Mirna 374b 5p mimic

Naïve CD4⁺ T cells (5 × 10⁵ cells per well) were grown in a 96-well plate containing T cell expansion medium (Thermo-Fisher) at 37 °C and 5% CO₂, and then transfected with miRNA-374b-5p inhibitor (200 nmol/mL; Qiagen) and miRNA-374b-5p mimic (50 nmol/mL; Qiagen) using the Hiperfect Transfection Reagent (Qiagen). MiScript NC siRNA (Qiagen) was employed as a control. After 4 h, T cell differentiation was performed in using 48-well plates different cytokine regimens according to a previous method [23 (link)]. For Th1, the transfected cells were incubated with complete RPMI, plate-bound 1 μg/mL anti-CD3 and 1 μg/mL soluble CD28 antibodies, 20 ng/mL IL-2, 10 ng/mL anti-IL-4 and 50 ng/mL IL-12 antibodies (BD Biosciences) for 96 h. For Th2, the cells were exposed to 1 μg/mL plate-bound anti-CD3 and 1 μg/mL anti-CD28 antibodies, 10 ng/mL IL-4, 20 ng/mL IL-2 and 10 ng/mL anti-IFN-γ antibodies (BD Biosciences) for 96 h. For Th17 cell differentiation, the transfected cells were incubated with complete RPMI, 1 μg/mL plate-bound anti-CD3 and 0.2 μg/mL soluble CD28 antibodies, 5 ng/mL TGF-β, 100 ng/mL IL-6, 50 ng/mL IL-23, 10 ng/mL anti-IL-4, and 10 ng/mL anti-IFN-γ antibodies (BD Biosciences) for 96 h.

Serum samples and peripheral blood mononuclear cells (PBMCs) were isolated from the EDTA-anticoagulated whole blood specimens of IBD patients and control subjects via Ficoll-Paque™ Plus density centrifugation (GE Healthcare Bio-Science, USA). Human-lymphocyte-cell-separation-medium kit (Solarbio Life Sciences, China) was used to further isolate PBMCs by following the kit’s protocol. Then, CD4⁺T cell isolation was performed by magnetically activated cell sorting using the human naïve CD4⁺T cell isolation kit (Miltenyi Biotec, Germany). Flow cytomerty was conducted to assess the purity of CD4⁺ T cells (> 95%). The cells were then cultured in T-cell expansion medium (Thermo-Fisher) containing 15% FBS and 100 g/mL penicillin–streptomycin. The naïve CD4⁺ T cells were assigned to miRNA-374b-5p mimic (50 nmol/mL; Qiagen, 219,600) group, miRNA-374b-5p inhibitor (200 nmol/mL; Qiagen, 219,300) group, negative control (NC) group and blank group, and transfected for 4 h with Hiperfect Transfection Reagent (Qiagen) by following the kit’s protocols. Finally, the transfected cells were exposed to 1 μg/mL anti-CD28 and 1 μg/mL anti-CD3(eBioscience) at 37 °C for 48 h.