Figure 3.

Confirmation of Target Site for miR-200b/c in IL-33 3′ UTR

(A) Predicted binding site for miR-200b/c in 3′ UTR of IL-33. (B) Relative luciferase activity in Jurkat cells cotransfected with control firefly luciferase vector (pMIR-Report), or a firefly luciferase reporter vector containing the 3′ UTR of IL-33, or a firefly luciferase vector with perfect miR-200b/c binding site in the 3′ UTR (pMIR-200), and either the pre-miR-200 expression vector (pMIRNA1-Pre-miR-200) or control vector (pMIRNA1-Control). Firefly luciferase activity was normalized to the Renilla luciferase activity and then to the average of the control firefly luciferase reporter. n = 4 per group; data are representative of three experiments. An unpaired t test was used to compare reporter gene activity. (C) Jurkat, Raji, THP-1, and A549 cells were transfected with pMIR-REPORT-IL-33 3′ UTR to correlate the expression of miR-200b/c with IL-33 3′ UTR regulatory activity. (D) MRC5 cells were transfected with a miR-200b/c expression vector; 24 hr later, cells were stimulated with IL-13 to induce production of IL-33. Overexpression of miR-200b and miR-200c greatly reduced the induction of IL-33 production. (E) Silencing the endogenous miR-200b/c via transfection with inhibitory oligonucleotides increased IL-33 production. **p < 0.01 (Student’s unpaired and two-tailed t test).