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.

  • Letter
  • Published:

RNA interference–induced hepatotoxicity results from loss of the first synthesized isoform of microRNA-122 in mice

Abstract

Small RNAs can be engineered to target and eliminate expression of disease-causing genes or infectious viruses, resulting in the preclinical and clinical development of RNA interference (RNAi) therapeutics using these small RNAs1. To ensure the success of RNAi therapeutics, small hairpin RNAs (shRNAs) must co-opt sufficient quantities of the endogenous microRNA machinery to elicit efficient gene knockdown without impeding normal cellular function. We previously observed liver toxicity—including hepatocyte turnover, loss of gene repression and lethality2—in mice receiving high doses of a recombinant adeno-associated virus (rAAV) vector expressing shRNAs (rAAV-shRNAs); however the mechanism by which toxicity ensues has not been elucidated. Using rAAV-shRNAs we have now determined that hepatotoxicity arises when exogenous shRNAs exceed 12% of the total amount of liver microRNAs. After this threshold was surpassed, shRNAs specifically reduced the initially synthesized 22-nucleotide isoform of microRNA (miR)-122-5p without substantially affecting other microRNAs, resulting in functional de-repression of miR-122 target mRNAs. Delivery of a rAAV-shRNA vector expressing mature miR-122-5p could circumvent toxicity, despite the exogenous shRNA accounting for 70% of microRNAs. Toxicity was also not observed in Mir122–knockout mice regardless of the level or sequence of the shRNA. Our study establishes limits to the microRNA machinery that is available for therapeutic siRNAs and suggests new paradigms for the role of miR-122 in liver homeostasis in mice.

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: Evaluation of toxicity relative to efficacy of delivered shRNAs.
Figure 2: shRNAs specifically outcompete the 22-nt isoform of miR-122-5p.
Figure 3: miR-122 targets are specifically de-repressed in livers receiving toxic shRNAs.
Figure 4: Toxicity is abrogated by delivering a U6 construct expressing miR-122-5p or a U6-shRNA in Mir122-knockout mice.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

Referenced accessions

Gene Expression Omnibus

References

  1. Kay, M.A. State-of-the-art gene-based therapies: the road ahead. Nat. Rev. Genet. 12, 316–328 (2011).

    Article  CAS  PubMed  Google Scholar 

  2. Grimm, D. et al. Fatality in mice due to oversaturation of cellular microRNA–short hairpin RNA pathways. Nature 441, 537–541 (2006).

    Article  CAS  PubMed  Google Scholar 

  3. Lares, M.R., Rossi, J.J. & Ouellet, D.L. RNAi and small interfering RNAs in human disease therapeutic applications. Trends Biotechnol. 28, 570–579 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Maguire, A.M. et al. Age-dependent effects of RPE65 gene therapy for Leber's congenital amaurosis: a phase 1 dose-escalation trial. Lancet 374, 1597–1605 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Rodino-Klapac, L.R., Chicoine, L.G., Kaspar, B.K. & Mendell, J.R. Gene therapy for Duchenne muscular dystrophy: expectations and challenges. Arch. Neurol. 64, 1236–1241 (2007).

    Article  PubMed  Google Scholar 

  6. Nakai, H. et al. Large-scale molecular characterization of adeno-associated virus vector integration in mouse liver. J. Virol. 79, 3606–3614 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Grimm, D. et al. Argonaute proteins are key determinants of RNAi efficacy, toxicity and persistence in the adult mouse liver. J. Clin. Invest. 120, 3106–3119 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Sekine, S. et al. Disruption of Dicer1 induces dysregulated fetal gene expression and promotes hepatocarcinogenesis. Gastroenterology 136, 2304–2315.e1-4 (2009).

    Article  CAS  PubMed  Google Scholar 

  9. Hand, N.J., Master, Z.R., Le Lay, J. & Friedman, J.R. Hepatic function is preserved in the absence of mature microRNAs. Hepatology 49, 618–626 (2009).

    Article  CAS  PubMed  Google Scholar 

  10. Hsu, S.H. et al. Essential metabolic, anti-inflammatory and antitumorigenic functions of miR-122 in liver. J. Clin. Invest. 122, 2871–2883 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Tsai, W.C. et al. MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis. J. Clin. Invest. 122, 2884–2897 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Gu, S. et al. The loop position of shRNAs and pre-miRNAs is critical for the accuracy of dicer processing in vivo. Cell 151, 900–911 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Lagos-Quintana, M. et al. Identification of tissue-specific microRNAs from mouse. Curr. Biol. 12, 735–739 (2002).

    Article  CAS  PubMed  Google Scholar 

  14. Chang, J. et al. miR-122, a mammalian liver-specific microRNA, is processed from hcr mRNA and may downregulate the high-affinity cationic amino acid transporter CAT-1. RNA Biol. 1, 106–113 (2004).

    Article  CAS  PubMed  Google Scholar 

  15. Landgraf, P. et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 129, 1401–1414 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Katoh, T. et al. Selective stabilization of mammalian microRNAs by 3′ adenylation mediated by the cytoplasmic poly(A) polymerase GLD-2. Genes Dev. 23, 433–438 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Cross, N.C., Tolan, D.R. & Cox, T.M. Catalytic deficiency of human aldolase B in hereditary fructose intolerance caused by a common missense mutation. Cell 53, 881–885 (1988).

    Article  CAS  PubMed  Google Scholar 

  18. Luna, J.M. et al. Hepatitis C virus RNA functionally sequesters miR-122. Cell 160, 1099–1110 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Castoldi, M. et al. The liver-specific microRNA miR-122 controls systemic iron homeostasis in mice. J. Clin. Invest. 121, 1386–1396 (2011).

    Article  CAS  PubMed  PubMed Central  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  PubMed  Google Scholar 

  21. Gatfield, D. et al. Integration of microRNA miR-122 in hepatic circadian gene expression. Genes Dev. 23, 1313–1326 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  23. Xie, J. et al. Long-term, efficient inhibition of microRNA function in mice using rAAV vectors. Nat. Methods 9, 403–409 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hommes, F.A. & Draisma, M.I. The development of L- and M-type aldolases in rat liver. Biochim. Biophys. Acta 222, 251–252 (1970).

    Article  CAS  PubMed  Google Scholar 

  25. Numazaki, M., Tsutsumi, K., Tsutsumi, R. & Ishikawa, K. Expression of aldolase isozyme mRNAs in fetal rat liver. Eur. J. Biochem. 142, 165–170 (1984).

    Article  CAS  PubMed  Google Scholar 

  26. Bhattacharyya, S.N., Habermacher, R., Martine, U., Closs, E.I. & Filipowicz, W. Relief of microRNA-mediated translational repression in human cells subjected to stress. Cell 125, 1111–1124 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Kandathil, A.J. et al. Use of laser-capture microdissection to map hepatitis C virus–positive hepatocytes in human liver. Gastroenterology 145, 1404–13.e1-10 (2013).

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  29. Helwak, A., Kudla, G., Dudnakova, T. & Tollervey, D. Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding. Cell 153, 654–665 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Loeb, G.B. et al. Transcriptome-wide miR-155 binding map reveals widespread noncanonical microRNA targeting. Mol. Cell 48, 760–770 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Betel, D., Koppal, A., Agius, P., Sander, C. & Leslie, C. Comprehensive modeling of microRNA targets predicts functional nonconserved and noncanonical sites. Genome Biol. 11, R90 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Jenkins, D.D. et al. Donor-derived, liver-specific protein expression after bone marrow transplantation. Transplantation 78, 530–536 (2004).

    Article  CAS  PubMed  Google Scholar 

  33. 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  PubMed  Google Scholar 

  34. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Anders, S. & Huber, W. Differential expression analysis for sequence-count data. Genome Biol. 11, R106 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Selitsky, S.R. et al. Small tRNA-derived RNAs are increased and more abundant than microRNAs in chronic hepatitis B and C. Sci. Rep. 5, 7675 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Trapnell, C. et al. Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Huang, W., Sherman, B.T. & Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

    Article  CAS  Google Scholar 

  40. Huang, W., Sherman, B.T. & Lempicki, R.A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1–13 (2009).

    Article  CAS  Google Scholar 

  41. Bartonicek, N. & Enright, A.J. SylArray: a web server for automated detection of miRNA effects from expression data. Bioinformatics 26, 2900–2901 (2010).

    Article  CAS  PubMed  Google Scholar 

  42. Paraskevopoulou, M.D. et al. DIANA-microT web server v5.0: service integration into miRNA functional-analysis workflows. Nucleic Acids Res. 41, W169–W173 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Lu, J., Zhang, F. & Kay, M.A. A mini-intronic plasmid (MIP): a novel robust transgene expression vector in vivo and in vitro. Mol. Ther. 21, 954–963 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Bogerd, H.P., Whisnant, A.W., Kennedy, E.M., Flores, O. & Cullen, B.R. Derivation and characterization of Dicer- and microRNA-deficient human cells. RNA 20, 923–937 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by NIH grants R01DK078424 (M.A.K.), R01AI071068 (M.A.K.) and R01CA193244 (K.G.) and a Banting Postdoctoral Fellowship from the Canadian Institutes of Health Research (P.N.V.). We would like to thank all members of the Kay laboratory for input and suggestions, and H. Vogel for EM image analysis. Dicer1-knockout cells were kindly provided by B. Cullen (Duke University). We would like to thank the Stanford Functional Genomics Facility and the Stanford Center for Genomics and Personalized Medicine for high-throughput sequencing services.

Author information

Authors and Affiliations

Authors

Contributions

P.N.V., S.G., L.L. and M.A.K. conceived the project and designed the experiments. P.N.V., S.G., K.C., L.J., F.Z., Y.H. and L.L. performed the experiments. Y.Z. provided bioinformatics support. H.K. and K.G. provided the Mir122-knockout mice. P.N.V., S.G., E.M.M. and L.L. analyzed the data. P.N.V. and M.A.K. wrote the manuscript with critical review from S.G., E.M.M., K.G. and L.L., and input from all other coauthors.

Corresponding author

Correspondence to Mark A Kay.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Tables 1, 6–7 (PDF 2789 kb)

Supplementary Table 2

Total small RNA read counts per million mapped microRNAs (XLSX 97 kb)

Supplementary Table 3

Ago2 immunoprecipitated small RNA read counts per million mapped microRNAs (XLSX 88 kb)

Supplementary Table 4

RNA-seq FPKM values for U6-rAAV-shRNA mouse livers relative to control and H1-rAAV-shRNA livers (XLSX 987 kb)

Supplementary Table 5

RNA-seq FPKM values for additional U6-rAAV-shRNA mouse livers relative to control liver (XLSX 1241 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Valdmanis, P., Gu, S., Chu, K. et al. RNA interference–induced hepatotoxicity results from loss of the first synthesized isoform of microRNA-122 in mice. Nat Med 22, 557–562 (2016). https://doi.org/10.1038/nm.4079

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.4079

This article is cited by

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research