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

miR-30b enhances IL-10 and NO by targeting Notch1. In order to further investigate the biological significance of the upregulated miR-30b expression in DCreg, we went further to investigate the effects of miR-30b in cytokine production of DCreg. As we reported previously 15, 32 , IL-10 and NO were preferentially produced in DCreg, acting as important negative regulators of immune response. Further, we found that miR-30b overexpression could enhance IL-10 and NO production in DCreg, whereas miR-30b inhibition could decrease their production (Fig. 6a) , indicating that miR-30b may contribute to the preferential IL-10 and NO production in DCreg. On a per-cell basis, similar results of IL-10 production were observed, as determined by intracellular cytokine staining (Fig. 6b) , further indicating that miR-30b influences IL-10 production by regulatory DCs. As miRNAs function mainly through repressing their targets, we examined the targets of miR-30b, which might modulate the preferential IL-10 and NO production in DCreg. By targets prediction of miR-30b via TargetScan (http://www.targetscan.org), we found that mouse Notch1 had one putative conserved miR-30b target site (Fig. 6c) . To certify whether Notch1 was regulated post-transcriptionally by miR-30b, we constructed reporter plasmids containing the 3 0 UTR region of mouse Notch1, and found that its expression was significantly decreased by miR-30b co-transfection (Fig. 6d ). Furthermore, Notch1 protein level was decreased by miR-30b overexpression, whereas elevated by miR-30b inhibition in DCs (Fig. 6e) , confirming that miR-30b targets Notch1. Similar results were observed in flow cytometry analysis of the surface expression of Notch1 on DCs after miR30b manipulation (Fig. 6f) , further confirming that miR-30b targets Notch1.