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PNAS 104 (5): 1604-1609

Copyright © 2007 by the National Academy of Sciences.

MicroRNA-155 is induced during the macrophage inflammatory response

Ryan M. O'Connell*, Konstantin D. Taganov*, Mark P. Boldin*, Genhong Cheng{dagger}, and David Baltimore*,{ddagger}

*Department of Biology, California Institute of Technology, 330 Braun, 1200 East California Boulevard, Pasadena, CA 91125; and {dagger}Departments of Microbiology, Immunology, and Molecular Genetics, University of California, 650 Charles East Young Drive South, Los Angeles, CA 90095


Figure 1
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Fig. 1. Microarray analysis of miRNAs induced during the macrophage antiviral response. (A) WT murine macrophages were stimulated with medium (m), 2 µg/ml poly(I:C) [p(I:C)], or 1,000 units/ml IFN-beta for 6 h. RNA was extracted and used to conduct a microarray analysis to determine the expression levels of 200 mammalian miRNAs. Data are presented on a scatter plot showing log10-transformed signal intensities for each probe on both channels for the Cy3-labeled media controls and samples stimulated with Cy5-labeled IFN-beta (Left) or poly(I:C) (Right). (B) RNA used in A was analyzed by qPCR to assay expression of miR-155 and in a separate experiment by Northern blot analysis under the same conditions. (C) RNA used in A was assayed by qPCR to detect expression of the small nuclear RNA U6 as a loading control or IP10 mRNA to ensure equivalent stimulation by poly(I:C) and IFN-beta. (D) A portion of the macrophages generated in A were stimulated with medium, poly(I:C), or IFN-beta and assayed for CD11b and CD86 expression by using FACS to ensure proper macrophage development and activation, respectively.

 

Figure 2
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Fig. 2. Kinetics of poly(I:C) and IFN-beta induction of BIC mRNA and mature miR-155. (A) Unscaled depiction of the genomic structure of the human BIC noncoding RNA gene and location of miR-155 (155) in exon 3. E, exon; I, intron. (B) After stimulation with 2 µg/ml poly(I:C) or 1,000 units/ml IFN-beta, macrophage expression of BIC mRNA was analyzed over a 48-h time course by reverse transcription with an oligonucleotide dT primer followed by detection using PCR and agarose gel electrophoresis. Primers were designed to target BIC sequences extending outside of miR-155. L32 mRNA detection is included as a control. (C) RNA from B was also used to assay mature miR-155 by qPCR over a 48-h time course.

 

Figure 3
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Fig. 3. TLRs induce miR-155 expression through MyD88- or TRIF-dependent signaling pathways. (A) WT (Wt) murine macrophages were stimulated with medium (m), 2 µg/ml poly(I:C) [p(I:C)], 5 ng/ml LPS, 2 µg/ml Pam3CSK4 (P3C), or 100 nM CpG for 6 h and assayed by Northern blot analysis with a miR-155-specific probe. (B) WT, MyD88–/–, or TRIF–/– macrophages were stimulated with medium, poly(I:C), LPS, CpG, or Pam3CSK4 for 6 h, and miR-155 expression was assayed by qPCR. (C) WT or IFNAR–/– macrophages were stimulated with medium, poly(I:C), LPS, CpG, or Pam3CSK4 for 6 h, and miR-155 expression was assayed by qPCR.

 

Figure 4
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Fig. 4. IFNs induce miR-155 expression through TNF-{alpha} autocrine/paracrine signaling. (A) WT (Wt) and TNFR1–/– murine macrophages were stimulated with medium (m), 1,000 units/ml IFN-beta, 50 ng/ml IFN-{gamma}, or 10 ng/ml TNF-{alpha} for 6 h, and miR-155 was assayed by qPCR. (B) (Left) WT macrophages were stimulated with medium, IFN-beta, or IFN-{gamma} for 6 h, and TNF-{alpha} mRNA was analyzed by qPCR. (Right) WT and TNFR1–/– macrophages were stimulated with IFN-beta for 6 h and assayed for IP10 mRNA expression by qPCR. (C) (Left) WT macrophages were stimulated with medium or 2 µg/ml poly(I:C) for 6 h and assayed for TNF-{alpha} expression by qPCR. (Right) WT and TNFR1–/– macrophages were stimulated with medium or poly(I:C) for 6 h, and miR-155 was assayed by qPCR.

 

Figure 5
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Fig. 5. Pharmacological inhibition of JNK blocks poly(I:C) and TNF-{alpha} induction of miR-155. WT murine macrophages were pretreated for 30 min with DMSO or sp600125 at 5 or 25 µg/ml and subsequently stimulated with medium, 2 µg/ml poly(I:C), or 10 ng/ml TNF-{alpha} in the presence of the vehicle or inhibitor. After 4 h, RNA was collected, and miR-155 expression was assayed by qPCR.

 


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