miR-146a is an NF-κB-dependent gene. (A) Schematic diagrams of miR-146a (Upper) and miR-146b (Lower) genomic loci on human chromosomes 5 and 10, respectively. Putative binding sites of NF-κB (black), IRF3/7 (gray), and C/EBPβ (white, partially overlaps with IRF3/7) transcriptional factors are shown as boxes. Genomic regions indicated by bars above the miR-146a locus were analyzed in promoter luciferase reporter assay (C). Mutations, disrupting transcription factor binding introduced in the A547 construct, are indicated by X, and the sequences of mutated sites are shown under the locus diagram. (B) Northern blot analysis of pri-miR-146a and pri-miR-146b expression in the THP-1 cell line. Cells were starved for 24 h and then stimulated with LPS for 8 h. Northern blots were hybridized with radiolabeled pre-miR-146a or pre-miR-146b probes, respectively. β-Actin (Northern blot) and 18S rRNA (methylene blue staining) levels are shown as loading controls. (C) miR-146a promoter analysis. Promoter constructs containing genomic fragments of miR-146a locus were transfected into the 293/IL-1R/TLR4/MD2 cell line. Cells were stimulated with TNFα (white bars), IL-1β (gray bars), or LPS (black bars) for 8 h and analyzed by luciferase reporter assay. Promoter regions used in this experiment are depicted schematically in A.

miRNA miR-146a is an immediate early-response gene induced by various microbial components and proinflammatory mediators. (A) Microarray analysis of miRNA expression in THP-1 cells after stimulation with LPS (E. coli strain 055:B5). The scatter plot shows averaged (n = 4) background-subtracted raw intensities for each probe on both channels for Cy3-labeled control and Cy5-labeled 8-h LPS-treated samples. Each dot represents one miRNA probe. (B) Kinetics of miR-146a up-regulation by LPS. THP-1 cells were stimulated with LPS for the indicated times, and miR-146 expression was analyzed by qPCR and normalized by using 5S RNA levels. (C) Northern blot analysis of miR-146a/b expression. Indicated human cell lines were treated with LPS (E. coli strain 055:B5) for 8 h and probed with DNA oligonucleotides complementary to mature miR-146a (Top) and miR-146b (Middle). To assess the specificity of the probes, we used total RNA from 293 cells transiently transfected with pcDNA3 vector or expression plasmids for either pre-miR-146a or pre-miR-146b (marked as C, A, and B, respectively). (The faint bands in the lanes probed for miR-146b are cross-reactions with mirR-146a bands; the dark band in lane 6 represents miR-146b.) The membrane was reprobed for miR-16 as a loading control (Bottom). Radiolabeled RNA decade marker was used as a molecular weight reference. (D) Analysis of miR-146 expression in response to a panel of innate immunity ligands. THP-1 cells were stimulated with the indicated stimuli for 8 h. miR-146 expression was analyzed by qPCR and normalized by using 5S RNA levels.

IRAK1 and TRAF6 may be molecular targets of miR-146 posttranscriptional repression. Shown is a sequence alignment of miR-146a and its target sites in 3′ UTRs of TRAF6 (A) and IRAK1 (B). Also shown is an analysis of expression of TRAF6-UTR (A) and IRAK1-UTR (B) luciferase reporters in the presence of miR-146a/b or the irrelevant control miR-21. Filled bars correspond to reporter constructs with wild-type miR-146 targeting sites, and open bars correspond to constructs with 4-nt substitutions disrupting base-pairing with the “seed region” of miR-146. In IRAK1-UTR-mut both miR-146-binding sites were mutated as shown.