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

Upregulation of miR-30b in regulatory DCs by TGF-b/Smad3. Interestingly, multiple differentially expressed miRNAs were detected at different stages of DC development and differentiation, thus suggesting that many miRNAs may function in this process. We found that, compared with maDCs, miR-30b expression significantly increased in DCreg (Fig. 5a) . As we reported previously, endothelial-like splenic stromal cells can secrete TGF-b to drive the differentiation from maDC to DCreg 15 . Therefore, we wondered whether the neutralizing antibody against TGF-b could influence miR-30b expression in DCreg. As shown in Fig. 5b , the addition of neutralizing antibody against TGF-b in the co-cultural system could decrease miR-30b expression in DCreg. Further, Smad3 deficiency decreased miR-30b expression in DCreg (Fig. 5c ). Furthermore, as acetylation level of histone in promoter region is always elevated by Smad3 activation 29, 30 , ChIP assays showed the upregulated histone H3 acetylation level and Smad3-binding activity in promoter region of miR-30b in DCreg compared with maDCs ( Fig. 5d-f ). To delineate whether Smad3 specifically binds miR-30b promoter in response to TGF-b, we performed silico analysis (rVista 2.0, http://rvista.dcode.org/) 31 to search for potential Smad-binding sites in miR-30b promoter. In this study, we found two potential Smad-binding sites in miR-30b promoter (Fig. 5g) . To confirm this finding, we used luciferase reporter assay to determine the interaction of Smad3 with the miR-30b promoter region in RAW264.7 cells. As shown in Fig. 5h , TGF-b could successfully upregulate luciferase activity containing the potential 28 Mock TSA Pre-miR-1-1