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Real-Time PCR Assay for the Analysis of Alternative Splicing of Immune Mediators in Cancer

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Immune Mediators in Cancer

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2108))

Abstract

Alternative splicing evolved as a very efficient way to generate proteome diversity and to regulate cell homeostasis from a limited number of genes. Moreover, changes in the relative amounts of different splice variants derived from the same pre-mRNA are a hallmark in cancer, and aberrant expression of alternatively spliced mRNAs has been linked to cancer initiation and progression. Therefore, splice variants are critical tools to assess disease progression and clinical prognosis, and hold great promise as potential targets for therapeutic intervention. In order to understand the role that such splice variants play in cancer, it is vital to be able to accurately quantify their expression levels in different cell types and organs, both in normal conditions and in disease. In this chapter we describe a protocol to efficiently detect, analyze, and quantify alternative splicing patterns of immune mediators such as chemokines, cytokine and their receptors and ligands in cancer by quantitative PCR.

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References

  1. Quackenbush J (2001) The power of public access: the human genome project and the scientific process. Nat Genet 29:4–6

    Article  CAS  PubMed  Google Scholar 

  2. Venter JC, Adams MD, Myers EW et al (2001) The sequence of the human genome. Science 291:1304–1351

    Article  CAS  PubMed  Google Scholar 

  3. Samuels DC, Han L, Li J et al (2013) Finding the lost treasures in exome sequencing data. Trends Genet 29:593–599

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Wang ET, Sandberg R, Luo S et al (2008) Alternative isoform regulation in human tissue transcriptomes. Nature 456:470–476

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Pan Q, Shai O, Lee LJ et al (2008) Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet 40:1413–1415

    Article  CAS  PubMed  Google Scholar 

  6. Nilsen TW, Graveley BR (2010) Expansion of the eukaryotic proteome by alternative splicing. Nature 463:457–463

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Modrek B, Resch A, Grasso C et al (2001) Genome-wide detection of alternative splicing in expressed sequences of human genes. Nucleic Acids Res 29:2850–2859

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Black DL (2003) Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem 72:291–336

    Article  CAS  PubMed  Google Scholar 

  9. López-Bigas N, Audit B, Ouzounis C et al (2005) Are splicing mutations the most frequent cause of hereditary disease? FEBS Lett 579:1900–1903

    Article  PubMed  CAS  Google Scholar 

  10. Cooper TA, Wan L, Dreyfuss G (2009) RNA and disease. Cell 136:777–793

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Singh RK, Cooper TA (2012) Pre-mRNA splicing in disease and therapeutics. Trends Mol Med 18:472–482

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Lynch KW (2004) Consequences of regulated pre-mRNA splicing in the immune system. Nat Rev Immunol 4:931–940

    Article  CAS  PubMed  Google Scholar 

  13. Martinez NM, Pan Q, Cole BS et al (2012) Alternative splicing networks regulated by signaling in human T cells. RNA 18:1029–1040

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Martinez NM, Lynch KW (2013) Control of alternative splicing in immune responses: many regulators, many predictions, much still to learn. Immunol Rev 253:216–236

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Yabas M, Elliott H, Hoyne GF (2015) The role of alternative splicing in the control of immune homeostasis and cellular differentiation. Int J Mol Sci 17:E3

    Article  PubMed  CAS  Google Scholar 

  16. Schaub A, Glasmacher E (2017) Splicing in immune cells-mechanistic insights and emerging topics. Int Immunol 29:173–181

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kim E, Goren A, Ast G (2008) Insights into the connection between cancer and alternative splicing. Trends Genet 24:7–10

    Article  CAS  PubMed  Google Scholar 

  18. Bruikman CS, Zhang H, Kemper AM et al (2019) Netrin family: role for protein isoforms in cancer. J Nucleic Acids 2019:3947123

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Li D, Harlan-Williams LM, Kumaraswamy E et al (2019) BRCA1-no matter how you splice it. Cancer Res 79:2091–2098

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Hagen RM, Ladomery MR (2012) Role of splice variants in the metastatic progression of prostate cancer. Biochem Soc Trans 40:870–874

    Article  CAS  PubMed  Google Scholar 

  21. Wang BD, Lee NH (2018) Aberrant RNA splicing in cancer and drug resistance. Cancers (Basel) 10:E458

    Article  CAS  Google Scholar 

  22. Coltri PP, Dos Santos MGP, da Silva GHG (2019) Splicing and cancer: challenges and opportunities. Wiley Interdiscip Rev RNA 10:e1527

    Article  PubMed  Google Scholar 

  23. Angiolini F, Belloni E, Giordano M et al (2019) A novel L1CAM isoform with angiogenic activity generated by NOVA2-mediated alternative splicing. Elife 8:e44305

    Article  PubMed  PubMed Central  Google Scholar 

  24. Stevens M, Oltean S (2019) Modulation of receptor tyrosine kinase activity through alternative splicing of ligands and receptors in the VEGF-A/VEGFR axis. Cells 8:E288

    Article  PubMed  Google Scholar 

  25. Itoh N, Yonehara S, Ishii A et al (1991) The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 66:233–243

    Article  CAS  PubMed  Google Scholar 

  26. Cascino I, Papoff G, Eramo A et al (1996) Soluble Fas/Apo-1 splicing variants and apoptosis. Front Biosci 1:d12–d18

    Article  CAS  PubMed  Google Scholar 

  27. Villamizar O, Chambers CB, Riberdy JM et al (2016) Long noncoding RNA Saf and splicing factor 45 increase soluble Fas and resistance to apoptosis. Oncotarget 7:13810–13826

    Article  PubMed  PubMed Central  Google Scholar 

  28. Fu Y, Wang Y (2018) SRSF7 knockdown promotes apoptosis of colon and lung cancer cells. Oncol Lett 15:5545–5552

    PubMed  PubMed Central  Google Scholar 

  29. Cheng J, Zhou T, Liu C et al (1994) Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule. Science 263:1759–1762

    Article  CAS  PubMed  Google Scholar 

  30. Cascino I, Fiucci G, Papoff G et al (1995) Three functional soluble forms of the human apoptosis-inducing Fas molecule are produced by alternative splicing. J Immunol 154:2706–2713

    CAS  PubMed  Google Scholar 

  31. Kischkel FC, Hellbardt S, Behrmann I et al (1995) Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO J 14:5579–5588

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Ashkenazi A, Dixit VM (1999) Apoptosis control by death and decoy receptors. Curr Opin Cell Biol 11:255–260

    Article  CAS  PubMed  Google Scholar 

  33. Ueno T, Toi M, Tominaga T (1999) Circulating soluble Fas concentration in breast cancer patients. Clin Cancer Res 5:3529–3533

    CAS  PubMed  Google Scholar 

  34. Sheen-Chen SM, Chen HS, Eng HL et al (2003) Circulating soluble Fas in patients with breast cancer. World J Surg 27:10–13

    Article  PubMed  Google Scholar 

  35. Natoli G, Ianni A, Costanzo A et al (1995) Resistance to Fas-mediated apoptosis in human hepatoma cells. Oncogene 11:1157–1164

    CAS  PubMed  Google Scholar 

  36. Owen-Schaub LB, Angelo LS, Radinsky R et al (1995) Soluble Fas/APO-1 in tumor cells: a potential regulator of apoptosis. Cancer Lett 94:1–8

    Article  CAS  PubMed  Google Scholar 

  37. Lasagni L, Francalanci M, Annunziato F et al (2003) An alternatively spliced variant of CXCR3 mediates the inhibition of endothelial cell growth induced by IP-10, Mig, and I-TAC, and acts as functional receptor for platelet factor 4. J Exp Med 197:1537–1549

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Dagan-Berger M, Feniger-Barish R, Avniel S et al (2006) Role of CXCR3 carboxyl terminus and third intracellular loop in receptor-mediated migration, adhesion and internalization in response to CXCL11. Blood 107:3821–3831

    Article  CAS  PubMed  Google Scholar 

  39. Romagnani P, Annunziato F, Lazzeri E et al (2001) Interferon-inducible protein 10, monokine induced by interferon gamma, and interferon-inducible T-cell alpha chemoattractant are produced by thymic epithelial cells and attract T-cell receptor (TCR) alphabeta+ CD8+ single-positive T cells, TCRgammadelta+ T cells, and natural killer-type cells in human thymus. Blood 97:601–607

    Article  CAS  PubMed  Google Scholar 

  40. Martín-Fontecha A, Thomsen LL, Brett S et al (2004) Induced recruitment of NK cells to lymph nodes provides IFN-gamma for T(H)1 priming. Nat Immunol 5:1260–1265

    Article  PubMed  CAS  Google Scholar 

  41. Bodnar RJ, Yates CC, Wells A (2006) IP-10 blocks vascular endothelial growth factor-induced endothelial cell motility and tube formation via inhibition of calpain. Circ Res 98:617–625

    Article  CAS  PubMed  Google Scholar 

  42. Wu Q, Dhir R, Wells A (2012) Altered CXCR3 isoform expression regulates prostate cancer cell migration and invasion. Mol Cancer 11:3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Berchiche YA, Sakmar TP (2016) CXC chemokine receptor 3 alternative splice variants selectively activate different signaling pathways. Mol Pharmacol 90:483–495

    Article  CAS  PubMed  Google Scholar 

  44. Smit MJ, Verdijk P, van der Raaij-Helmer EM et al (2003) CXCR3-mediated chemotaxis of human T cells is regulated by a Gi- and phospholipase C-dependent pathway and not via activation of MEK/p44/p42 MAPK nor Akt/PI-3 kinase. Blood 102:1959–1965

    Article  CAS  PubMed  Google Scholar 

  45. Satish L, Blair HC, Glading A et al (2005) Interferon-inducible protein 9 (CXCL11)-induced cell motility in keratinocytes requires calcium flux-dependent activation of mu-calpain. Mol Cell Biol 25:1922–1941

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Kouroumalis A, Nibbs RJ, Aptel H et al (2005) The chemokines CXCL9, CXCL10, and CXCL11 differentially stimulate G alpha i-independent signaling and actin responses in human intestinal myofibroblasts. J Immunol 175:5403–5411

    Article  CAS  PubMed  Google Scholar 

  47. Thompson BD, Jin Y, Wu KH et al (2007) Inhibition of G alpha i2 activation by G alpha i3 in CXCR3-mediated signaling. J Biol Chem 282:9547–9555

    Article  CAS  PubMed  Google Scholar 

  48. Petrai I, Rombouts K, Lasagni L et al (2008) Activation of p38(MAPK) mediates the angiostatic effect of the chemokine receptor CXCR3-B. Int J Biochem Cell Biol 40:1764–1774

    Article  CAS  PubMed  Google Scholar 

  49. Kruse JP, Gu W (2009) Modes of p53 regulation. Cell 137:609–622

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Yin Y, Stephen CW, Luciani MG et al (2002) p53 stability and activity is regulated by Mdm2-mediated induction of alternative p53 translation products. Nat Cell Biol 4:462–467

    Article  CAS  PubMed  Google Scholar 

  51. Courtois S, Verhaegh G, North S et al (2002) DeltaN-p53, a natural isoform of p53 lacking the first transactivation domain, counteracts growth suppression by wild-type p53. Oncogene 21:6722–6728

    Article  CAS  PubMed  Google Scholar 

  52. Maier B, Gluba W, Bernier B et al (2004) Modulation of mammalian life span by the short isoform of p53. Genes Dev 18:306–319

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Ghosh A, Stewart D, Matlashewski G (2004) Regulation of human p53 activity and cell localization by alternative splicing. Mol Cell Biol 24:7987–7997

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Ponta H, Sherman L, Herrlich PA (2003) CD44: from adhesion molecules to signalling regulators. Nat Rev Mol Cell Biol 4:33–45

    Article  CAS  PubMed  Google Scholar 

  55. Yin T, Wang G, He S et al (2016) Human cancer cells with stem cell-like phenotype exhibit enhanced sensitivity to the cytotoxicity of IL-2 and IL-15 activated natural killer cells. Cell Immunol 300:41–45

    Article  CAS  PubMed  Google Scholar 

  56. Chen C, Zhao S, Karnad A et al (2018) The biology and role of CD44 in cancer progression: therapeutic implications. J Hematol Oncol 11:64

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. Tölg C, Hofmann M, Herrlich P et al (1993) Splicing choice from ten variant exons establishes CD44 variability. Nucleic Acids Res 21:1225–1229

    Article  PubMed  PubMed Central  Google Scholar 

  58. Screaton GR, Bell MV, Jackson DG et al (1992) Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. Proc Natl Acad Sci U S A 89:12160–12164

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Screaton GR, Bell MV, Bell JI et al (1993) The identification of a new alternative exon with highly restricted tissue expression in transcripts encoding the mouse Pgp-1 (CD44) homing receptor. Comparison of all 10 variable exons between mouse, human, and rat. J Biol Chem 268:12235–12238

    CAS  PubMed  Google Scholar 

  60. Cheng C, Sharp PA (2006) Regulation of CD44 alternative splicing by SRm160 and its potential role in tumor cell invasion. Mol Cell Biol 26:362–370

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Yae T, Tsuchihashi K, Ishimoto T et al (2012) Alternative splicing of CD44 mRNA by ESRP1 enhances lung colonization of metastatic cancer cell. Nat Commun 3:883

    Article  PubMed  CAS  Google Scholar 

  62. Prochazka L, Tesarik R, Turanek J (2014) Regulation of alternative splicing of CD44 in cancer. Cell Signal 26:2234–2239

    Article  CAS  PubMed  Google Scholar 

  63. Orian-Rousseau V, Chen L, Sleeman JP et al (2002) CD44 is required for two consecutive steps in HGF/c-Met signaling. Genes Dev 16:3074–3086

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Todaro M, Gaggianesi M, Catalano V et al (2014) CD44v6 is a marker of constitutive and reprogrammed cancer stem cells driving colon cancer metastasis. Cell Stem Cell 14:342–356

    Article  CAS  PubMed  Google Scholar 

  65. Kaufmann M, Heider KH, Sinn HP et al (1995) CD44 variant exon epitopes in primary breast cancer and length of survival. Lancet 345:615–619

    Article  CAS  PubMed  Google Scholar 

  66. Magyarosy E, Sebestyén A, Timár J (2001) Expression of metastasis associated proteins, CD44v6 and NM23-H1, in pediatric acute lymphoblastic leukemia. Anticancer Res 21:819–823

    CAS  PubMed  Google Scholar 

  67. Shakola F, Byrne S, Javed K et al (2014) Radiolabeled semi-quantitative RT-PCR assay for the analysis of alternative splicing of interleukin genes. Methods Mol Biol 1172:343–362

    Article  PubMed  CAS  Google Scholar 

  68. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  69. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Rao X, Huang X, Zhou Z et al (2013) An improvement of the 2(−delta delta CT) method for quantitative real-time polymerase chain reaction data analysis. Biostat Bioinform Biomath 3:71–85

    Google Scholar 

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Acknowledgment

This work was supported by NIH grant 1R15GM119099-01 to M. Ruggiu.

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Correspondence to Matteo Ruggiu .

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Wang, R., Hossain, M.F., Mirkovic, J., Sabzanov, S., Ruggiu, M. (2020). Real-Time PCR Assay for the Analysis of Alternative Splicing of Immune Mediators in Cancer. In: Vancurova, I., Zhu, Y. (eds) Immune Mediators in Cancer. Methods in Molecular Biology, vol 2108. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0247-8_21

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  • DOI: https://doi.org/10.1007/978-1-0716-0247-8_21

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