Figure 5. TGF-β/Smad3-dependent pathways are responsible for the increased miR-30b expression in DCreg.

(a) miR-30b expression in mouse maDCs and DCreg were detected by qRT–PCR. *P<0.0001 (two-tailed Student’s t-test). Data are shown as means ±s.d. (n=3). (b) Neutralizing antibody against mouse TGF-β (5μg ml⁻¹) or isotype antibody was added into the co-culture system between endothelial-like splenic stromal cells and maDCs. After 7 days, maDCs and DCreg were collected, and miR-30b expression was detected by qRT–PCR. Data are shown as means ±s.d. (n=3). *P=0.0014, **P=0.0021 (two-tailed Student’s t-test). (c) maDCs and DCreg from wild-type or Smad3-deficient mice were collected, and miR-30b expression was detected by qRT–PCR. Data are shown as means ±s.d. (n=3). *P=0.0236 (two-tailed Student’s t-test). (d) Two sets of primers were used in the qPCR analysis to span the potential promoter region of miR-30b. (e) qPCR analysis of histone H3 acetylation in the promoter region of miR-30b by ChIP assay in maDCs and DCreg. Data are shown as mean±s.d.(n=3). *P=0.0228, **P=0.0374 (two-tailed Student’s t-test). (f) qPCR analysis of Smad3 binding in the promoter region of miR-30b by ChIP assay in maDCs and DCreg. Data are shown as mean±s.d. (n=4). *P=0.0153, **P=0.0286 (two-tailed Student’s t-test). (g) Two predicted Smad3-binding sites (S1 and S2) were indicated in the potential promoter region of miR-30b. Three sequences including S1 or S2 sites were individually, or in combination cloned into pGL3 luciferase reporter plasmid and indicated in the potential promoter region of miR-30b. (h) Transcriptional activation from the predicted Smad3 binding sites was induced by TGF-β in RAW cells. Cells were co-transfected with luciferase reporter including Smad3-binding sites and pRL-TK. After 48 h, cells were treated with TGF-β for 4 h and luciferase values were measured. pRL-TK was co-transfected as an internal control to correct the differences in both transfection and harvest efficiencies. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity. Data are shown as mean±s.d. (n=4). Similar results were obtained in three independent experiments. *P=0.0008, **P=0.0002, ***P=0.0006 (two-tailed Student’s t-test).