 (E) mRNA levels as described in Materials and Methods. Inset: The predicted miR-146b and miR-125b binding sites located in the 3′-UTR of SOCS-1 and MyD88, respectively. The sequence alignment of miR-146b/SOCS-1 and miR-125b/MyD88 is shown, and perfect matches are indicated by a line. (F) Raw264.7 macrophages were transfected with a luciferase construct containing the 3′ UTR of SOCS1, 3’ UTR of MyD88 and empty vector expressing luciferase reporter plasmid followed by the microRNA mimics miR-155, miR-146b and miR-125b for 24 h and the luciferase activity was determined as described in the Material and Methods. (G) WT macrophages were stimulated with 100 nM LTB₄ for different time points and the expression of miR-155, SOCS-1 and MyD88 were determined by qPCR. Data represent mean ± SEM from at least 3 individual experiments, each performed in triplicate. *P < 0.05 versus unstimulated WT; ^#P < 0.05 versus stimulated WT.