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Identification of E6/E7-Dependent MicroRNAs in HPV-Positive Cancer Cells

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MicroRNA and Cancer

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1699))

Abstract

Oncogenic types of human papillomaviruses (HPVs) are closely linked to the development of anogenital and head and neck cancers . The expression of the viral E6 and E7 genes is crucial for the transforming activities of HPVs. There is accumulating evidence that the HPV E6/E7 oncogenes can profoundly affect the cellular microRNA (miRNA) composition. Since alterations of miRNA expression levels can contribute to cancer development and maintenance, it will be important to understand in depth the crosstalk between the HPV oncogenes and the cellular miRNA network . Here, we describe a method to identify E6/E7-dependent intracellular miRNAs by small RNA deep sequencing , upon silencing of endogenous E6/E7 expression in HPV-positive cancer cells in vitro. In addition, we provide a protocol to identify E6/E7-dependent miRNA alterations in exosomes that are secreted by HPV-positive cancer cells in vitro.

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References

  1. Schiller JT, Lowy DR (2012) Understanding and learning from the success of prophylactic human papillomavirus vaccines. Nat Rev Microbiol 10:681–692

    Article  CAS  PubMed  Google Scholar 

  2. American Cancer Society (2015) Global cancer facts and figures, 3rd edn. American Cancer Society, Atlanta

    Google Scholar 

  3. zur Hausen H (2002) Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2:342–350

    Article  CAS  PubMed  Google Scholar 

  4. McLaughlin-Drubin ME, Münger K (2009) Oncogenic activities of human papillomaviruses. Virus Res 143:195–208

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hoppe-Seyler F, Hoppe-Seyler K (2011) Emerging topics in human tumor virology. Int J Cancer 129:1289–1299

    Article  CAS  PubMed  Google Scholar 

  6. Di Leva G, Garofalo M, Croce CM (2014) MicroRNAs in cancer. Annu Rev Pathol 9:287–314

    Article  PubMed  Google Scholar 

  7. Schwarzenbach H, Nishida N, Calin GA et al (2014) Clinical relevance of circulating cell-free microRNAs in cancer. Nat Rev Clin Oncol 11:145–156

    Article  CAS  PubMed  Google Scholar 

  8. Kincaid RP, Sullivan CS (2012) Virus-encoded microRNAs: an overview and a look to the future. PLoS Pathog 8:e1003018. doi:10.1371/journal.ppat.1003018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Honegger A, Schilling D, Bastian S et al (2015) Dependence of intracellular and exosomal microRNAs on viral E6/E7 oncogene expression in HPV-positive tumor cells. PLoS Pathog 11:e1004712. doi:10.1371/journal.ppat.1004712

    Article  PubMed  PubMed Central  Google Scholar 

  10. Thierry F (2009) Transcriptional regulation of the papillomavirus oncogenes by cellular and viral transcription factors in cervical carcinoma. Virology 384:375–379

    Article  CAS  PubMed  Google Scholar 

  11. Colombo M, Raposo G, Théry C (2014) Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol 30:255–289

    Article  CAS  PubMed  Google Scholar 

  12. Valadi H, Ekström K, Bossios A et al (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9:654–659

    Article  CAS  PubMed  Google Scholar 

  13. Zhang J, Li S, Li L et al (2015) Exosome and exosomal microRNA: trafficking, sorting, and function. Genomics Proteomics Bioinformatics 13:17–24

    Article  PubMed  PubMed Central  Google Scholar 

  14. Properzi F, Logozzi M, Fais S (2013) Exosomes: the future of biomarkers in medicine. Biomark Med 7:769–778

    Article  CAS  PubMed  Google Scholar 

  15. Friedlander MR, Mackowiak SD, Li N et al (2012) miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades. Nucleic Acids Res 40:37–52

    Article  PubMed  Google Scholar 

  16. Griffiths-Jones S (2004) The microRNA registry. Nucleic Acids Res 32:D109–D111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Griffiths-Jones S, Grocock RJ, van Dongen S et al (2006) miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res 34:D140–D144

    Article  CAS  PubMed  Google Scholar 

  18. Griffiths-Jones S, Saini HK, van Dongen S et al (2008) miRBase: tools for microRNA genomics. Nucleic Acids Res 36:D154–D158

    Article  CAS  PubMed  Google Scholar 

  19. Kozomara A, Griffiths-Jones S (2011) miRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res 39:D152–D157

    Article  CAS  PubMed  Google Scholar 

  20. Butz K, Ristriani T, Hengstermann A et al (2003) siRNA targeting of the viral E6 oncogene efficiently kills human papillomavirus-positive cancer cells. Oncogene 22:5938–5945

    Article  CAS  PubMed  Google Scholar 

  21. Parsons BD, Schindler A, Evans DH et al (2009) A direct phenotypic comparison of siRNA pools and multiple individual duplexes in a functional assay. PLoS One 4:e8471

    Article  PubMed  PubMed Central  Google Scholar 

  22. Jackson AL, Linsley PS (2010) Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. Nat Rev Drug Discov 9:57–67

    Article  CAS  PubMed  Google Scholar 

  23. Scheffner M, Werness BA, Huibregtse JM et al (1990) The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63:1129–1136

    Article  CAS  PubMed  Google Scholar 

  24. el-Deiry WS, Tokino T, Velculescu VE et al (1993) WAF1, a potential mediator of p53 tumor suppression. Cell 75:817–825

    Article  CAS  PubMed  Google Scholar 

  25. McLaughlin-Drubin ME, Munger K (2009) The human papillomavirus E7 oncoprotein. Virology 384:335–344

    Article  CAS  PubMed  Google Scholar 

  26. Thery C, Amigorena S, Raposo G et al (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol 3(11.) 1-3.22.29

    Google Scholar 

  27. Honegger A, Leitz J, Bulkescher J et al (2013) Silencing of human papillomavirus (HPV) E6/E7 oncogene expression affects both the contents and the amounts of extracellular microvesicles released from HPV-positive cancer cells. Int J Cancer 133:1631–1642

    Article  CAS  PubMed  Google Scholar 

  28. Weischenfeldt J, Simon R, Feuerbach L et al (2013) Integrative genomic analyses reveal an androgen-driven somatic alteration landscape in early-onset prostate cancer. Cancer Cell 23:159–170

    Article  CAS  PubMed  Google Scholar 

  29. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  30. Witwer KW, Buzás EI, Bemis LT et al (2013) Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles 2:20360. doi:10.3402/jev.v2i0.20360

    Article  Google Scholar 

  31. Taylor DD, Gercel-Taylor C (2008) MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol Oncol 110:13–21

    Article  CAS  PubMed  Google Scholar 

  32. Mullokandov G, Baccarini A, Ruzo A et al (2012) High-throughput assessment of microRNA activity and function using microRNA sensor and decoy libraries. Nat Methods 9:840–846

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

The authors thank Julia Bulkescher, Claudia Lohrey, and Sandra Bastian for expert technical assistance, and Felicitas Bossler for helpful comments on the manuscript.

Conflict of interest: The authors declare no conflict of interest.

Author information

Authors and Affiliations

  1. Molecular Therapy of Virus-Associated Cancers (F065), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, Heidelberg, 69120, Germany

    Anja Honegger, Karin Hoppe-Seyler & Felix Hoppe-Seyler

  2. Cancer Genome Research (B063), German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Heidelberg, Germany

    Daniela Schilling & Holger Sültmann

Authors
  1. Anja Honegger
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  2. Daniela Schilling
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  3. Holger Sültmann
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  4. Karin Hoppe-Seyler
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  5. Felix Hoppe-Seyler
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Corresponding author

Correspondence to Felix Hoppe-Seyler .

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Editors and Affiliations

  1. Department of Medicine, Helen Diller Family Comprehensive Cancer Center, University of California in San Francisco, San Francisco, California, USA

    Wei Wu

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Honegger, A., Schilling, D., Sültmann, H., Hoppe-Seyler, K., Hoppe-Seyler, F. (2018). Identification of E6/E7-Dependent MicroRNAs in HPV-Positive Cancer Cells. In: Wu, W. (eds) MicroRNA and Cancer. Methods in Molecular Biology, vol 1699. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7435-1_10

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  • DOI: https://doi.org/10.1007/978-1-4939-7435-1_10

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7433-7

  • Online ISBN: 978-1-4939-7435-1

  • eBook Packages: Springer Protocols

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