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In Vitro Evaluation of Candidate Gene Targets for Cancer Therapy

  • Xing Fei Tan
  • Wei Xuan Teo
  • George W. YipEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1974)

Abstract

Discovery and development of gene targets for cancer therapeutics are lengthy and highly costly processes. Identification and evaluation of candidate gene targets are of fundamental importance. RNA interference allows candidate genes to be specifically and effectively knocked down in cancer cells. This tool can be easily incorporated into a loss-of-function approach in the initial evaluation of candidate gene targets for cancer treatment prior to moving on to animal studies and clinical trials. This chapter describes a relatively simple and straightforward protocol that makes use of small interfering RNA to achieve knockdown of the candidate gene target and to evaluate the resultant effects on four aspects of cancer cell behavior: migration, invasion, proliferation, and adhesion.

Keywords

siRNA Breast cancer Migration Invasion Proliferation Adhesion 

Notes

Acknowledgment

We would like to thank S. L. Bay for her help in preparing the illustrations and C. X. Ng for contributing images for Fig. 3. Work in the laboratory of G. W. Yip is supported by Grants NMRC/CSA/0041/2012 and NMRC/CIRG/1436/2015 from the National Medical Research Council, Singapore.

References

  1. 1.
    Dana H, Chalbatani GM, Mahmoodzadeh H, Karimloo R, Rezaiean O, Moradzadeh A et al (2017) Molecular mechanisms and biological functions of siRNA. Int J Biomed Sci 13(2):48–57PubMedPubMedCentralGoogle Scholar
  2. 2.
    Iravani O, Bay BH, Yip GW (2017) Silencing HS6ST3 inhibits growth and progression of breast cancer cells through suppressing IGF1R and inducing XAF1. Exp Cell Res 350(2):380–389CrossRefPubMedGoogle Scholar
  3. 3.
    Schlabach MR, Luo J, Solimini NL, Hu G, Xu Q, Li MZ et al (2008) Cancer proliferation gene discovery through functional genomics. Science 319(5863):620–624CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Ahmadzada T, Reid G, McKenzie DR (2018) Fundamentals of siRNA and miRNA therapeutics and a review of targeted nanoparticle delivery systems in breast cancer. Biophys Rev 10(1):69–86CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Chen X, Mangala LS, Rodriguez-Aguayo C, Kong X, Lopez-Berestein G, Sood AK (2018) RNA interference-based therapy and its delivery systems. Cancer Metastasis Rev 37(1):107–124CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Lai Y, Lim D, Tan PH, Leung TK, Yip GW, Bay BH (2010) Silencing the metallothionein-2A gene induces entosis in adherent MCF-7 breast cancer cells. Anat Rec (Hoboken) 293(10):1685–1691CrossRefGoogle Scholar
  7. 7.
    Vijaya Kumar A, Salem Gassar E, Spillmann D, Stock C, Sen YP, Zhang T et al (2014) HS3ST2 modulates breast cancer cell invasiveness via MAP kinase- and Tcf4 (Tcf7l2)-dependent regulation of protease and cadherin expression. Int J Cancer 135(11):2579–2592CrossRefPubMedGoogle Scholar
  8. 8.
    Roussos ET, Condeelis JS, Patsialou A (2011) Chemotaxis in cancer. Nat Rev Cancer 11(8):573–587CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Hall DM, Brooks SA (2014) In vitro invasion assay using matrigel: a reconstituted basement membrane preparation. Methods Mol Biol 1070:1–11CrossRefPubMedGoogle Scholar
  10. 10.
    Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144(5):646–674CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100(1):57–70CrossRefPubMedGoogle Scholar
  12. 12.
    Cory AH, Owen TC, Barltrop JA, Cory JG (1991) Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Commun 3(7):207–212CrossRefPubMedGoogle Scholar
  13. 13.
    Oskarsson T (2013) Extracellular matrix components in breast cancer progression and metastasis. Breast 22(Suppl 2):S66–S72CrossRefPubMedGoogle Scholar
  14. 14.
    (2003) Whither RNAi? Nat Cell Biol 5(6):489–490Google Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Department of Anatomy, Yong Loo Lin School of MedicineNational University of SingaporeSingaporeSingapore

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