Fig. 7.

Notch1-induced miR-142a-3p impedes Treg differentiation by targeting TGFBR1.

(A) Prediction of the binding site of miR-142a-3p in TGFBR1 by miRWalk and miRDB (upper panel) and luciferase assay revealing binding of miR-142a-3p to the TGFBR1 3′UTR (lower panel). MFI of TGFBR1 in T cells was detected by flow cytometry after (B) naïve CD4⁺ T cells transfected with NC or a miR-142a-3p mimic were co-cultured with BMDMs (three/group), (C) naïve CD4⁺ T cells transfected with NC or miR-142a-3p inhibitor were co-cultured with BMDMs pretreated with LPS (100 ng/mL) and PA (250 μM) for 12 h (three/group), (D) naïve CD4⁺ T cells co-cultured with BMDMs from Notch1^FL/FL and Notch1^M-KO mice pretreated with LPS (100 ng/ml) and PA (250 μM) or PBS for 12 h (four/group), and (E) naïve CD4⁺ T cells co-cultured with LPS (100 ng/ml) and PA (250 μM) or PBS-pretreated BMDMs transfecting pEF-Flag-NICD or control plasmids for 12 h (four/group). The level of miR-142a-3p (F) and MFI of TGFBR1 in CD4⁺ T cells (G) in the liver tissues of Notch1^FL/FL mice and Notch1^M-KO mice fed with NCD or HFD for 6 weeks (five/group) were detected. Values represent means ± SD. Statistical analysis was performed by (A, D–G) one-way ANOVA and Tukey's test, or (B, C) 2-tailed unpaired Student t test. ∗∗p <0.01, ∗∗∗p <0.001. Abbreviations: BMDM, bone marrow-derived macrophage; HFD, high-fat diet; LPS, lipopolysaccharide; MFI, mean fluorescence intensity; NC, negative control; NCD, normal chow diet; NICD, Notch1 intracellular domain; Notch1^FL/FL, floxed Notch1; Notch1^M-KO, myeloid-specific Notch1-knockout; PA, palmitic acid; TGFBR1, transforming growth factor beta receptor 1; UTR, untranslated region.