Mechanisms of allergy and clinical immunologyLet-7 microRNA–mediated regulation of IL-13 and allergic airway inflammation
Section snippets
In silico identification of microRNA binding sites
A database search for all human microRNAs (miRBase v9) was made against the human IL13 3′ untranslated region (UTR) using miRanda, RNAhybrid, and TargetScan.16, 17, 18 A consensus approach was used, as described earlier19 and listed in Table I, to minimize false-positive hits.
Plasmids
The human IL13 3′UTR was amplified from genomic DNA, cloned in pMIR-REPORT vector (Ambion, Austin, Tex), and designated as pMIR-REPORT-IL13 3′UTR. PCR-based site-directed mutagenesis was performed as described previously
Let-7 targets IL13 3′ UTR
Consensus prediction using 3 independent software programs indicated potential binding of 11 microRNAs to the IL13 3′UTR. Five of these belonged to the let-7 family (namely miR-98, let-7d, let-7f, let-7g, and let-7i), showing a high degree of conservation in the let-7 family26 and its predicted targeting of IL13 (Table I). The entire IL13 3′UTR was cloned into a luciferase reporter system and cotransfected with individual microRNA mimics (10 nmol/L) in A549 cells to experimentally validate
Discussion
IL-13 is a key effector TH2 cytokine. Because of its well-established role in modulating TH2 immune responses, including asthma, efforts are being made to therapeutically modulate IL-13 function. For instance, the anti–IL-13 antibodies, such as QAX576, were under clinical trials for the treatment of IL-13–related disorders32; the human anti–IL-13 IgG4 mAb CAT-354 has shown a significant reduction in airway eosinophilia and airway reactivity in a murine model of airway and esophageal
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Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest.