Skip to main content
. 2014 Dec;20(12):1878–1889. doi: 10.1261/rna.045633.114

FIGURE 2.

FIGURE 2.

Let-7i enhances IL-2 promoter activity in human and mouse primary CD4+ T cells. (A) Predicted binding between let-7i and IL-2 core promoter region. TATA-box motif was highlighted. (B) Northern blot demonstrating the relative nuclear and cytoplasmic abundance of endogenous let-7i in Sup-T1 cells. U78 small nucleolar RNA and valine-tRNA (tRNAval) served as controls for the nuclear and cytoplasmic fractionation. (C) The let-7i mimic or negative control was co-transfected with the IL-2 promoter-driven firefly luciferase (FL) construct into HEK293T cells, a CMV-renilla luciferase (RL) plasmid served as a transfection control. Dual-luciferase assay was performed at 24 h after transfection. (D) The effect of overexpressing let-7i on IL-2 mRNA expression. The PLKO-let-7i cells which expressed let-7i miRNA plus GFP and PLKO cells which just expressed GFP were generated by infecting Jurkat cells with lentiviral vector containing let-7i precursor or the PLKO empty vector and sorted with FACS. The gfp-positive cells were subsequently treated with (left) and without (right) phorbolmyristate acetate (PMA, 50 ng/mL) and ionomycin (1 μM) for 12 h. IL-2 mRNA was then evaluated by qRT-PCR and normalized to GAPDH mRNA. (E) Levels of IL-2 mRNA in primary human CD4+ T cells after treatment with agomir-let-7i. The CD4+ T-cells were treated with agomir-let-7i or control for 48 h, and then cells were activated with anti-CD3 and anti-CD28 antibodies. The levels of IL-2 mRNA were evaluated by qRT-PCR and normalized to β-actin mRNA. (F) Levels of IL-7 and IL-15 mRNA in primary human CD4+ T cells after the treatment of agomir-let-7i as described above. (G) Cellular IL-2 protein was determined by ELISA. The samples were collected from the cell lysates of primary human CD4+ T cells transfected with let-7i mimic or negative control and activated as described above. (H) IL-2 mRNA levels in primary human CD4+ T cells transfected with let-7i miRNA inhibitor or negative control. The CD4+ T cells were transfected with let-7i inhibitor or negative control at a final concentration of 200 nM and then activated as described above. The levels of IL-2 mRNA were evaluated by qRT-PCR and normalized to GAPDH mRNA. (I) Intracellular staining of IL-2 in the CD+ cells transfected with let-7i miRNA inhibitor or negative control. The cells were transfected with let-7i miRNA inhibitor or negative control at 200 nM and activated with anti-CD3/anti-CD28 for 48 h before IL-2 intracellular staining and flow cytometry assay. These data represent three independent experiments. (J) IL-2 mRNA levels in mouse CD4+ T cells transfected with mmu-let-7i mimic or negative control. (K) Quantification of serum IL-2 levels of mice treated with agomir-mmu-let-7i or negative control (25 nmol per animal) for 48 h by ELISA. P-values were calculated using the two-tailed unpaired Student's t-test with equal variances, n = 3 biological replicates unless specified. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001.