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Knockdown Strategies for the Study of Proprotein Convertases and Proliferation in Prostate Cancer Cells

  • François D’Anjou
  • Frédéric Couture
  • Roxane Desjardins
  • Robert Day
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1103)

Abstract

Gene silencing strategies targeting mRNA are suitable methods to validate the functions of specific genes. In this chapter, we sought to compare two knockdown strategies for the study of proprotein convertases and proliferation in prostate cancer cells. We used both SOFA-HDV ribozyme and lentiviral-mediated shRNA delivery system to reduce PACE4 mRNA levels and validate its implication in the proliferation of DU145 prostate cancer cells. The cellular effects of PACE4 knockdown were assessed (1) in vitro using two tetrazolium salts (MTT and XTT assays) and (2) in vivo using a tumor xenograft approach in immunodeficient mice (Nu/Nu). Our results confirm the unique role of the proprotein convertase PACE4 in prostate cancer cell proliferation while demonstrating advantages and disadvantages of each approach. Achieving target validation in an effective manner is critical, as further development using a drug development approach is highly laborious and requires enormous resources.

Key words

HDV-ribozyme shRNA Lentivirus MTT XTT Xenografts Proprotein convertases Prostate cancer 

Notes

Acknowledgments

This work is funded by grants from the Canadian Institutes of Health Research (CIHR) and the Ministère du Développement Économique de l’Innovation et de l’Exportation (MDEIE) to RD. RD is a member of the Centre de Recherche Clinique Etienne-Le Bel (Sherbrooke, QC, Canada). FC holds a Graduate Scholarship from the Fonds de la Recherche Santé Québec (FRSQ).

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Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • François D’Anjou
    • 1
  • Frédéric Couture
    • 1
  • Roxane Desjardins
    • 1
  • Robert Day
    • 1
  1. 1.Département de chirurgie et service de urologie, Institut de pharmacologie de SherbrookeUniversité de SherbrookeSherbrookeCanada

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