Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Technical Report
  • Published:

Silencing of microRNA families by seed-targeting tiny LNAs

Subjects

Abstract

The challenge of understanding the widespread biological roles of animal microRNAs (miRNAs) has prompted the development of genetic and functional genomics technologies for miRNA loss-of-function studies. However, tools for exploring the functions of entire miRNA families are still limited. We developed a method that enables antagonism of miRNA function using seed-targeting 8-mer locked nucleic acid (LNA) oligonucleotides, termed tiny LNAs. Transfection of tiny LNAs into cells resulted in simultaneous inhibition of miRNAs within families sharing the same seed with concomitant upregulation of direct targets. In addition, systemically delivered, unconjugated tiny LNAs showed uptake in many normal tissues and in breast tumors in mice, coinciding with long-term miRNA silencing. Transcriptional and proteomic profiling suggested that tiny LNAs have negligible off-target effects, not significantly altering the output from mRNAs with perfect tiny LNA complementary sites. Considered together, these data support the utility of tiny LNAs in elucidating the functions of miRNA families in vivo.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Schematic overview of the miRNA silencing approach using seed-targeting tiny LNAs.
Figure 2: Inhibition of miR-21 function by tiny antimiR-21.
Figure 3: Silencing of miRNA families by seed-targeting tiny LNAs in cultured cells.
Figure 4: Silencing of miR-21 in vivo by tiny antimiR-21.
Figure 5: Silencing of miR-122 in the mouse liver by seed-targeting tiny LNA.
Figure 6: Off-target analysis.

Similar content being viewed by others

Accession codes

Accessions

ArrayExpress

References

  1. Filipowicz, W., Bhattacharyya, S.N. & Sonenberg, N. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat. Rev. Genet. 9, 102–114 (2008).

    Article  CAS  Google Scholar 

  2. Bartel, D.P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233 (2009).

    Article  CAS  Google Scholar 

  3. Bushati, N. & Cohen, S.M. microRNA functions. Annu. Rev. Cell Dev. Biol. 23, 175–205 (2007).

    Article  CAS  Google Scholar 

  4. Gottwein, E. & Cullen, B.R. Viral and cellular microRNAs as determinants of viral pathogenesis and immunity. Cell Host Microbe 3, 375–387 (2008).

    Article  CAS  Google Scholar 

  5. Williams, A.H., Liu, N., van Rooij, E. & Olson, E.N. MicroRNA control of muscle development and disease. Curr. Opin. Cell Biol. 21, 461–469 (2009).

    Article  CAS  Google Scholar 

  6. Ventura, A. & Jacks, T. MicroRNAs and cancer: short RNAs go a long way. Cell 136, 586–591 (2009).

    Article  CAS  Google Scholar 

  7. Lim, L.P. et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433, 769–773 (2005).

    Article  CAS  Google Scholar 

  8. Baek, D. et al. The impact of microRNAs on protein output. Nature 455, 64–71 (2008).

    Article  CAS  Google Scholar 

  9. Selbach, M. et al. Widespread changes in protein synthesis induced by microRNAs. Nature 455, 58–63 (2008).

    Article  CAS  Google Scholar 

  10. Abbott, A.L. et al. The let-7 MicroRNA family members mir-48, mir-84, and mir-241 function together to regulate developmental timing in Caenorhabditis elegans. Dev. Cell 9, 403–414 (2005).

    Article  CAS  Google Scholar 

  11. Sokol, N.S. & Ambros, V. Mesodermally expressed Drosophila microRNA-1 is regulated by Twist and is required in muscles during larval growth. Genes Dev. 19, 2343–2354 (2005).

    Article  CAS  Google Scholar 

  12. Miska, E.A. et al. Most Caenorhabditis elegans microRNAs are individually not essential for development or viability. PLoS Genet. 3, e215 (2007).

    Article  Google Scholar 

  13. Ventura, A. et al. Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters. Cell 132, 875–886 (2008).

    Article  CAS  Google Scholar 

  14. van Rooij, E. et al. Control of stress-dependent cardiac growth and gene expression by a microRNA. Science 316, 575–579 (2007).

    Article  CAS  Google Scholar 

  15. Mu, P. et al. Genetic dissection of the miR-1792 cluster of microRNAs in Myc-induced B-cell lymphomas. Genes Dev. 23, 2806–2811 (2009).

    Article  CAS  Google Scholar 

  16. van Rooij, E. et al. A family of microRNAs encoded by myosin genes governs myosin expression and muscle performance. Dev. Cell 17, 662–673 (2009).

    Article  CAS  Google Scholar 

  17. Ebert, M.S., Neilson, J.R. & Sharp, P.A. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat. Methods 4, 721–726 (2007).

    Article  CAS  Google Scholar 

  18. Loya, C.M., Lu, C.S., Van, V.D. & Fulga, T.A. Transgenic microRNA inhibition with spatiotemporal specificity in intact organisms. Nat. Methods 6, 897–903 (2009).

    Article  CAS  Google Scholar 

  19. Gentner, B. et al. Stable knockdown of microRNA in vivo by lentiviral vectors. Nat. Methods 6, 63–66 (2009).

    Article  CAS  Google Scholar 

  20. Esau, C. et al. miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metab. 3, 87–98 (2006).

    Article  CAS  Google Scholar 

  21. Elmen, J. et al. Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver. Nucleic Acids Res. 36, 1153–1162 (2008).

    Article  CAS  Google Scholar 

  22. Horwich, M.D. & Zamore, P.D. Design and delivery of antisense oligonucleotides to block microRNA function in cultured Drosophila and human cells. Nat. Protoc. 3, 1537–1549 (2008).

    Article  CAS  Google Scholar 

  23. Krutzfeldt, J. et al. Silencing of microRNAs in vivo with 'antagomirs'. Nature 438, 685–689 (2005).

    Article  Google Scholar 

  24. Elmen, J. et al. LNA-mediated microRNA silencing in non-human primates. Nature 452, 896–899 (2008).

    Article  CAS  Google Scholar 

  25. Worm, J. et al. Silencing of microRNA-155 in mice during acute inflammatory response leads to derepression of c/ebp Beta and down-regulation of G-CSF. Nucleic Acids Res. 37, 5784–5792 (2009).

    Article  CAS  Google Scholar 

  26. Lanford, R.E. et al. Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science 327, 198–201 (2010).

    Article  CAS  Google Scholar 

  27. Ma, L. et al. Therapeutic silencing of miR-10b inhibits metastasis in a mouse mammary tumor model. Nat. Biotechnol. 28, 341–347 (2010).

    Article  CAS  Google Scholar 

  28. Robertson, B. et al. Specificity and functionality of microRNA inhibitors. Silence 1, 10 (2010).

    Article  Google Scholar 

  29. Zheng, G., Ambros, V. & Li, W.H. Inhibiting miRNA in Caenorhabditis elegans using a potent and selective antisense reagent. Silence 1, 9 (2010).

    Article  Google Scholar 

  30. Krichevsky, A.M. & Gabriely, G. miR-21: a small multi-faceted RNA. J. Cell. Mol. Med. 13, 39–53 (2009).

    Article  CAS  Google Scholar 

  31. Frankel, L.B. et al. Programmed cell death 4 (PDCD4) is an important functional target of the microRNA miR-21 in breast cancer cells. J. Biol. Chem. 283, 1026–1033 (2008).

    Article  CAS  Google Scholar 

  32. Liu, M. et al. Regulation of the cell cycle gene, BTG2, by miR-21 in human laryngeal carcinoma. Cell Res. 19, 828–837 (2009).

    Article  CAS  Google Scholar 

  33. Stein, C.A. et al. Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents. Nucleic Acids Res. 38, e3 (2010).

    Article  CAS  Google Scholar 

  34. le Sage, C. et al. Regulation of the p27(Kip1) tumor suppressor by miR-221 and miR-222 promotes cancer cell proliferation. EMBO J. 26, 3699–3708 (2007).

    Article  CAS  Google Scholar 

  35. Galardi, S. et al. miR-221 and miR-222 expression affects the proliferation potential of human prostate carcinoma cell lines by targeting p27Kip1. J. Biol. Chem. 282, 23716–23724 (2007).

    Article  CAS  Google Scholar 

  36. Reinhart, B.J. et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403, 901–906 (2000).

    Article  CAS  Google Scholar 

  37. Johnson, C.D. et al. The let-7 microRNA represses cell proliferation pathways in human cells. Cancer Res. 67, 7713–7722 (2007).

    Article  CAS  Google Scholar 

  38. Mayr, C., Hemann, M.T. & Bartel, D.P. Disrupting the pairing between let-7 and Hmga2 enhances oncogenic transformation. Science 315, 1576–1579 (2007).

    Article  CAS  Google Scholar 

  39. Pulaski, B.A., Clements, V.K., Pipeling, M.R. & Ostrand-Rosenberg, S. Immunotherapy with vaccines combining MHC class II/CD80+ tumor cells with interleukin-12 reduces established metastatic disease and stimulates immune effectors and monokine induced by interferon gamma. Cancer Immunol. Immunother. 49, 34–45 (2000).

    Article  CAS  Google Scholar 

  40. van Dongen, S., Abreu-Goodger, C. & Enright, A.J. Detecting microRNA binding and siRNA off-target effects from expression data. Nat. Methods 5, 1023–1025 (2008).

    Article  CAS  Google Scholar 

  41. Wang, Y., Sheng, G., Juranek, S., Tuschl, T. & Patel, D.J. Structure of the guide-strand-containing argonaute silencing complex. Nature 456, 209–213 (2008).

    Article  CAS  Google Scholar 

  42. Wang, Y. et al. Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex. Nature 456, 921–926 (2008).

    Article  CAS  Google Scholar 

  43. Vester, B. & Wengel, J. LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. Biochemistry 43, 13233–13241 (2004).

    Article  CAS  Google Scholar 

  44. Grimson, A. et al. MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol. Cell 27, 91–105 (2007).

    Article  CAS  Google Scholar 

  45. Chi, S.W., Zang, J.B., Mele, A. & Darnell, R.B. Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 460, 479–486 (2009).

    Article  CAS  Google Scholar 

  46. Kumar, M.S. et al. Suppression of non-small cell lung tumor development by the let-7 microRNA family. Proc. Natl. Acad. Sci. USA 105, 3903–3908 (2008).

    Article  CAS  Google Scholar 

  47. Miyoshi, K., Uejima, H., Nagami-Okada, T., Siomi, H. & Siomi, M.C. In vitro RNA cleavage assay for Argonaute-family proteins. Methods Mol. Biol. 442, 29–43 (2008).

    Article  CAS  Google Scholar 

  48. Broom, O.J., Zhang, Y., Oldenborg, P.A., Massoumi, R. & Sjolander, A. CD47 regulates collagen I-induced cyclooxygenase-2 expression and intestinal epithelial cell migration. PLoS ONE 4, e6371 (2009).

    Article  Google Scholar 

  49. Straarup, E.M. et al. Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates. Nucleic Acids Res. 38, 7100–7111 (2010).

    Article  CAS  Google Scholar 

  50. Dai, M. et al. Evolving gene/transcript definitions significantly alter the interpretation of GeneChip data. Nucleic Acids Res. 33, e175 (2005).

    Article  Google Scholar 

  51. Ross, P.L. et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell. Proteomics 3, 1154–1169 (2004).

    Article  CAS  Google Scholar 

  52. Horth, P., Miller, C.A., Preckel, T. & Wenz, C. Efficient fractionation and improved protein identification by peptide OFFGEL electrophoresis. Mol. Cell. Proteomics 5, 1968–1974 (2006).

    Article  Google Scholar 

  53. Taylor, P. et al. Automated 2D peptide separation on a 1D nano-LC-MS system. J. Proteome Res. 8, 1610–1616 (2009).

    Article  CAS  Google Scholar 

  54. Bantscheff, M. et al. Robust and sensitive iTRAQ quantification on an LTQ Orbitrap mass spectrometer. Mol. Cell. Proteomics 7, 1702–1713 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors wish to thank A. Koustrup, H. Brostrøm, A. Konge, J.J. Jørgensen, M. R. Møller, L. Bang and M. Meldgaard for excellent technical assistance, and A. Lund and T. Bou-Kheir for reagents. This study was supported by grants from the Danish National Advanced Technology Foundation and the Danish Medical Research Council to S.K.

Author information

Authors and Affiliations

Authors

Contributions

S.O., C.O.d.S., A.P., M.H., O.B., C.R., C.F. and E.M.S. performed experiments and contributed data. H.F.H. performed synthesis of oligonucleotides. S.O., C.O.d.S., A.P., M.H., M.L., J.S., T.K., D.P., G.J.H. and S.K. designed experiments and discussed the data. S.K. supervised the study and wrote the manuscript together with S.O. and with input from other authors.

Corresponding author

Correspondence to Sakari Kauppinen.

Ethics declarations

Competing interests

S.O., O.B., A.P., M.H., M.L., J.S., E.M.S., H.F.H., T.K. and S.K. are employees of Santaris Pharma, a clinical stage biopharmaceutical company that develops RNA-based therapeutics.

Supplementary information

Supplementary Text and Figures

Supplementary Note, Supplementary Figures 1–14 and Supplementary Tables 1–5. (PDF 2588 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Obad, S., dos Santos, C., Petri, A. et al. Silencing of microRNA families by seed-targeting tiny LNAs. Nat Genet 43, 371–378 (2011). https://doi.org/10.1038/ng.786

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng.786

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing