miRNomes of haematopoietic stem cells and dendritic cells identify miR-30b as a regulator of Notch1

Su, Xiaoping; Qian, Cheng; Zhang, Qian; Hou, Jin; Gu, Yan; Han, Yanmei; Chen, Yongjian; Jiang, Minghong; Cao, Xuetao

miRNAs are spatially and temporally expressed in multiple types of immune cells, suggesting their specific roles in the development and function of these immune cells. Based on analysis of miRNomes, we went further to find that, compared with maDCs, miR-30b expression was significantly enriched in | miR-30b promotes preferential IL-10 and NO production in DCreg through targeting Notch1. (a) maDCs and DCreg (5 Â 10 5 per ml) were transfected with control mimics or miR-30b mimics, control inhibitor or miR-30b inhibitor as indicated. After 48 h, IL-10 (left) and NO (right) in supernatants was measured by ELISA and Griess assay, respectively. Data are shown as means ± s.d. (n ¼ 3) . Similar results were obtained in three independent experiments. *P ¼ 0.0005, **P ¼ 0.0007, ***P ¼ 0.0004, ****P ¼ 0.0034 (two-tailed Student's t-test). (b) Flow cytometry analysis of intracellular staining for IL-10 expression of DCs transfected as in (a). Numbers in the histograms indicate the geometric mean fluorescence of the test samples. Similar results were obtained in at least three independent experiments. (c) Mouse Notch1 might be molecular target of miR-30b. Shown is a sequence alignment of miR-30b and its target sites in 3 0 UTR of Notch1, which was downloaded from TargetScan (http://www.targetscan.org). (d) HEK293 cells (1 Â 10 4 ) were co-transfected with pGL3 firefly luciferase reporter plasmids or pGL3-Notch1 3 0 UTR firefly luciferase reporter plasmids, pTK-Renillaluciferase plasmids, together with ctrl mimics or miR-30b mimics as indicated. After 24 h, firefly luciferase activity was measured and normalized by Renilla luciferase activity. Data are shown as means ± s.d. (n ¼ 3). Similar results were obtained in three independent experiments. *P ¼ 0.0033 (two-tailed Student's t-test). (e) maDCs and DCreg (1 Â 10 6 ) were transfected as described in (a). After 48 h, Notch1 and b-actin were detected. (f) maDCs and DCreg were transfected with miR-30b mimics or miR-30b inhibitor as in (e). The surface expression of Notch1 expression was detected by flow cytometry. Numbers in the histograms indicate the geometric mean fluorescence of the test samples. (g) Cells were treated with mouse Delta 4 (0.5 mg ml À 1 ) or g-SiX (10 mM) for 18 h. IL-10 (left) and NO (right) in supernatants were measured by ELISA and Griess assay, respectively. Data are shown as means ± s.d. (n ¼ 3). Similar results were obtained in three independent experiments. *P ¼ 0.0095, **P ¼ 0.0031, ***P ¼ 0.0125, ****P ¼ 0.0132 (two-tailed Student's t-test). (h) maDCs and DCreg were treated as in (g) as indicated. Expression of IL-10 was detected by intracellular staining. Numbers in the histograms indicate the geometric mean fluorescence of the test samples. Similar results were obtained in at least three independent experiments. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3903 ARTICLE DCreg in vitro and in vivo. We further investigated the underlying mechanism for the miR-30b enrichment in DCreg. In our previous report, endothelial-like splenic stromal cells can induce the differentiation from maDCs to DCreg, which is dependent on TGF-b production 15 . TGF-b is a pleiotropic growth factor that modulates immune response by controlling cell proliferation, cell differentiation and apoptosis. Upon activation, TGF-b activates Smad3, which subsequently translocates into the nucleus and initiates transcription of target genes. Despite the known roles of TGF-b/Smad3 signaling in regulating immune