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

Discussion miRNAs have been shown to regulate mammalian immune cell differentiation, development and influence the outcome of immune responses, and also they are spatially and temporally regulated in a panel of mammalian tissues and cell types 33 . Whereas DCs are of vital importance in linking innate and adaptive immune responses, the function of intrinsic miRNAs in DCs remains elusive. In order to elucidate the alteration of individual miRNA, several methods, including qRT-PCR 34 and microarray 35 techniques have been widely used in qualitative miRNA studies. However, these approaches have low throughput Empty S2 S1+S2 S1 0 pre-miR-30b S2 S1 S1+S2 500 bp S1 S2 Neutralizing antibody against mouse TGF-b (5mg ml À 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. (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.