RNA Interference-Mediated Gene Silencing in Esophageal Adenocarcinoma

  • Farhadul Islam
  • Vinod Gopalan
  • Alfred K. Lam
Part of the Methods in Molecular Biology book series (MIMB, volume 1756)


RNA interference (RNAi) is a normal physiological mechanism in which a short effector antisense RNA molecule regulates target gene expression. It is a powerful tool to silence a particular gene of interest in a sequence-specific manner and can be used to target against various molecular pathways in esophageal adenocarcinoma by designing RNAi targeting key pathogenic genes. RNAi-based therapeutics against esophageal adenocarcinoma can be developed using different strategies including inhibition of overexpressed oncogenes, blocking cell division by interfering cyclins and related genes or enhancing apoptosis by suppressing anti-apoptotic genes. In addition, RNAi against multidrug resistance genes or chemo-resistance targets may provide promising cancer therapeutic options. Here, we describe RNAi technology using MET, a proto-oncogene in esophageal adenocarcinoma cells, as a model target. Lentiviral particles expressing MET shRNA was used to silence MET genes. Then, Western blot analysis was performed to confirm MET knockdown.

Key words

RNAi Esophageal adenocarcinoma Western blot siRNA shRNAs 


  1. 1.
    Izquierdo M (2005) Short interfering RNAs as a tool for cancer gene therapy. Cancer Gene Ther 12:217–227CrossRefGoogle Scholar
  2. 2.
    Li CX, Parker A, Menocal E et al (2006) Delivery of RNA interference. Cell Cycle 5:2103–2109CrossRefGoogle Scholar
  3. 3.
    Saurabh S, Vidyarthi AS, Prasad D (2014) RNA interference: concept to reality in crop improvement. Planta 239:543–564CrossRefGoogle Scholar
  4. 4.
    Takeshita F, Ochiya T (2006) Therapeutic potential of RNA interference against cancer. Cancer Sci 97:689–696CrossRefGoogle Scholar
  5. 5.
    Fire A, Xu S, Montgomery MK et al (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811CrossRefGoogle Scholar
  6. 6.
    Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286:950–952CrossRefGoogle Scholar
  7. 7.
    Hayashi R, Schnabl J, Handler D et al (2016) Genetic and mechanistic diversity of piRNA 3′-end formation. Nature 539:588–592CrossRefGoogle Scholar
  8. 8.
    Siomi MC, Sato K, Pezic D et al (2011) PIWI-interacting small RNAs: the vanguard of genome defence. Nat Rev Mol Cell Biol 12:246–258CrossRefGoogle Scholar
  9. 9.
    Ahlquist P (2002) RNA-dependent RNA polymerases, viruses, and RNA silencing. Science 296:1270–1273CrossRefGoogle Scholar
  10. 10.
    Liu Q, Rand TA, Kalidas S et al (2003) R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway. Science 301:1921–1925CrossRefGoogle Scholar
  11. 11.
    Weh KM, Howell AB, Kresty LA (2016) Expression, modulation, and clinical correlates of the autophagy protein Beclin-1 in esophageal adenocarcinoma. Mol Carcinog 55:1876–1885CrossRefGoogle Scholar
  12. 12.
    Myers AL, Lin L, Nancarrow DJ et al (2015) IGFBP2 modulates the chemoresistant phenotype in esophageal adenocarcinoma. Oncotarget 6:25897–25916CrossRefGoogle Scholar
  13. 13.
    Beales IL, Ogunwobi OO (2010) Microsomal prostaglandin E synthase-1 inhibition blocks proliferation and enhances apoptosis in esophageal adenocarcinoma cells without affecting endothelial prostacyclin production. Int J Cancer 126:2247–2255PubMedGoogle Scholar
  14. 14.
    Hong J, Resnick M, Behar J et al (2011) Role of Rac1 in regulation of NOX5-S function in Barrett’s esophageal adenocarcinoma cells. Am J Physiol Cell Physiol 301:C413–C420CrossRefGoogle Scholar
  15. 15.
    Beales IL, Ogunwobi OO (2009) Glycine-extended gastrin inhibits apoptosis in Barrett’s esophageal and esophageal adenocarcinoma cells through JAK2/STAT3 activation. J Mol Endocrinol 42:305–318CrossRefGoogle Scholar
  16. 16.
    Kebenko M, Drenckhan A, Gros SJ et al (2015) ErbB2 signaling activates the Hedgehog pathway via PI3K-Akt in human esophageal adenocarcinoma: identification of novel targets for concerted therapy concepts. Cell Signal 27:373–381CrossRefGoogle Scholar
  17. 17.
    MacFarlane LA, Gu Y, Casson AG et al (2010) Regulation of fibroblast growth factor-2 by an endogenous antisense RNA and by argonaute-2. Mol Endocrinol 24:800–812CrossRefGoogle Scholar
  18. 18.
    Pierini R, Kroon PA, Guyot S et al (2008) The procyanidin-mediated induction of apoptosis and cell-cycle arrest in esophageal adenocarcinoma cells is not dependent on p21(Cip1/WAF1). Cancer Lett 270:234–241CrossRefGoogle Scholar
  19. 19.
    Rees JR, Onwuegbusi BA, Save VE (2006) In vivo and in vitro evidence for transforming growth factor-beta1-mediated epithelial to mesenchymal transition in esophageal adenocarcinoma. Cancer Res 66:9583–9590CrossRefGoogle Scholar
  20. 20.
    Wang Z, Hao Y, Lowe AW (2008) The adenocarcinoma-associated antigen, AGR2, promotes tumor growth, cell migration, and cellular transformation. Cancer Res 68:492–497CrossRefGoogle Scholar
  21. 21.
    Gentile A, Trusolino L, Comoglio PM (2008) The Met tyrosine kinase receptor in development and cancer. Cancer Metastasis Rev 27:85–94CrossRefGoogle Scholar
  22. 22.
    Tuynman JB, Lagarde SM, Ten Kate FJ et al (2008) Met expression is an independent prognostic risk factor in patients with esophageal adenocarcinoma. Br J Cancer 98:1102–1108CrossRefGoogle Scholar
  23. 23.
    Hack SP, Bruey JM, Koeppen H (2014) HGF/MET-directed therapeutics in gastresophageal cancer: a review of clinical and biomarker development. Oncotarget 5:2866–2880CrossRefGoogle Scholar
  24. 24.
    Jardim DL, de MeloGagliato D, Falchook GS et al (2014) MET aberrations and c-MET inhibitors in patients with gastric and esophageal cancers in a phase I unit. Oncotarget 5:1837–1845CrossRefGoogle Scholar
  25. 25.
    Mesteri I, Schoppmann SF, Preusser M et al (2014) Overexpression of CMET is associated with signal transducer and activator of transcription 3 activation and diminished prognosis in esophageal adenocarcinoma but not in squamous cell carcinoma. Eur J Cancer 50:1354–1360CrossRefGoogle Scholar
  26. 26.
    Lennerz JK, Kwak EL, Ackerman A et al (2011) MET amplification identifies a small and aggressive subgroup of esophagogastric adenocarcinoma with evidence of responsiveness to crizotinib. J Clin Oncol 29:4803–4810CrossRefGoogle Scholar
  27. 27.
    Watson GA, Zhang X, Stang MT et al (2006) Inhibition of c-Met as a therapeutic strategy for esophageal adenocarcinoma. Neoplasia 8:949–955CrossRefGoogle Scholar
  28. 28.
    Anderson MR, Harrison R, Atherfold PA et al (2006) Met receptor signaling: a key effector in esophageal adenocarcinoma. Clin Cancer Res 12:5936–5943CrossRefGoogle Scholar
  29. 29.
    Herrera LJ, El-Hefnawy T, Queiroz de Oliveira PE (2005) The HGF receptor c-Met is overexpressed in esophageal adenocarcinoma. Neoplasia 7:75–84CrossRefGoogle Scholar
  30. 30.
    Lockwood WW, Thu KL, Lin L et al (2012) Integrative genomics identified RFC3 as an amplified candidate oncogene in esophageal adenocarcinoma. Clin Cancer Res 18:1936–1946CrossRefGoogle Scholar
  31. 31.
    Lyros O, Rafiee P, Nie L et al (2015) Wnt/β-Catenin signaling activation beyond robust nuclear β-Catenin accumulation in non dysplastic Barrett’s esophagus: regulation via Dickkopf-1. Neoplasia 17:598–611CrossRefGoogle Scholar
  32. 32.
    Hong YS, Kim J, Pectasides E et al (2014) Src mutation induces acquired lapatinib resistance in ERBB2-amplified human gastresophageal adenocarcinoma models. PLoS One 9:e109440CrossRefGoogle Scholar
  33. 33.
    Aichler M, Elsner M, Ludyga N et al (2013) Clinical response to chemotherapy in esophageal adenocarcinoma patients is linked to defects in mitochondria. J Pathol 230:410–419CrossRefGoogle Scholar
  34. 34.
    Sims-Mourtada J, Izzo JG, Apisarnthanarax S et al (2006) Hedgehog: an attribute to tumor regrowth after chemoradiotherapy and a target to improve radiation response. Clin Cancer Res 12:6565–6572CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Farhadul Islam
    • 1
    • 2
  • Vinod Gopalan
    • 1
  • Alfred K. Lam
    • 1
  1. 1.Cancer Molecular Pathology of School of MedicineGriffith UniversityGold CoastAustralia
  2. 2.Department of Biochemistry and Molecular BiologyUniversity of RajshahiRajshahiBangladesh

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