Skip to main content
SpringerLink
Log in

Characterization of MicroRNAs Regulating FOXO Expression

  • Protocol
  • First Online:
FOXO Transcription Factors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1890))

Abstract

MicroRNAs are critical post-transcriptional regulators of a majority of genes, of which the FOXO family of transcription factors is no exception. Here, we describe generalizable methods, including 3’ UTR reporter assays and western blotting after microRNA manipulation, to test if a candidate miRNA (miR-182) directly targets a candidate (FOXO3) gene product. We also provide guidance on candidate miRNA selection and unbiased miRNA-target identification methods.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Hannenhalli S, Kaestner KH (2009) The evolution of Fox genes and their role in development and disease. Nat Rev Genet 10(4):233–240. https://doi.org/10.1038/nrg2523

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Link W, Fernandez-Marcos PJ (2017) FOXO transcription factors at the interface of metabolism and cancer. Int J Cancer 141(12):2379–2391. https://doi.org/10.1002/ijc.30840

    Article  CAS  PubMed  Google Scholar 

  3. Jackson BC, Carpenter C, Nebert DW, Vasiliou V (2010) Update of human and mouse forkhead box (FOX) gene families. Hum Genomics 4(5):345–352

    Article  CAS  Google Scholar 

  4. Eijkelenboom A, Burgering BM (2013) FOXOs: signalling integrators for homeostasis maintenance. Nat Rev Mol Cell Biol 14(2):83–97. https://doi.org/10.1038/nrm3507

    Article  CAS  PubMed  Google Scholar 

  5. Urbanek P, Klotz LO (2017) Posttranscriptional regulation of FOXO expression: microRNAs and beyond. Br J Pharmacol 174(12):1514–1532. https://doi.org/10.1111/bph.13471

    Article  CAS  PubMed  Google Scholar 

  6. Jonas S, Izaurralde E (2015) Towards a molecular understanding of microRNA-mediated gene silencing. Nat Rev Genet 16(7):421–433. https://doi.org/10.1038/nrg3965

    Article  CAS  PubMed  Google Scholar 

  7. Segura MF, Hanniford D, Menendez S, Reavie L, Zou X, Alvarez-Diaz S, Zakrzewski J, Blochin E, Rose A, Bogunovic D, Polsky D, Wei J, Lee P, Belitskaya-Levy I, Bhardwaj N, Osman I, Hernando E (2009) Aberrant miR-182 expression promotes melanoma metastasis by repressing FOXO3 and microphthalmia-associated transcription factor. Proc Natl Acad Sci U S A 106(6):1814–1819. https://doi.org/10.1073/pnas.0808263106

    Article  PubMed  PubMed Central  Google Scholar 

  8. Fendler A, Jung M, Stephan C, Erbersdobler A, Jung K, Yousef GM (2013) The antiapoptotic function of miR-96 in prostate cancer by inhibition of FOXO1. PLoS One 8(11):e80807. https://doi.org/10.1371/journal.pone.0080807

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Guo Y, Liu H, Zhang H, Shang C, Song Y (2012) miR-96 regulates FOXO1-mediated cell apoptosis in bladder cancer. Oncol Lett 4(3):561–565. https://doi.org/10.3892/ol.2012.775

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Haflidadottir BS, Larne O, Martin M, Persson M, Edsjo A, Bjartell A, Ceder Y (2013) Upregulation of miR-96 enhances cellular proliferation of prostate cancer cells through FOXO1. PLoS One 8(8):e72400. https://doi.org/10.1371/journal.pone.0072400

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hudson MB, Rahnert JA, Zheng B, Woodworth-Hobbs ME, Franch HA, Price SR (2014) miR-182 attenuates atrophy-related gene expression by targeting FoxO3 in skeletal muscle. Am J Physiol Cell Physiol 307(4):C314–C319. https://doi.org/10.1152/ajpcell.00395.2013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kim KM, Park SJ, Jung SH, Kim EJ, Jogeswar G, Ajita J, Rhee Y, Kim CH, Lim SK (2012) miR-182 is a negative regulator of osteoblast proliferation, differentiation, and skeletogenesis through targeting FoxO1. J Bone Miner Res 27(8):1669–1679. https://doi.org/10.1002/jbmr.1604

    Article  CAS  PubMed  Google Scholar 

  13. Liu Z, Liu J, Segura MF, Shao C, Lee P, Gong Y, Hernando E, Wei JJ (2012) MiR-182 overexpression in tumourigenesis of high-grade serous ovarian carcinoma. J Pathol 228(2):204–215. https://doi.org/10.1002/path.4000

    Article  CAS  PubMed  Google Scholar 

  14. Stittrich AB, Haftmann C, Sgouroudis E, Kuhl AA, Hegazy AN, Panse I, Riedel R, Flossdorf M, Dong J, Fuhrmann F, Heinz GA, Fang Z, Li N, Bissels U, Hatam F, Jahn A, Hammoud B, Matz M, Schulze FM, Baumgrass R, Bosio A, Mollenkopf HJ, Grun J, Thiel A, Chen W, Hofer T, Loddenkemper C, Lohning M, Chang HD, Rajewsky N, Radbruch A, Mashreghi MF (2010) The microRNA miR-182 is induced by IL-2 and promotes clonal expansion of activated helper T lymphocytes. Nat Immunol 11(11):1057–1062. https://doi.org/10.1038/ni.1945

    Article  CAS  PubMed  Google Scholar 

  15. Wallis CJ, Gordanpour A, Bendavid JS, Sugar L, Nam RK, Seth A (2015) MiR-182 Is associated with growth, migration and invasion in prostate cancer via suppression of FOXO1. J Cancer 6(12):1295–1305. https://doi.org/10.7150/jca.13176

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Xu X, Wu J, Li S, Hu Z, Xu X, Zhu Y, Liang Z, Wang X, Lin Y, Mao Y, Chen H, Luo J, Liu B, Zheng X, Xie L (2014) Downregulation of microRNA-182-5p contributes to renal cell carcinoma proliferation via activating the AKT/FOXO3a signaling pathway. Mol Cancer 13:109. https://doi.org/10.1186/1476-4598-13-109

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Choi WY, Giraldez AJ, Schier AF (2007) Target protectors reveal dampening and balancing of Nodal agonist and antagonist by miR-430. Science 318(5848):271–274. https://doi.org/10.1126/science.1147535

    Article  CAS  PubMed  Google Scholar 

  18. Chi SW, Zang JB, Mele A, Darnell RB (2009) Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 460(7254):479–486. https://doi.org/10.1038/nature08170

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Hafner M, Landthaler M, Burger L, Khorshid M, Hausser J, Berninger P, Rothballer A, Ascano M Jr, Jungkamp AC, Munschauer M, Ulrich A, Wardle GS, Dewell S, Zavolan M, Tuschl T (2010) Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141(1):129–141. https://doi.org/10.1016/j.cell.2010.03.009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Konig J, Zarnack K, Rot G, Curk T, Kayikci M, Zupan B, Turner DJ, Luscombe NM, Ule J (2010) iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol 17(7):909–915. https://doi.org/10.1038/nsmb.1838

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Licatalosi DD, Mele A, Fak JJ, Ule J, Kayikci M, Chi SW, Clark TA, Schweitzer AC, Blume JE, Wang X, Darnell JC, Darnell RB (2008) HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 456(7221):464–469. https://doi.org/10.1038/nature07488

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Grosswendt S, Filipchyk A, Manzano M, Klironomos F, Schilling M, Herzog M, Gottwein E, Rajewsky N (2014) Unambiguous identification of miRNA:target site interactions by different types of ligation reactions. Mol Cell 54(6):1042–1054. https://doi.org/10.1016/j.molcel.2014.03.049

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Helwak A, Kudla G, Dudnakova T, Tollervey D (2013) Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding. Cell 153(3):654–665. https://doi.org/10.1016/j.cell.2013.03.043

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pathology, New York University Langone Health, New York, NY, USA

    Doug Hanniford & Eva Hernando

  2. Interdisciplinary Melanoma Cooperative Group, New York University Langone Health, New York, NY, USA

    Doug Hanniford & Eva Hernando

Authors
  1. Doug Hanniford
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eva Hernando
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Eva Hernando .

Editor information

Editors and Affiliations

  1. Instituto de Investigaciones Biomédicas “Alberto Sols” (CSIC-UAM), Madrid, Spain

    Wolfgang Link

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Hanniford, D., Hernando, E. (2019). Characterization of MicroRNAs Regulating FOXO Expression. In: Link, W. (eds) FOXO Transcription Factors. Methods in Molecular Biology, vol 1890. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-8900-3_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8900-3_2

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-8899-0

  • Online ISBN: 978-1-4939-8900-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation