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siRNA and shRNA as Anticancer Agents in a Cervical Cancer Model

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Book cover RNAi

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 442))

Summary

We describe the protocols of using siRNAs, or shRNAs delivered by a lentiviral vector, as a means to silence cancer-causing genes. We use cervical cancer as a model to demonstrate the inhibition of the human papillomavirus (HPV) oncogenes E6 and E7 in cervical cancer cells by RNAi and inhibition of the cell growth in vitro and tumor growth in mouse models. The protocols include methods on siRNA and shRNA design, production of lentiviral-vectored shRNA, transfection or transduction of cervical cancer cells with siRNA or shRNA, and detection of the inhibitory effects of siRNA or shRNA both in vitro and in vitro.

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References

  1. Bitko, V., Musiyenko, A., Shulyayeva, O., and Barik, S. (2005). Inhibition of respiratory viruses by nasally administered siRNA. Nat. Med. 11, 50–55.

    Article  CAS  PubMed  Google Scholar 

  2. Capodici, J., Kariko, K., and Weissman, D. (2002). Inhibition of HIV-1 infection by small interfering RNA-mediated RNA interference. J. Immunol. 169, 5196–5201.

    PubMed  Google Scholar 

  3. Dasgupta, R., and Perrimon, N. (2004). Using RNAi to catch Drosophila genes in a web of interactions: Insights into cancer research. Oncogene 23, 8359–8365.

    Article  CAS  PubMed  Google Scholar 

  4. Hannon, G. J., and Rossi, J. J. (2004). Unlocking the potential of the human genome with RNA interference. Nature 431, 371–378.

    Article  CAS  PubMed  Google Scholar 

  5. Jacque, J. M., Triques, K., and Stevenson, M. (2002). Modulation of HIV-1 replication by RNA interference. Nature 418, 435–438.

    Article  CAS  PubMed  Google Scholar 

  6. Meister, G., and Tuschl, T. (2004). Mechanisms of gene silencing by double-stranded RNA. Nature 431, 343–349.

    Article  CAS  PubMed  Google Scholar 

  7. Check, E. (2005). Pioneering HIV treatment would use interference and gene therapy. Nature 437, 601.

    Article  CAS  PubMed  Google Scholar 

  8. Humeau, L. M., Binder, G. K., Lu, X., et al. (2004). Efficient lentiviral vector-mediated control of HIV-1 replication in CD4 lymphocytes from diverse HIV+ infected patients grouped according to CD4 count and viral load. Mol. Ther. 9, 902–913.

    Article  CAS  PubMed  Google Scholar 

  9. Scherr, M., and Eder, M. (2002). Gene transfer into hematopoietic stem cells using lentiviral vectors. Curr. Gene Ther. 2, 45–55.

    Article  CAS  PubMed  Google Scholar 

  10. Check, E. (2005). A crucial test. Nat. Med. 11, 243–244.

    Article  CAS  PubMed  Google Scholar 

  11. Raoul, C., Abbas-Terki, T., Bensadoun, J. C., et al. (2005). Lentiviral-mediated silencing of SOD1 through RNA interference retards disease onset and progression in a mouse model of ALS. Nat. Med. 11, 423–428.

    Article  CAS  PubMed  Google Scholar 

  12. Ralph, G. S., Radcliffe, P. A., Day, D. M., et al. (2005). Silencing mutant SOD1 using RNAi protects against neurodegeneration and extends survival in an ALS model. Nat. Med. 11, 429–433.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  14. DiPaolo, J. A., and Alvarez-Salas, L. M. (2004). Advances in the development of therapeutic nucleic acids against cervical cancer. Expert Opin. Biol. Ther. 4, 1251–1264.

    CAS  Google Scholar 

  15. Parkin, D. M., Bray, F. I., and Devesa, S. S. (2001). Cancer burden in the year 2000. The global picture. Eur. J. Cancer 37, S4–S66.

    Article  PubMed  Google Scholar 

  16. Goodwin, E. C., and DiMaio, D. (2000). Repression of human papillomavirus oncogenes in HeLa cervical carcinoma cells causes the orderly reactivation of dormant tumor suppressor pathways. Proc. Natl. Acad. Sci. USA 97, 12513–12518.

    Google Scholar 

  17. Munger, K., Werness, B. A., Dyson, N., Phelps, W. C., Harlow, E., and Howley, P. M. (1989). Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product. EMBO J. 8, 4099–4105.

    CAS  PubMed  Google Scholar 

  18. von Knebel Doeberitz, M., Oltersdorf, T., Schwarz, E., and Gissmann, L. (1988). Correlation of modified human papilloma virus early gene expression with altered growth properties in C4–1 cervical carcinoma cells. Cancer Res. 48, 3780–3786.

    Google Scholar 

  19. Putral, L., Bywater, M., Gu, W., et al. (2005). RNAi against HPV oncogenes in cervical cancer cells results in increased sensitivity to cisplatin. Mol. Pharmacol. 68, 1311–1319.

    Article  CAS  PubMed  Google Scholar 

  20. Butz, K., Ristriani, T., Hengstermann, A., Denk, C., Scheffner, M., and Hoppe-Seyler, F. (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. Hall, A. H., and Alexander, K. A. (2003). RNA interference of human papillomavirus type 18 E6 and E7 induces senescence in HeLa cells. J. Virol. 77, 6066–6069.

    Article  CAS  PubMed  Google Scholar 

  22. Jiang, M., and Milner, J. (2002). Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference. Oncogene 21, 6041–6048.

    Article  CAS  PubMed  Google Scholar 

  23. Jiang, M., Rubbi, C. P., and Milner, J. (2004). Gel-based application of siRNA to human epithelial cancer cells induces RNAi-dependent apoptosis. Oligonucleotides 14, 239–248.

    Article  CAS  PubMed  Google Scholar 

  24. Yoshinouchi, M, Yamada, T., Kizaki, M., et al. (2003). In vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by E6 siRNA. Mol. Ther. 8, 762–768.

    Article  CAS  PubMed  Google Scholar 

  25. Yamato, K., Fen, J., Kobuchi, H., et al. (2006). Induction of cell death in human papillomavirus 18-positive cervical cancer cells by E6 siRNA. Cancer Gene Ther. 13, 234–241.

    Article  CAS  PubMed  Google Scholar 

  26. Schiffelers, R. M., Ansari, A., Xu, J., et al. (2004). Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. Nucleic Acids Res. 32, e149.

    Article  PubMed  Google Scholar 

  27. Zimmermann, T. S., Lee, A. C., Akinc, A., et al. (2006). RNAi-mediated gene silencing in non-human primates. Nature 441, 111–114.

    Article  CAS  PubMed  Google Scholar 

  28. Sioud, M., and Sorensen, D.R. (2003). Cationic liposome-mediated delivery of siRNAs in adult mice. Biochem. Biophys. Res. Commun. 312, 1220–1225.

    Article  CAS  PubMed  Google Scholar 

  29. Bai, Y., Soda, Y., Izawa, K., et al. (2003). Effective transduction and stable transgene expression in human blood cells by a third-generation lentiviral vector. Gene Ther. 10, 1446–1457.

    Article  CAS  PubMed  Google Scholar 

  30. Van den Haute, C., Eggermont, K., Nuttin, B., Debyser, Z., and Baekelandt. V. (2003). Lentiviral vector-mediated delivery of short hairpin RNA results in persistent knockdown of gene expression in mouse brain. Hum. Gene Ther. 14, 1799–1807.

    Article  PubMed  Google Scholar 

  31. Kafri, T., van Praag, H., Gage, F. H., and Verma, I. M. (2000). Lentiviral vectors: Regulated gene expression. Mol. Ther. 1, 516–521.

    Article  CAS  PubMed  Google Scholar 

  32. Miyoshi, H., Blomer, U., Takahashi, M., Gage, F. H., and Verma, I. M. (1998). Development of a self-inactivating lentivirus vector. J. Virol. 72, 8150–8157.

    CAS  PubMed  Google Scholar 

  33. Naldini, L., and Verma, I. M. (2000). Lentiviral vectors. Adv. Virus Res. 55, 599–609.

    Article  CAS  PubMed  Google Scholar 

  34. van der Wegen, P., Louwen, R., Imam, A. M., et al. (2006). Successful treatment of UGT1A1 deficiency in a rat model of Crigler–Najjar disease by intravenous administration of a liver-specific lentiviral vector. Mol. Ther. 13, 374–381.

    Article  PubMed  Google Scholar 

  35. Morizono, K., Xie, Y., Ringpis, G. E., et al. (2005). Lentiviral vector retargeting to P-glycoprotein on metastatic melanoma through intravenous injection. Nat. Med. 11, 346–352.

    Article  CAS  PubMed  Google Scholar 

  36. Yanez-Munoz, R. J., Balaggan, K. S., MacNeil, A., et al. (2006). Effective gene therapy with nonintegrating lentiviral vectors. Nat. Med. 12, 348–353.

    Article  CAS  PubMed  Google Scholar 

  37. Kimchi-Sarfaty, C., Brittain, S., Garfield, S., Caplen, N. J., Tang, Q., and Gottesman, M. M. (2005). Efficient delivery of RNA interference effectors via in vitro-packaged SV40 pseudovirions. Hum. Gene Ther. 16, 1110–1115.

    Article  CAS  PubMed  Google Scholar 

  38. Dull, T., Zufferey, R., Kelly, M., et al. (1998). A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72, 8463–8471.

    CAS  PubMed  Google Scholar 

  39. Brummelkamp, T. R., Bernards, R., and Agami, R. (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550–553.

    Article  CAS  PubMed  Google Scholar 

  40. Jazag, A., Kanai, F., Ijichi, H., et al. (2005). Single small-interfering RNA expression vector for silencing multiple transforming growth factor-beta pathway components. Nucleic Acids Res. 33, e131.

    Article  PubMed  Google Scholar 

  41. Gu, W., Putral, L., Hengst, K., et al. (2006). Inhibition of cervical cancer cell growth in vitro and in vivo with lentiviral-vector delivered short hairpin RNA targeting human papillomavirus E6 and E7 oncogenes. Cancer Gene Ther. 13, 1023–1032.

    Article  CAS  PubMed  Google Scholar 

  42. Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.

    Article  CAS  PubMed  Google Scholar 

  43. Gu, W., Janssens, P., Holland, M., Seamark, R., and Kerr, P. (2005). Lymphocytes and MHC class II positive cells in the female rabbit reproductive tract before and after ovulation. Immunol. Cell Biol. 83, 596–606.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgment

The research work was supported by a grant from the National Health and Medical Research Council and by a Peter Doherty Postdoctoral Fellowship award.

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© 2008 Humana Press, a part of Springer Science+Business Media, LLC

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Gu, W., Putral, L., McMillan, N. (2008). siRNA and shRNA as Anticancer Agents in a Cervical Cancer Model. In: Barik, S. (eds) RNAi. Methods in Molecular Biology™, vol 442. Humana Press. https://doi.org/10.1007/978-1-59745-191-8_12

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  • DOI: https://doi.org/10.1007/978-1-59745-191-8_12

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-874-4

  • Online ISBN: 978-1-59745-191-8

  • eBook Packages: Springer Protocols

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