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Evaluation of MicroRNA Delivery In Vivo

  • Rikki A. M. Brown
  • Kirsty L. Richardson
  • Felicity C. Kalinowski
  • Michael R. Epis
  • Jessica L. Horsham
  • Tasnuva D. Kabir
  • Marisa H. De Pinho
  • Dianne J. Beveridge
  • Lisa M. Stuart
  • Larissa C. Wintle
  • Peter J. LeedmanEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1699)

Abstract

MicroRNAs (miRNAs) are a family of short noncoding RNA molecules that fine-tune expression of mRNAs. Often their altered expression is associated with a number of diseases, including cancer. Given that miRNAs target multiple genes and “difficult to drug” oncogenes, they present attractive candidates to manipulate as an anti-cancer strategy. MicroRNA-7 (miR-7) is a tumor suppressor miRNA that has been shown to target oncogenes overexpressed in cancers, such as the epidermal growth factor receptor (EGFR) and the nuclear factor-κ B subunit, RelA. Here, we describe methods for evaluating systemic delivery of miR-7 using a lipid nanoparticle formulation in an animal model. The microRNA is delivered three times, over 1 week and tissues collected 24 h after the last injection. RNA and protein are extracted from snap frozen tissues and processed to detect miRNA distribution and subsequent assessment of downstream targets and signaling mediators, respectively. Importantly, variability in efficiency of miRNA delivery will be observed between organs of the same animal and also between animals. Additionally, delivering the microRNA to organs other than the liver, particularly the brain, remains challenging. Furthermore, large variation in miRNA targets is seen both within tissues and across tissues depending on the lysis buffer used for protein extraction. Therefore, analyzing protein expression is dependent upon the method used for isolation and requires optimization for each individual application. Together, these methods will provide a foundation for those planning on assessing the efficacy of delivery of a miRNA in vivo.

Key words

MicroRNA miR-7 Systemic microRNA delivery EGFR RelA Akt signaling RNA isolation Protein extraction Mouse tissues 

Notes

Acknowledgments

This work was supported by the National Health and Medical Research Council of Australia and the Cancer Council of Western Australia.

References

  1. 1.
    Lin H, Hannon GJ (2004) MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 5(7):522–531CrossRefGoogle Scholar
  2. 2.
    Kloosterman WP, Plasterk RHA (2006) The diverse functions of microRNAs in animal development and disease. Dev Cell 11(4):441–450CrossRefPubMedGoogle Scholar
  3. 3.
    Zhang B, Pan X, Cobb GP, Anderson TA (2007) microRNAs as oncogenes and tumor suppressors. Developmental biol 302(1):1–12CrossRefGoogle Scholar
  4. 4.
    Yamaguchi K, Carr BI, Nalesnik MA (1995) Concomitant expression of TGFα and EGF-R in human hepatoma. J Surg Oncol 58(4):240–245CrossRefPubMedGoogle Scholar
  5. 5.
    Ang KK, Berkey BA, Tu X, Zhang HZ, Katz R, Hammond EH, Fu KK, Milas L (2002) Impact of epidermal growth factor receptor expression on survival and pattern of relapse in patients with advanced head and neck carcinoma. Cancer Res 62(24):7350–7356PubMedGoogle Scholar
  6. 6.
    Normanno N, De Luca A, Bianco C, Strizzi L, Mancino M, Maiello MR, Carotenuto A, De Feo G, Caponigro F, Salomon DS (2006) Epidermal growth factor receptor (EGFR) signaling in cancer. Gene 366(1):2–16CrossRefPubMedGoogle Scholar
  7. 7.
    Tai DI, Tsai SL, Chang YH, Huang SN, Chen TC, Chang KS, Liaw YF (2000) Constitutive activation of nuclear factor κB in hepatocellular carcinoma. Cancer 89(11):2274–2281CrossRefPubMedGoogle Scholar
  8. 8.
    Ueda Y, Richmond A (2006) NF-κB activation in melanoma. Pigment Cell Res 19(2):112–124CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Dhawan P, Singh AB, Ellis DL, Richmond A (2002) Constitutive activation of Akt/protein kinase B in melanoma leads to up-regulation of nuclear factor-κB and tumor progression. Cancer Res 62(24):7335–7342PubMedGoogle Scholar
  10. 10.
    Kalinowski FC, Brown RA, Ganda C, Giles KM, Epis MR, Horsham J, Leedman PJ (2014) microRNA-7: a tumor suppressor miRNA with therapeutic potential. Int J Biol Chem 54:312–317Google Scholar
  11. 11.
    Horsham JL, Kalinowski FC, Epis MR, Ganda C, Brown RA, Leedman PJ (2015) Clinical potential of microRNA-7 in cancer. J Clin Med 4(9):1668–1687CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Kalinowski FC, Giles KM, Candy PA, Ali A, Ganda C, Epis MR, Webster RJ, Leedman PJ (2012) Regulation of epidermal growth factor receptor signaling and erlotinib sensitivity in head and neck cancer cells by miR-7. PLoS One 7(10):e47067CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Giles KM, Brown RA, Ganda C, Podgorny MJ, Candy PA, Wintle LC, Richardson KL, Kalinowski FC, Stuart LM, Epis MR, Haass NK, Herlyn M, Leedman PJ (2016) microRNA-7-5p inhibits melanoma cell proliferation and metastasis by suppressing RelA/NF-κB. Oncotarget 7(22):31663–31680CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Krützfeldt J (2016) Strategies to use microRNAs as therapeutic targets. Best Pract Res Clin Endocrinol Metab 30(5):551–561CrossRefPubMedGoogle Scholar
  15. 15.
    Robb T, Reid G, Blenkiron C (2017) Exploiting microRNAs as cancer therapeutics. Target Oncol:1–16Google Scholar
  16. 16.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 25(4):402–408CrossRefPubMedGoogle Scholar
  17. 17.
    Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4254CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Parkinson CM, O’Brien A, Albers TM, Simon MA, Clifford CB, Pritchett-Corning KR (2011) Diagnostic necropsy and selected tissue and sample collection in rats and mice. JoVE 54:2966. doi: 10.3791/2966 Google Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Rikki A. M. Brown
    • 1
  • Kirsty L. Richardson
    • 1
  • Felicity C. Kalinowski
    • 1
  • Michael R. Epis
    • 1
  • Jessica L. Horsham
    • 1
  • Tasnuva D. Kabir
    • 1
  • Marisa H. De Pinho
    • 1
  • Dianne J. Beveridge
    • 1
  • Lisa M. Stuart
    • 1
  • Larissa C. Wintle
    • 1
  • Peter J. Leedman
    • 1
    • 2
    Email author
  1. 1.Laboratory for Cancer Medicine, Harry Perkins Institute of Medical ResearchUniversity of Western Australia Centre for Medical ResearchNedlandsAustralia
  2. 2.School of Medicine and PharmacologyThe University of Western AustraliaNedlandsAustralia

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