Figure 3

Modulation of miR-17-92 expression by IL-4 signaling. (A) Immuno-magnetically isolated mouse splenic CD4⁺ T cells were cultured with 5 μg/ml plated anti-CD3, feeder cells and 100 U/ml hIL-2 ("Neutral" condition). Anti-IL-4 (2.5 μg/ml) or isotype control mAb was added to the appropriate wells and cultured for 5 days prior to extraction of total RNA. Statistical analysis was carried out using the student t test. The blockade of IL-4 up-regulated miR-17-5p and miR-92 significantly with p < .001 and p < .005, respectively. (B), CD4⁺ T cells were cultured with anti-CD3, feeder cells, and hIL-2 and varying amounts of IL-4 for 5 days. Total RNA was extracted and analyzed by RT-PCR for miR-17-5p expression. The dose dependent decrease of miR-17-92 expression was analyzed using post test for linear trend and was significant (p < .001). (C), Th1 and Th2 cells were induced from splenic CD4⁺ T cells isolated from either wild-type or STAT6^-/- mice. Total RNA was extracted and RT-PCR was performed using specific primers against miR-17-5p and miR-92. Columns represent the mean of triplicates from one of 2 two experiments with similar results, and error bars represent standard deviations. STAT6^-/- cells demonstrated significantly higher levels of miR-17-5p and miR-92 compared with wild type (WT) cells in both Th1 and Th2 conditions (p < .001) using the student t test.