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Identification of Immune Modulatory miRNAs by miRNA Enrichment via RNA Affinity Purification

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Immune Checkpoint Blockade

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1913))

Abstract

Immune escape by cancer cells can be triggered by aberrant expression of immunological key players, which can be achieved by distinct molecular mechanisms including immune modulatory miRNAs. One suitable method to identify miRNAs that specifically target immune relevant molecules is the miRNA enrichment via RNA affinity purification method named miTRAP (miRNA trapping by RNA in vitro affinity purification). Here, we present a detailed protocol for construct preparation, RNA immobilization via MS2BP-MBP to beads, miRNA enrichment, and elution followed by analysis of the obtained miRNA candidates via qRT-PCR.

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References

  1. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144(5):646–674. https://doi.org/10.1016/j.cell.2011.02.013

    Article  CAS  PubMed  Google Scholar 

  2. Eichmüller SB, Osen W, Mandelboim O et al (2017) Immune modulatory microRNAs involved in tumor attack and tumor immune escape. J Natl Cancer Inst 109(10). https://doi.org/10.1093/jnci/djx034

  3. Seliger B (2017) Immune modulatory microRNAs as a novel mechanism to revert immune escape of tumors. Cytokine Growth Factor Rev 36:49–56. https://doi.org/10.1016/j.cytogfr.2017.07.001

    Article  CAS  PubMed  Google Scholar 

  4. Braun J, Misiak D, Busch B et al (2014) Rapid identification of regulatory microRNAs by miTRAP (miRNA trapping by RNA in vitro affinity purification). Nucleic Acids Res 42(8):e66. https://doi.org/10.1093/nar/gku127

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Gao F, Zhao Z, Zhao W et al (2013) miR-9 modulates the expression of interferon-regulated genes and MHC class I molecules in human nasopharyngeal carcinoma cells. Biochem Biophys Res Commun 431(3):610–616. https://doi.org/10.1016/j.bbrc.2012.12.097

    Article  CAS  PubMed  Google Scholar 

  6. Wang B, Wang Q, Wang Z et al (2014) Metastatic consequences of immune escape from NK cell cytotoxicity by human breast cancer stem cells. Cancer Res 74(20):5746–5757. https://doi.org/10.1158/0008-5472.CAN-13-2563

    Article  CAS  PubMed  Google Scholar 

  7. Xie J, Liu M, Li Y et al (2014) Ovarian tumor-associated microRNA-20a decreases natural killer cell cytotoxicity by downregulating MICA/B expression. Cell Mol Immunol 11(5):495–502. https://doi.org/10.1038/cmi.2014.30

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Wang X, Li J, Dong K et al (2015) Tumor suppressor miR-34a targets PD-L1 and functions as a potential immunotherapeutic target in acute myeloid leukemia. Cell Signal 27(3):443–452. https://doi.org/10.1016/j.cellsig.2014.12.003

    Article  CAS  PubMed  Google Scholar 

  9. Kulkarni S, Qi Y, O'hUigin C et al (2013) Genetic interplay between HLA-C and MIR148A in HIV control and Crohn disease. Proc Natl Acad Sci U S A 110(51):20705–20710. https://doi.org/10.1073/pnas.1312237110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Jasinski-Bergner S, Stoehr C, Bukur J et al (2015) Clinical relevance of miR-mediated HLA-G regulation and the associated immune cell infiltration in renal cell carcinoma. Oncoimmunology 4(6):e1008805. https://doi.org/10.1080/2162402X.2015.1008805

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Cheng Z, Ma R, Tan W et al (2014) MiR-152 suppresses the proliferation and invasion of NSCLC cells by inhibiting FGF2. Exp Mol Med 46:e112. https://doi.org/10.1038/emm.2014.51

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Chen L, Gibbons DL, Goswami S et al (2014) Metastasis is regulated via microRNA-200/ZEB1 axis control of tumour cell PD-L1 expression and intratumoral immunosuppression. Nat Commun 5:5241. https://doi.org/10.1038/ncomms6241

    Article  CAS  PubMed  Google Scholar 

  13. Johansson HE, Liljas L, Uhlenbeck OC (1997) RNA recognition by the MS2 phage coat protein. Semin Virol 8(3):176–185. https://doi.org/10.1006/smvy.1997.0120

    Article  CAS  Google Scholar 

  14. Chamberlin M, Ring J (1973) Characterization of T7-specific ribonucleic acid polymerase. 1. General properties of the enzymatic reaction and the template specificity of the enzyme. J Biol Chem 248(6):2235–2244

    CAS  PubMed  Google Scholar 

  15. Jurica MS, Licklider LJ, Gygi SR et al (2002) Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis. RNA 8(4):426–439

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9(7):671–675

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Wang D, Zhang Z, O'Loughlin E et al (2012) Quantitative functions of Argonaute proteins in mammalian development. Genes Dev 26(7):693–704. https://doi.org/10.1101/gad.182758.111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Agarwal V, Bell GW, Nam J et al (2015) Predicting effective microRNA target sites in mammalian mRNAs. Elife 4. https://doi.org/10.7554/eLife.05005

  19. Kertesz M, Iovino N, Unnerstall U et al (2007) The role of site accessibility in microRNA target recognition. Nat Genet 39(10):1278–1284. https://doi.org/10.1038/ng2135

    Article  CAS  PubMed  Google Scholar 

  20. Betel D, Koppal A, Agius P et al (2010) Comprehensive modeling of microRNA targets predicts functional non-conserved and non-canonical sites. Genome Biol 11(8):R90. https://doi.org/10.1186/gb-2010-11-8-r90

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kramer MF (2011) Stem-loop RT-qPCR for miRNAs. Curr Protoc Mol Biol Chapter 15: Unit 15.10. https://doi.org/10.1002/0471142727.mb1510s95

    Article  Google Scholar 

  22. Chen C, Ridzon DA, Broomer AJ et al (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33(20):e179. https://doi.org/10.1093/nar/gni178

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Summer H, Grämer R, Dröge P (2009) Denaturing urea polyacrylamide gel electrophoresis (urea PAGE). J Vis Exp 32:1485. https://doi.org/10.3791/1485

    Article  Google Scholar 

  24. Kedzierski W, Porter JC (1991) A novel non-enzymatic procedure for removing DNA template from RNA transcription mixtures. Biotechniques 10(2):210–214

    CAS  PubMed  Google Scholar 

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Correspondence to Barbara Seliger .

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Tretbar, U.S., Friedrich, M., Lazaridou, MF., Seliger, B. (2019). Identification of Immune Modulatory miRNAs by miRNA Enrichment via RNA Affinity Purification. In: Pico de Coaña, Y. (eds) Immune Checkpoint Blockade. Methods in Molecular Biology, vol 1913. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8979-9_6

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  • DOI: https://doi.org/10.1007/978-1-4939-8979-9_6

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8978-2

  • Online ISBN: 978-1-4939-8979-9

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