Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.
Subscribe

Logo for

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
View larger version (30K):
[in this window]
[in a new window]

  		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
View larger version (42K):
[in this window]
[in a new window]

  		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
View larger version (36K):
[in this window]
[in a new window]

  		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
View larger version (45K):
[in this window]
[in a new window]

  		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
View larger version (34K):
[in this window]
[in a new window]

  		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.

 

To Advertise     Find Products

Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882