MiR-31 is critical for the activation, survival, and differentiation of CD4 T cells in a hypoxic environment via targeting FIH1. (A) A highly conserved binding site of miR-31 seed sequence in the 3′ untranslated region (UTR) of FIH1 was identified using the TargetScan Web site. (B-E) CD4⁺CD25⁻ WT or KO cells were isolated and stimulated with plate-bound anti-CD3 (4 μg/mL) and soluble anti-CD28 (2 μg/mL) in 3% or 21% oxygen (O₂) conditions. Three days later, expression of FIH1 protein was determined by western blot (B), and expression of HIF1α (C) and CD69 and ICOS (D) in gated live CD4 T cells was determined by flow cytometry. CD4⁺CD25⁻ WT or KO cells were stimulated with anti-CD3/CD28 in 3% O₂ with 0.1 mM dimethyloxallyl glycine (DMOG) dissolved in phosphate-buffered saline (PBS) or vehicle control. Frequency of annexin V⁺7AAD⁺ dead cells is shown in gated CD4 T cells on day 3 (E). (F) CD4⁺CD25⁻ WT of KO cells were stimulated with anti-CD3/CD28 together with anti–IFN-γ (1 μg/mL), transforming growth factor β (2 ng/mL), and IL-6 (1 ng/mL) in 3% O₂. Expression of RORγt and Foxp3 in gated live CD4 T cells was determined by flow cytometry on day 3. Data shown are from 1 representative of 3 individual experiments. *P < .05, **P < .01, ***P < .001, ****P < .0001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.