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Evaluation of Post-transcriptional Gene Regulation in Pancreatic Cancer Cells: Studying RNA Binding Proteins and Their mRNA Targets

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Pancreatic Cancer

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1882))

Abstract

Post-transcriptional regulation of gene expression through interaction between RNA binding proteins (RBPs) and target mRNAs have gained considerable interest over the last decade. Altered expression of RBPs as detected in pancreatic ductal adenocarcinoma (PDAC) cells alters mRNA processing, and in turn, the entire transcriptome and proteome. Thus, this gene regulatory mechanism can regulate important pro-oncogenic signaling pathways (e.g., TP53, WEE1, and c-MYC) in PDAC cells. Ribonucleoprotein immunoprecipitation assays (RNP-IP or RIP) are a modified immunoprecipitation method to study physical interactions between RBPs and their mRNA targets. As a first step to explore RBP interactomes and define novel therapeutic targets and dysregulated pathways in disease, RIPs are a sensitive and established molecular biology technique used to isolate and differentiate bound transcripts to RBPs in a variety of experimental conditions. This chapter describes an up-to-date, detailed protocol for performing this assay in mammalian cytoplasmic extracts (i.e., PDAC cells), and reviews current methods to validate target binding sites such as electrophoretic mobility shift assay (EMSA) and cross-linking immunoprecipitation polymerase chain reaction (CLIP-PCR).

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References

  1. Pishvaian MJ, Brody JR (2017) Therapeutic implications of molecular subtyping for pancreatic Cancer. Oncology (Williston Park). 31(3):159–166, 168

    Google Scholar 

  2. Blanco FF, Jimbo M, Wulfkuhle J, Gallagher I, Deng J, Enyenihi L, Meisner-Kober N, Londin E, Rigoutsos I, Sawicki JA, Risbud MV, Witkiewicz AK, McCue PA, Jiang W, Rui H, Yeo CJ, Petricoin E, Winter JM, Brody JR (2016) The mRNA-binding protein HuR promotes hypoxia-induced chemoresistance through posttranscriptional regulation of the proto-oncogene PIM1 in pancreatic cancer cell. Oncogene 35(19):2529–2541

    Article  CAS  Google Scholar 

  3. Zarei M, Lal S, Parker SJ et al (2017) Posttranscriptional upregulation of IDH1 by HuR establishes a powerful survival phenotype in pancreatic cancer cells. Cancer Res 77(16):4460–4471

    Article  CAS  Google Scholar 

  4. Abdelmohsen K, Gorospe M (2010) Posttranscriptional regulation of cancer traits by HuR. Wiley Interdiscip Rev RNA 1(2):214–229

    Article  CAS  Google Scholar 

  5. Fei T, Chen Y, Xiao T, Li W, Cato L, Zhang P, Cotter MB, Bowden M, Lis RT, Zhao SG, Wu Q, Feng FY, Loda M, He HH, Liu XS, Brown M (2017) Genome-wide CRISPR screen identifies HNRNPL as a prostate cancer dependency regulating RNA splicing. Proc Natl Acad Sci U S A 114(26):E5207–E5215

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Heinonen M, Bono P, Narko K, Chang SH, Lundin J, Joensuu H, Furneaux H, Hla T, Haglund C, Ristimäki A (2005) Cytoplasmic HuR expression is a prognostic factor in invasive ductal breast carcinoma. Cancer Res 65(6):2157–2161

    Article  CAS  Google Scholar 

  7. King CE et al (2011) LIN28B promotes colon cancer progression and metastasis. Cancer Res 71:4260–4268

    Article  CAS  Google Scholar 

  8. Richards NG, Rittenhouse DW, Freydin B, Cozzitorto JA, Grenda D, Rui H, Gonye G, Kennedy EP, Yeo CJ, Brody JR, Witkiewicz AK (2010) HuR status is a powerful marker for prognosis and response to gemcitabine-based chemotherapy for resected pancreatic ductal adenocarcinoma patients. Ann Surg 252(3):499–505 discussion 505-6

    PubMed  Google Scholar 

  9. Pereira B, Billaud M, Almeida R (2017) RNA-binding proteins in cancer: old players and new actors. Trends Cancer 3(7):506–528

    Article  Google Scholar 

  10. Harvey R, Dezi V, Pizzinga M, Willis AE (2017) Post-transcriptional control of gene expression following stress: the role of RNA-binding proteins. Biochem Soc Trans 45(4):1007–1014

    Article  CAS  Google Scholar 

  11. Lal S, Burkhart RA, Beeharry N, Bhattacharjee V, Londin ER, Cozzitorto JA, Romeo C, Jimbo M, Norris ZA, Yeo CJ, Sawicki JA, Winter JM, Rigoutsos I, Yen TJ, Brody JR (2014) HuR posttranscriptionally regulates WEE1: implications for the DNA damage response in pancreatic cancer cells. Cancer Res 74:1128–1140

    Article  CAS  Google Scholar 

  12. Chand SN, Zarei M, Schiewer MJ, Kamath AR, Romeo C, Lal S, Cozzitorto JA, Nevler A, Scolaro L, Londin E, Jiang W, Meisner-Kober N, Pishvaian MJ, Knudsen KE, Yeo CJ, Pascal JM, Winter JM, Brody JR (2017) Posttranscriptional regulation of PARG mRNA by HuR facilitates DNA repair and resistance to PARP inhibitors. Cancer Res 77(18):5011–5025

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Cozzitorto JA, Jimbo M, Chand S, Blanco F, Lal S, Gilbert M, Winter JM, Gorospe M, Brody JR (2015) Studying RNA-binding protein interactions with target mRNAs in eukaryotic cells: native ribonucleoprotein immunoprecipitation (RIP) assays. Methods Mol Biol 1262:239–246

    Article  CAS  Google Scholar 

  14. Piskounova E, Viswanathan SR, Janas M et al (2008) Determinants of MicroRNA processing inhibition by the developmentally regulated RNA-binding protein Lin28. J Biol Chem 283(31):21310–21314

    Article  CAS  Google Scholar 

  15. Sengupta S, Jang BC, Wu MT, Paik JH, Furneaux H, Hla T (2003) The RNA-binding protein HuR regulates the expression of cyclooxygenase-2. J Biol Chem 278(27):25227–25233

    Article  CAS  Google Scholar 

  16. Kaku S, Iwahashi Y, Kuraishi A et al (2001) Binding to the naturally occurring double p53 binding site of the Mdm2 promoter alleviates the requirement for p53 C-terminal activation. Nucleic Acids Res 29(9):1989–1993

    Article  CAS  Google Scholar 

  17. Finkin S, Yuan D, Stein I et al (2015) Ectopic lymphoid structures function as microniches for tumor progenitor cells in hepatocellular carcinoma. Nat Immunol 16(12):1235–1244

    Article  CAS  Google Scholar 

  18. Kloetgen A, Borkhardt A, Hoell JI, McHardy AC (2016) The PARA-suite: PAR-CLIP specific sequence read simulation and processing. Papaleo E, ed. PeerJ 4:e2619

    Article  Google Scholar 

  19. Weyn-Vanhentenryck SM, Mele A, Yan Q et al (2014) HITS-CLIP and integrative modeling define the Rbox splicing-regulatory network linked to brain development and autism. Cell Rep 6(6):1139–1152

    Article  CAS  Google Scholar 

  20. Darnell RB (2010) HITS-CLIP: panoramic views of protein-RNA regulation in living cells. Wiley interdisciplinary reviews RNA 1(2):266–286

    Article  CAS  Google Scholar 

  21. McAllister F, Pineda DM, Jimbo M, Lal S, Burkhart RA, Moughan J, Winter KA, Abdelmohsen K, Gorospe M, Acosta Ade J, Lankapalli RH, Winter JM, Yeo CJ, Witkiewicz AK, Iacobuzio-Donahue CA, Laheru D, Brody JR (2014) dCK expression correlates with 5-fluorouracil efficacy and HuR cytoplasmic expression in pancreatic cancer: a dual-institutional follow-up with the RTOG 9704 trial. Cancer Biol Ther 15(6):688–698

    Article  Google Scholar 

  22. Huppertz I, Attig J, D’Ambrogio A et al (2014) iCLIP: protein–RNA interactions at nucleotide resolution. Methods (San Diego, Calif) 65(3):274–287

    Article  CAS  Google Scholar 

  23. Li X, Song J, Yi C (2014) Genome-wide mapping of cellular protein–RNA interactions enabled by chemical crosslinking. Genomics Proteomics Bioinformatics 12(2):72–78

    Article  CAS  Google Scholar 

  24. Uhl M, Houwaart T, Corrado G, Wright PR, Backofen R (2017) Computational analysis of CLIP-seq data. Methods 118-119:60–72

    Article  CAS  Google Scholar 

  25. Maragkakis M, Alexiou P, Nakaya T, Mourelatos Z (2016) CLIPSeqTools--a novel bioinformatics CLIP-seq analysis suite. RNA 22(1):1–9

    Article  CAS  Google Scholar 

  26. Liu Q, Zhong X, Madison BB, Rustgi AK, Shyr Y (2015) Assessing computational steps for CLIP-Seq data analysis. Biomed Res Int 2015:196082

    PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Division of Surgical Research, Department of Surgery, Jefferson Pancreas, Biliary and Related Cancer Center, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, USA

    Aditi Jain, Samantha Z. Brown, Henry L. Thomsett & Jonathan R. Brody

  2. Computational Medicine Center, Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, PA, USA

    Eric Londin

Authors
  1. Aditi Jain
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  2. Samantha Z. Brown
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  3. Henry L. Thomsett
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  4. Eric Londin
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  5. Jonathan R. Brody
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Corresponding author

Correspondence to Jonathan R. Brody .

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Editors and Affiliations

  1. Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY, USA

    Gloria H. Su

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Jain, A., Brown, S.Z., Thomsett, H.L., Londin, E., Brody, J.R. (2019). Evaluation of Post-transcriptional Gene Regulation in Pancreatic Cancer Cells: Studying RNA Binding Proteins and Their mRNA Targets. In: Su, G. (eds) Pancreatic Cancer. Methods in Molecular Biology, vol 1882. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8879-2_22

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  • DOI: https://doi.org/10.1007/978-1-4939-8879-2_22

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8878-5

  • Online ISBN: 978-1-4939-8879-2

  • eBook Packages: Springer Protocols

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