Use of RNA Interference to Investigate Cytokine Signal Transduction in Pancreatic Beta Cells

  • Fabrice MooreEmail author
  • Daniel A. Cunha
  • Hindrik Mulder
  • Decio L. Eizirik
Part of the Methods in Molecular Biology book series (MIMB, volume 820)


Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by immune infiltration of the pancreatic islets resulting in an inflammatory reaction named insulitis and subsequent beta cell apoptosis. During the course of insulitis beta cell death is probably caused by direct contact with activated macrophages and T-cells, and/or exposure to soluble mediators secreted by these cells, including cytokines, nitric oxide, and free oxygen radicals. In vitro exposure of beta cells to the cytokines interleukin(IL)-1β + interferon(IFN)-γ or to tumor necrosis factor(TNF)-α + IFN-γ induces beta cell dysfunction and ultimately apoptosis. The transcription factors NF-κB and STAT1 are key regulators of cytokine-induced beta cell death. However, little is known about the gene networks regulated by these (or other) transcription factors that trigger beta cell apoptosis. The recent development of RNA interference (RNAi) technology offers a unique opportunity to decipher the cytokine-activated molecular pathways responsible for beta cell death. Use of RNAi has been hampered by technical difficulties in transfecting primary beta cells, but in recent years we have succeeded in developing reliable and reproducible protocols for RNAi in beta cells. This chapter details the methods and settings used to achieve efficient and nontoxic transfection of small interfering RNA in immortal and primary beta cells.

Key words

Small interfering RNA siRNA Pancreatic beta cells Apoptosis Gene knockdown Inducible nitric oxide synthase Interleukin-1β Interferon-γ Tumor necrosis factor-α 



This work has been supported by grants from the Fonds National de la Recherche Scientifique (FNRS – FRSM) Belgium, the Communauté Française de Belgique – Actions de Recherche Concertées (ARC), the European Union (STREP Savebeta, contract no. 036903; in the Framework Programme 6 of the European Community) and the Belgium Program on Interuniversity Poles of Attraction initiated by the Belgium State (IUAP P6/40). F.M. is the recipient of a Post-Doctoral Fellowship from FNRS, Belgium. The authors have no duality of interest associated with this manuscript. We thank M.A. Neef, G. Vandenbroeck, M. Urbain, J. Schoonheydt, R. Leeman, A. M. Musuaya, and S. Mertens from the Laboratory of Experimental Medicine, ULB, for excellent technical support, Dr. Fernanda Ortis (Laboratory of Experimental Medicine) for helpful comments and Dr. Piero Marchetti (Department of Endocrinology and Metabolism, Metabolic Unit, University of Pisa, Pisa, Italy) for providing the human islets used for siRNA testing.


  1. 1.
    Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., Mello, C.C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806–811.PubMedCrossRefGoogle Scholar
  2. 2.
    Ghildiyal, M., Zamore, P.D. (2009) Small silencing RNAs: an expanding universe. Nat. Rev. Genet. 10: 94–108.PubMedCrossRefGoogle Scholar
  3. 3.
    Naqvi, A.R., Islam, M.N., Choudhury, N.R., Haq, Q.M. (2009) The fascinating world of RNA interference. Int. J. Biol. Sci. 5: 97–117.PubMedCrossRefGoogle Scholar
  4. 4.
    Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494–498.PubMedCrossRefGoogle Scholar
  5. 5.
    Wall, N.R., Shi, Y. (2003) Small RNA: can RNA interference be exploited for therapy? Lancet 362: 1401–1403.PubMedCrossRefGoogle Scholar
  6. 6.
    Whitehead, K.A., Langer, R., Anderson, D.G. (2009) Knocking down barriers: advances in siRNA delivery. Nat. Rev. Drug Discov. 8: 129–138.PubMedCrossRefGoogle Scholar
  7. 7.
    Chen, Y., Stamatoyannopoulos, G., Song, C.Z. (2003) Down-regulation of CXCR4 by inducible small interfering RNA inhibits breast cancer cell invasion in vitro. Cancer Res. 63: 4801–4804.PubMedGoogle Scholar
  8. 8.
    Cunha, D.A., Hekerman, P., Ladriere, L., Bazarra-Castro, A., Ortis, F., Wakeham, M.C. Moore, F., Rasschaert, J., Cardozo, A.K., Bellomo, E., Overbergh, L., Mathieu, C., Lupi, R., Hai, T., Herchuelz, A., Marchetti, P., Rutter, G.A., Eizirik, D.L., Cnop, M. (2008) Initiation and execution of lipotoxic ER stress in pancreatic beta-cells. J. Cell Sci. 121: 2308–2318.PubMedCrossRefGoogle Scholar
  9. 9.
    Xia, H., Mao, Q., Paulson, H.L., Davidson, B.L. (2002) siRNA-mediated gene silencing in vitro and in vivo. Nat. Biotechnol. 20: 1006–1010.PubMedCrossRefGoogle Scholar
  10. 10.
    Eizirik, D.L., Moore, F., Flamez, D., Ortis, F. (2008) Use of a systems biology approach to understand pancreatic beta-cell death in Type 1 diabetes. Biochem. Soc. Trans. 36: 321–327.PubMedCrossRefGoogle Scholar
  11. 11.
    Pipeleers, D.G., in’t Veld, P.A., Van de Winkel, M., Maes, E., Schuit, F.C., Gepts, W. (1985) A new in vitro model for the study of pancreatic A and B cells. Endocrinology 117: 806–816.Google Scholar
  12. 12.
    Rasschaert, J., Ladriere, L., Urbain, M., Dogusan, Z., Katabua, B., Sato, S., Akira, S, Gysemans, C., Mathieu, C., Eizirik, D.L. (2005) Toll-like receptor 3 and STAT-1 contribute to double-stranded RNA  +  interferon-gamma-induced apoptosis in primary pancreatic beta-cells. J. Biol. Chem. 280: 33984–33991.PubMedCrossRefGoogle Scholar
  13. 13.
    Ling, Z., Hannaert, J.C., Pipeleers, D. (1994) Effect of nutrients, hormones and serum on survival of rat islet beta cells in culture. Diabetologia 37: 15–21.PubMedCrossRefGoogle Scholar
  14. 14.
    Lupi, R., Dotta, F., Marselli, L., Del Guerra, S., Masini, M., Santangelo, C. Patané, G., Boggi, U., Piro, S., Anello, M., Bergamini, E., Mosca, F., Di Mario, U., Del Prato, S., Marchetti, P. (2002) Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets: evidence that beta-cell death is caspase mediated, partially dependent on ceramide pathway, and Bcl-2 regulated. Diabetes 51: 1437–1442.PubMedCrossRefGoogle Scholar
  15. 15.
    Delaney, C.A., Pavlovic, D., Hoorens, A., Pipeleers, D.G., Eizirik, D.L. (1997) Cytokines induce deoxyribonucleic acid strand breaks and apoptosis in human pancreatic islet cells. Endocrinology 138: 2610–2614.PubMedCrossRefGoogle Scholar
  16. 16.
    Pei, Y., Tuschl, T. (2006) On the art of identifying effective and specific siRNAs. Nat. Methods 3: 670–676.PubMedCrossRefGoogle Scholar
  17. 17.
    Birmingham, A., Anderson, E., Sullivan, K., Reynolds, A., Boese, Q., Leake, D., Karpilow, J., Khvorova, A. (2007) A protocol for designing siRNAs with high functionality and specificity. Nat. Protoc. 2: 2068–2078.PubMedCrossRefGoogle Scholar
  18. 18.
    Du, Q., Thonberg, H., Wang, J., Wahlestedt, C., Liang, Z. (2005) A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites. Nucleic Acids Res. 33: 1671–1677.PubMedCrossRefGoogle Scholar
  19. 19.
    Reynolds, A., Anderson, E.M., Vermeulen, A., Fedorov, Y., Robinson, K., Leake, D., Karpilow, J., Marshall, W.S., Khvorova, A. (2006) Induction of the interferon response by siRNA is cell type- and duplex length-dependent. RNA 12: 988–993.PubMedCrossRefGoogle Scholar
  20. 20.
    Dande, P., Prakash, T.P., Sioufi, N., Gaus, H., Jarres, R., Berdeja, A., Swayze, E.E., Griffey, R.H., Bhat, B. (2006) Improving RNA interference in mammalian cells by 4′-thio-modified small interfering RNA (siRNA): effect on siRNA activity and nuclease stability when used in combination with 2′-O-alkyl modifications. J. Med. Chem. 49: 1624–1634.PubMedCrossRefGoogle Scholar
  21. 21.
    Hall, A.H., Wan, J., Shaughnessy, E.E., Ramsay, S.B., Alexander, K.A. (2004) RNA interference using boranophosphate siRNAs: structure-activity relationships. Nucleic Acids Res. 32: 5991–6000.PubMedCrossRefGoogle Scholar
  22. 22.
    Zhang, N., Tan, C., Cai, P., Zhang, P., Zhao, Y., Jiang, Y. (2009) RNA interference in mammalian cells by siRNAs modified with morpholino nucleoside analogues. Bioorg. Med. Chem. 17: 2441–2446.PubMedCrossRefGoogle Scholar
  23. 23.
    Editorial Comment (2003) Whither RNAi? Nat. Cell Biol. 5: 489–490.Google Scholar
  24. 24.
    Moore, F., Colli, M.L., Cnop, M., Esteve, M.I., Cardozo, A.K., Cunha, D.A., Buglian, M., Marchetti, P., Eizirik, D.L. (2009) PTPN2, a candidate gene for type 1 diabetes, modulates interferon-gamma-induced pancreatic beta-cell apoptosis. Diabetes 58: 1283–1291.Google Scholar
  25. 25.
    Hoorens, A., Van de Casteele, M., Kloppel, G., Pipeleers, D. (1996) Glucose promotes survival of rat pancreatic beta cells by activating synthesis of proteins which suppress a constitutive apoptotic program. J. Clin. Invest. 98: 1568–1574.PubMedCrossRefGoogle Scholar
  26. 26.
    Eizirik, D.L., Mandrup-Poulsen, T. (2001) A choice of death – the signal-transduction of immune-mediated beta-cell apoptosis. Diabetologia 44: 2115–2133.PubMedCrossRefGoogle Scholar
  27. 27.
    Moore, F., Naamane, N., Colli, M.L., Bouckenooghe, T., Ortis, F., Gurzov, E.N., Igoillo-Esteve, M., Mathieu, C., Bontempi, G., Thykjaer, T., Ørntoft, T.F., Eizirik, D.L. (2011) STAT1 is a master regulator of pancreatic beta cell apoptosis and islet inflammation. J. Biol. Chem. 286: 929–941.Google Scholar
  28. 28.
    Callewaert, H.I., Gysemans, C.A., Ladriere, L., D’Hertog, W., Hagenbrock, J., Overbergh, L. Eizirik, D.L., Mathieu C. (2007) Deletion of STAT-1 pancreatic islets protects against streptozotocin-induced diabetes and early graft failure but not against late rejection. Diabetes 56: 2169–2173.PubMedCrossRefGoogle Scholar
  29. 29.
    Gysemans, C.A., Ladriere, L., Callewaert, H., Rasschaert, J., Flamez, D., Levy, D.E., Matthys, P., Eizirik, D.L., Mathieu, C. (2005) Disruption of the gamma-interferon signaling pathway at the level of signal transducer and activator of transcription-1 prevents immune destruction of beta-cells. Diabetes 54: 2396–2403.PubMedCrossRefGoogle Scholar
  30. 30.
    Echeverri, C.J., Perrimon, N. (2006) High-throughput RNAi screening in cultured cells: a user’s guide. Nat. Rev. Genet. 7: 373–384.PubMedCrossRefGoogle Scholar
  31. 31.
    Muller, P., Kuttenkeuler, D., Gesellchen, V., Zeidler, M.P., Boutros, M. (2005) Identification of JAK/STAT signalling components by genome-wide RNA interference. Nature 436: 871–875.PubMedCrossRefGoogle Scholar
  32. 32.
    Berns, K., Hijmans, E.M., Mullenders, J., Brummelkamp, T.R., Velds, A., Heimerikx, M. Kerkhoven, R.M., Madiredjo, M., Nijkamp, W., Weigelt, B., Agami, R., Ge, W., Cavet, G., Linsley, P.S., Beijersbergen, R.L., Bernards, R. (2004) A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428: 431–437.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Fabrice Moore
    • 1
    Email author
  • Daniel A. Cunha
    • 1
  • Hindrik Mulder
    • 2
  • Decio L. Eizirik
    • 1
  1. 1.Laboratory of Experimental MedicineUniversité Libre de BruxellesBrusselsBelgium
  2. 2.Unit of Molecular Metabolism, Department of Clinical Sciences in MalmöLund University Diabetes Center, Clinical Research Center 91:12MalmöSweden

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