Ranjan R, Lee Y G, Karpurapu M, Syed M A, Chung S, Deng J, Jeong J J, Zhao G, Xiao L, Sadikot R T, Weiss M J, Christman J W, Park G Y
Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois , Chicago, IL , USA.
Free Radic Res. 2015 Jan;49(1):25-34. doi: 10.3109/10715762.2014.974037. Epub 2014 Oct 30.
The production of microRNAs (miRNA) is influenced by various stimuli, including environmental stresses. We hypothesized that reactive oxygen species (ROS)-associated stress could regulate macrophage miRNA synthesis. miRNAs undergo unique steps of maturation processing through either one of two pathways of cytoplasmic processing. Unlike the canonical pathway, the regulation of alternative cytoplasmic processing of miRNA has not been fully elucidated yet. We cultured bone marrow derived macrophages (BMDM) from wild type (WT) and p47(phox-/-) mice and profiled miRNA expression using microarrays. We analyzed 375 miRNAs including four endogenous controls to normalize the data. At resting state, p47(phox-/-) BMDM has the markedly reduced expression of miR-451 compared to WT BMDM, without other significant differences. Unlike majority of miRNAs, miR-451 goes through the unique alternative processing pathway, in which Ago2 plays a key role. In spite of significant reduction of mature miR-451, however, its precursor form, pre-mir-451, was similar in both BMDMs, suggesting that the processing of pre-mir-451 is impaired in p47(phox-/-) BMDM. Moreover, p47(phox-/-) BMDM expressed significantly reduced level of Ago2. In contrast, Ago2 mRNA levels were similar in WT and p47(phox-/-) BMDM, suggesting a post-transcriptional defect of Ago2 production in p47(phox-/-) macrophages, which resulted in impaired processing of pre-miR-451. In order to examine the functional significance of miR-451 in macrophages, we cultured BMDMs from miR-451 knock-out mice. Of interest, miR-451-deficient BMDM exhibited reduced ROS generation upon zymosan stimulation, compared to WT BMDM. Our studies suggest functional crosstalk between ROS and miR-451 in the regulation of macrophage oxidant stress.
微小RNA（miRNA）的产生受包括环境应激在内的多种刺激影响。我们推测活性氧（ROS）相关应激可能调节巨噬细胞miRNA合成。miRNA通过细胞质加工的两条途径之一经历独特的成熟加工步骤。与经典途径不同，miRNA的替代性细胞质加工调控尚未完全阐明。我们培养了来自野生型（WT）和p47（phox-/-）小鼠的骨髓来源巨噬细胞（BMDM），并使用微阵列分析miRNA表达。我们分析了375种miRNA，包括四个内源性对照以标准化数据。在静息状态下，与WT BMDM相比，p47（phox-/-）BMDM中miR-451的表达明显降低，无其他显著差异。与大多数miRNA不同，miR-451通过独特的替代性加工途径，其中Ago2起关键作用。然而，尽管成熟的miR-451显著减少，但其前体形式pre-mir-451在两种BMDM中相似，表明p47（phox-/-）BMDM中pre-mir-451的加工受损。此外，p47（phox-/-）BMDM中Ago2的表达水平显著降低。相反，WT和p47（phox-/-）BMDM中Ago2 mRNA水平相似，表明p47（phox-/-）巨噬细胞中Ago2产生存在转录后缺陷，导致pre-miR-451加工受损。为了研究miR-451在巨噬细胞中的功能意义，我们培养了来自miR-451基因敲除小鼠的BMDM。有趣的是，与WT BMDM相比，miR-451缺陷的BMDM在酵母聚糖刺激后ROS生成减少。我们的研究表明ROS与miR-451在巨噬细胞氧化应激调节中存在功能性相互作用。
