Silencing of microRNAs in vivo with ‘antagomirs’ (original) (raw)

Nature volume 438, pages 685–689 (2005)Cite this article

Abstract

MicroRNAs (miRNAs) are an abundant class of non-coding RNAs that are believed to be important in many biological processes through regulation of gene expression1,2,3. The precise molecular function of miRNAs in mammals is largely unknown and a better understanding will require loss-of-function studies in vivo. Here we show that a novel class of chemically engineered oligonucleotides, termed ‘antagomirs’, are efficient and specific silencers of endogenous miRNAs in mice. Intravenous administration of antagomirs against miR-16, miR-122, miR-192 and miR-194 resulted in a marked reduction of corresponding miRNA levels in liver, lung, kidney, heart, intestine, fat, skin, bone marrow, muscle, ovaries and adrenals. The silencing of endogenous miRNAs by this novel method is specific, efficient and long-lasting. The biological significance of silencing miRNAs with the use of antagomirs was studied for miR-122, an abundant liver-specific miRNA. Gene expression and bioinformatic analysis of messenger RNA from antagomir-treated animals revealed that the 3′ untranslated regions of upregulated genes are strongly enriched in miR-122 recognition motifs, whereas downregulated genes are depleted in these motifs. Analysis of the functional annotation of downregulated genes specifically predicted that cholesterol biosynthesis genes would be affected by miR-122, and plasma cholesterol measurements showed reduced levels in antagomir-122-treated mice. Our findings show that antagomirs are powerful tools to silence specific miRNAs in vivo and may represent a therapeutic strategy for silencing miRNAs in disease.

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Acknowledgements

We thank J. Maraganore, V. Kotelianski and P. Sharp for discussion and suggestions. These studies were supported by NIH grants (to M.S., T.T. and N.R.), and an unrestricted grant from Bristol Myers Squibb (M.S.).

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Authors and Affiliations

  1. Laboratory of Metabolic Diseases, The Rockefeller University, 1230 York Avenue, New York, 10021, New York, USA
    Jan Krützfeldt & Markus Stoffel
  2. Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, 10021, New York, USA
    Thomas Tuschl
  3. Biology and Mathematics, Center for Comparative Functional Genomics, Department of Biology, New York University, New York, 10003, New York, USA
    Nikolaus Rajewsky
  4. Alnylam Pharmaceuticals Inc., 300 3rd Street, Massachusetts, 02142, Cambridge, USA
    Ravi Braich, Kallanthottathil G. Rajeev & Muthiah Manoharan

Authors

  1. Jan Krützfeldt
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  2. Nikolaus Rajewsky
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  3. Ravi Braich
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  4. Kallanthottathil G. Rajeev
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  5. Thomas Tuschl
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  6. Muthiah Manoharan
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  7. Markus Stoffel
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Corresponding author

Correspondence toMarkus Stoffel.

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Competing interests

M.S. serves on the Scientific Advisory Board and T.T. is a co-founder of Alnylam Pharmaceuticals, Inc. Some participating authors are employed by Alnylam Pharmaceuticals, Inc.

Supplementary information

Supplementary Figure 1

MiR-122 is quantitatively recovered from a duplex formed with antagomir-122. (PDF 64 kb)

Supplementary Figure 2

Annealing antagomir-122 to liver RNA in vitro does not alter miR-122 detection in formamide-containing gels. (PDF 126 kb)

Supplementary Figure 3

Sustained silencing of miR-122 after injection of antagomir-122 into mice. (PDF 217 kb)

Supplementary Figure 4

Antagomirs specifically target miRNAs that derive from the same primary transcript. (PDF 234 kb)

Supplementary Table 1

Affymetrix gene expression analysis of genes up-regulated ≥1.4-fold in livers of mice treated with antagomir-122 compared to control mm-antagomir-122. (XLS 123 kb)

Supplementary Table 2

Affymetrix gene expression analysis of genes down-regulated ≥1.4-fold in livers of mice treated with antagomir-122 compared to control mm-antagomir-122. Probe sets with "absent (A)" to "absent" changes were excluded. (XLS 98 kb)

Supplementary Notes

This contains Supplementary Figure Legends and Supplementary Notes. (DOC 24 kb)

Supplementary Methods

This file contains additional details of the methods used in this study. (DOC 33 kb)

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Krützfeldt, J., Rajewsky, N., Braich, R. et al. Silencing of microRNAs in vivo with ‘antagomirs’.Nature 438, 685–689 (2005). https://doi.org/10.1038/nature04303

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Editorial Summary

miRNAs acting naturally

The discovery of microRNAs (miRNAs), the non-coding RNAs thought to be involved in many biological processes, is changing our perception of gene regulation. Little is known about their function in mammalian systems in vivo, but a newly developed group of compounds that silences miRNAs in mice should provide a powerful tool for the study of their function — and a potential therapeutic strategy for silencing miRNAs in disease. These ‘antagomirs’ are chemically engineered oligonucleotides with sequences that complement natural miRNAs. Intravenous administration of antagomirs to miR-16, -122, -192 and -194 in mice caused a marked reduction of corresponding miRNA expression in liver, lung, kidney, heart, muscle, intestine, fat, skin, bone marrow, ovaries and adrenals. In worms and flies, miRNAs play important developmental roles in the embryo. In vertebrates, various developmental genes have been shown to be targets of miRNA regulation, but there were no examples of miRNAs playing specific roles in known developmental processes. Now one such example has been found: miR-196 acts in mouse embryos as a mechanism to ensure accurate expression of genes primarily regulated by Hoxb8 and Shh transcription factors. This supports the idea that vertebrate miRNAs may function as a secondary level of gene regulation.