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MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells

Abstract

MicroRNAs are predicted to regulate thousands of mammalian genes, but relatively few targets have been experimentally validated and few microRNA loss-of-function phenotypes have been assigned. As an alternative to chemically modified antisense oligonucleotides, we developed microRNA inhibitors that can be expressed in cells, as RNAs produced from transgenes. Termed 'microRNA sponges', these competitive inhibitors are transcripts expressed from strong promoters, containing multiple, tandem binding sites to a microRNA of interest. When vectors encoding these sponges are transiently transfected into cultured cells, sponges derepress microRNA targets at least as strongly as chemically modified antisense oligonucleotides. They specifically inhibit microRNAs with a complementary heptameric seed, such that a single sponge can be used to block an entire microRNA seed family. RNA polymerase II promoter (Pol II)-driven sponges contain a fluorescence reporter gene for identification and sorting of sponge-treated cells. We envision the use of stably expressed sponges in animal models of disease and development.

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Figure 1: Design of microRNA sponges.
Figure 2: Efficacy of microRNA sponges.
Figure 3: Specificity of microRNA sponges.
Figure 4: Validation of microRNA targets.
Figure 5: Effect of sponges on microRNA levels.

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References

  1. Lewis, B.P., Burge, C.B. & Bartel, D.P. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15–20 (2005).

    Article  CAS  Google Scholar 

  2. Lu, J. et al. MicroRNA expression profiles classify human cancers. Nature 435, 834–838 (2005).

    Article  CAS  Google Scholar 

  3. Li, Q.J. et al. miR-181a is an intrinsic modulator of T cell sensitivity and selection. Cell 129, 147–161 (2007).

    Article  CAS  Google Scholar 

  4. Cheng, H.Y. et al. MicroRNA modulation of circadian-clock period and entrainment. Neuron 54, 813–829 (2007).

    Article  CAS  Google Scholar 

  5. Chang, T.C. et al. Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis. Mol. Cell 26, 745–752 (2007).

    Article  CAS  Google Scholar 

  6. He, L. et al. A microRNA polycistron as a potential human oncogene. Nature 435, 828–833 (2005).

    Article  CAS  Google Scholar 

  7. Care, A. et al. MicroRNA-133 controls cardiac hypertrophy. Nat. Med. 13, 613–618 (2007).

    Article  CAS  Google Scholar 

  8. Hutvagner, G., Simard, M.J., Mello, C.C. & Zamore, P.D. Sequence-specific inhibition of small RNA function. PLoS Biol. 2, e98 (2004).

    Article  Google Scholar 

  9. Meister, G., Landthaler, M., Dorsett, Y. & Tuschl, T. Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. RNA 10, 544–550 (2004).

    Article  CAS  Google Scholar 

  10. Orom, U.A., Kauppinen, S. & Lund, A.H. LNA-modified oligonucleotides mediate specific inhibition of microRNA function. Gene 372, 137–141 (2006).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  12. Davis, S., Lollo, B., Freier, S. & Esau, C. Improved targeting of miRNA with antisense oligonucleotides. Nucleic Acids Res. 34, 2294–2304 (2006).

    Article  CAS  Google Scholar 

  13. Paul, C.P. et al. Localized expression of small RNA inhibitors in human cells. Mol. Ther. 7, 237–247 (2003).

    Article  CAS  Google Scholar 

  14. Doench, J.G., Petersen, C.P. & Sharp, P.A. siRNAs can function as miRNAs. Genes Dev. 17, 438–442 (2003).

    Article  CAS  Google Scholar 

  15. Barad, O. et al. MicroRNA expression detected by oligonucleotide microarrays: system establishment and expression profiling in human tissues. Genome Res. 14, 2486–2494 (2004).

    Article  CAS  Google Scholar 

  16. O'Donnell, K.A., Wentzel, E.A., Zeller, K.I., Dang, C.V. & Mendell, J.T. c-Myc-regulated microRNAs modulate E2F1 expression. Nature 435, 839–843 (2005).

    Article  CAS  Google Scholar 

  17. Neilson, J.R., Zheng, G.X., Burge, C.B. & Sharp, P.A. Dynamic regulation of miRNA expression in ordered stages of cellular development. Genes Dev. 21, 578–589 (2007).

    Article  CAS  Google Scholar 

  18. Lewis, B.P., Shih, I.H., Jones-Rhoades, M.W., Bartel, D.P. & Burge, C.B. Prediction of mammalian microRNA targets. Cell 115, 787–798 (2003).

    Article  CAS  Google Scholar 

  19. John, B. et al. Human microRNA targets. PLoS Biol. 2, 363 (2004).

    Article  Google Scholar 

  20. Doench, J.G. & Sharp, P.A. Specificity of microRNA target selection in translational repression. Genes Dev. 18, 504–511 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was funded by US Public Health Service grants U19-AI056900 from the National Cancer Institute, by an Integrative Cancer Biology Program Grant U54 CA112967 from the National Institutes of Health to P.A.S. and partially by Cancer Center Support (core) P30-CA14051 from the National Cancer Institute. M.S.E. is supported by a Howard Hughes Medical Institute Predoctoral Fellowship and a Paul and Cleo Schimmel Scholarship. J.R.N. is supported by the Cancer Research Institute. We thank A. Garfinkel and M. Kumar for luciferase reporter preparations, A. Leung for assistance with fluorescence in situ hybridization, D. Engelke (University of Michigan) for the U6 vector and members of the Sharp laboratory for helpful discussions.

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Contributions

M.S.E. and J.R.N. conceived the experimental design and made the sponge constructs. M.S.E. performed the experiments and wrote the manuscript. P.A.S. supervised the work.

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Correspondence to Phillip A Sharp.

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The authors declare no competing financial interests.

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Supplementary Figures 1–4, Supplementary Tables 1–2, Supplementary Methods (PDF 2033 kb)

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Ebert, M., Neilson, J. & Sharp, P. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat Methods 4, 721–726 (2007). https://doi.org/10.1038/nmeth1079

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