FIGURE 4.

IL-13 expression is suppressed by let-7a. A and B, let-7a suppresses mouse and human IL-13 in HEK293T cells. HEK293T cells were transfected with plasmids containing firefly luciferase under the control of the mouse (A) or human (B) il-13 3′-UTR or control 3′-UTR and simultaneously with plasmids expressing pre-mmu-miR-705, scrambled pre-miR, or pre-let-7a, as indicated. After 2 days, gene expression was quantitated as firefly relative light units after normalizing for transfection efficiency based on Renilla luciferase activity (Firefly/Renilla). C and D, anti-let-7a rescues mouse IL-13 expression. HEK293T cells were transfected simultaneously with mouse (C) or human (D) IL-13 3′-UTR and pre-mmu-let-7a plasmids as in A and additionally scrambled, irrelevant (anti-miR-705) or anti-let-7a locked nucleic acids. After 2 days, IL-13 expression was assessed as firefly/Renilla relative light units. E–G, let-7a suppresses IL-13 gene expression in primary T cells. E, mouse splenic CD4⁺ T cells were electroporated with FITC-labeled anti-let-7a LNAs (black curve) or sham (red curve), and the efficiency of transfection was assessed by flow cytometry. Additional T cells were transfected with control or anti-let-7a LNA, and relative expression of let-7a (F) and IL-13 (G) transcripts were determined by quantitative RT-PCR 24 h later. H, editing of let-7a to let-7e reduces efficiency of targeting of il-13. HEK293T cells were transfected with mouse IL-13 3′-UTR-containing luciferase plasmid, as in A, and either plasmids for expression of let-7a or edited let-7a(U→G) and either scrambled or anti-let-7a(U→G) LNA, as indicated, and the effect on il-13 gene expression was assessed as relative light units. I, pre-let-7a(U→G) is fully processed to let-7e. Quantitative RT-PCR quantitation of let-7e or let-7a in HEK293T cells transfected with either pre-let-7a or pre-let-7a(U→G) expression plasmids. Data are presented as means ± S.E. (error bars), n = 3 or 4 replicates/condition; *, p < 0.05 for the indicated comparisons.