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MicroRNA Regulation of Growth Factor Receptor Signaling in Human Cancer Cells

  • Keith M. Giles
  • Andrew Barker
  • Priscilla M. Zhang
  • Michael R. Epis
  • Peter J. Leedman
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 676)

Abstract

Aberrant expression of the epidermal growth factor receptor (EGFR) and/or human epidermal growth factor receptor 2 (HER2) is a feature of many human tumors and is associated with disease progression, treatment resistance, and poor prognosis. Protein kinase B/Akt, an important downstream effector of these receptor tyrosine kinases, induces signaling pathways that control cancer cell proliferation, invasion, angiogenesis, and apoptosis resistance. MicroRNAs (miRNAs), small noncoding RNAs that bind to the 3′-untranslated region of target mRNAs, are now recognized to play key roles in the regulation of gene expression, particularly in tumor development and metastasis. We have shown that miRNA-7 (miR-7) and miRNA-331-3p (miR-331-3p) directly regulate expression of EGFR and HER2, respectively, in glioblastoma and prostate cancer cell lines. As a consequence, miR-7 and miR-331-3p reduce Akt activity and thus have the capacity to regulate a signaling pathway critical to the development and progression of glioblastoma and prostate cancer. This chapter provides a detailed approach outlining how to confirm that a putative target of a miRNA is a direct target, and subsequent assessment of downstream signaling mediators.

Key words

MicroRNA EGFR HER2 miR-7 miR-331-3p Glioblastoma Prostate cancer Transfection Immunoblotting Luciferase reporter gene assay 

Notes

Acknowledgments

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

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Keith M. Giles
    • 1
  • Andrew Barker
    • 1
  • Priscilla M. Zhang
    • 1
  • Michael R. Epis
    • 1
  • Peter J. Leedman
    • 2
  1. 1.Laboratory for Cancer Medicine, Centre for Medical Research, Western Australian Institute for Medical ResearchUniversity of Western AustraliaPerthAustralia
  2. 2.Laboratory for Cancer Medicine, Centre for Medical Research, School of Medicine and Pharmacologcxxy, Western Australian Institute for Medical ResearchUniversity of Western AustraliaPerthAustralia

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