Advertisement

Mast Cells pp 287-304 | Cite as

MicroRNA Function in Mast Cell Biology: Protocols to Characterize and Modulate MicroRNA Expression

  • Steven MaltbyEmail author
  • Maximilian Plank
  • Catherine Ptaschinski
  • Joerg Mattes
  • Paul S. FosterEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1220)

Abstract

MicroRNAs (miRNAs) are small noncoding RNA molecules that can modulate mRNA levels through RNA-induced silencing complex (RISC)-mediated degradation. Recognition of target mRNAs occurs through imperfect base pairing between an miRNA and its target, meaning that each miRNA can target a number of different mRNAs to modulate gene expression. miRNAs have been proposed as novel therapeutic targets and many studies are aimed at characterizing miRNA expression patterns and functions within a range of cell types. To date, limited research has focused on the function of miRNAs specifically in mast cells; however, this is an emerging field. In this chapter, we will briefly overview miRNA synthesis and function and the current understanding of miRNAs in hematopoietic development and immune function, emphasizing studies related to mast cell biology. The chapter will conclude with fundamental techniques used in miRNA studies, including RNA isolation, real-time PCR and microarray approaches for quantification of miRNA expression levels, and antagomir design to interfere with miRNA function.

Key words

MicroRNA RNA isolation Microarray Real-time PCR Antagomir 

References

  1. 1.
    Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120(1): 15–20PubMedCrossRefGoogle Scholar
  2. 2.
    Berezikov E (2011) Evolution of microRNA diversity and regulation in animals. Nat Rev Genet 12(12):846–860PubMedCrossRefGoogle Scholar
  3. 3.
    Friedman RC et al (2009) Most mammalian mRNAs are conserved targets of microRNAs. Genome Res 19(1):92–105PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Lee Y et al (2004) MicroRNA genes are transcribed by RNA polymerase II. EMBO J 23(20):4051–4060PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Lee Y et al (2002) MicroRNA maturation: stepwise processing and subcellular localization. EMBO J 21(17):4663–4670PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Denli AM et al (2004) Processing of primary microRNAs by the Microprocessor complex. Nature 432(7014):231–235PubMedCrossRefGoogle Scholar
  7. 7.
    Hutvagner G et al (2001) A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 293(5531):834–838PubMedCrossRefGoogle Scholar
  8. 8.
    Ketting RF et al (2001) Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev 15(20):2654–2659PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2):281–297PubMedCrossRefGoogle Scholar
  10. 10.
    Weinmann L et al (2009) Importin 8 is a gene silencing factor that targets argonaute proteins to distinct mRNAs. Cell 136(3):496–507PubMedCrossRefGoogle Scholar
  11. 11.
    Hammond SM et al (2001) Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293(5532):1146–1150PubMedCrossRefGoogle Scholar
  12. 12.
    Mallory AC et al (2004) MicroRNA control of PHABULOSA in leaf development: importance of pairing to the microRNA 5′ region. EMBO J 23(16):3356–3364PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Liu J (2008) Control of protein synthesis and mRNA degradation by microRNAs. Curr Opin Cell Biol 20(2):214–221PubMedCrossRefGoogle Scholar
  14. 14.
    Lewis BP et al (2003) Prediction of mammalian microRNA targets. Cell 115(7):787–798PubMedCrossRefGoogle Scholar
  15. 15.
    Lim LP et al (2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433(7027): 769–773PubMedCrossRefGoogle Scholar
  16. 16.
    Wightman B, Ha I, Ruvkun G (1993) Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 75(5): 855–862PubMedCrossRefGoogle Scholar
  17. 17.
    Pillai RS et al (2005) Inhibition of translational initiation by Let-7 MicroRNA in human cells. Science 309(5740):1573–1576PubMedCrossRefGoogle Scholar
  18. 18.
    Nottrott S, Simard MJ, Richter JD (2006) Human let-7a miRNA blocks protein production on actively translating polyribosomes. Nat Struct Mol Biol 13(12):1108–1114PubMedCrossRefGoogle Scholar
  19. 19.
    Petersen CP et al (2006) Short RNAs repress translation after initiation in mammalian cells. Mol Cell 21(4):533–542PubMedCrossRefGoogle Scholar
  20. 20.
    Bagga S et al (2005) Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation. Cell 122(4):553–563PubMedCrossRefGoogle Scholar
  21. 21.
    Giraldez AJ et al (2006) Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs. Science 312(5770):75–79PubMedCrossRefGoogle Scholar
  22. 22.
    Wu L, Fan J, Belasco JG (2006) MicroRNAs direct rapid deadenylation of mRNA. Proc Natl Acad Sci U S A 103(11):4034–4039PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Liu J et al (2005) MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies. Nat Cell Biol 7(7):719–723PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Guo S et al (2010) MicroRNA miR-125a controls hematopoietic stem cell number. Proc Natl Acad Sci U S A 107(32):14229–14234PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Gruber JJ et al (2009) Ars2 links the nuclear cap-binding complex to RNA interference and cell proliferation. Cell 138(2):328–339PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Gerrits A et al (2012) Genetic screen identifies microRNA cluster 99b/let-7e/125a as a regulator of primitive hematopoietic cells. Blood 119(2):377–387PubMedCrossRefGoogle Scholar
  27. 27.
    O’Connell RM et al (2010) MicroRNAs enriched in hematopoietic stem cells differentially regulate long-term hematopoietic output. Proc Natl Acad Sci U S A 107(32): 14235–14240PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Georgantas RW 3rd et al (2007) CD34+ hematopoietic stem-progenitor cell microRNA expression and function: a circuit diagram of differentiation control. Proc Natl Acad Sci U S A 104(8):2750–2755PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Felli N et al (2005) MicroRNAs 221 and 222 inhibit normal erythropoiesis and erythroleukemic cell growth via kit receptor down-modulation. Proc Natl Acad Sci U S A 102(50): 18081–18086PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Lu J et al (2008) MicroRNA-mediated control of cell fate in megakaryocyte-erythrocyte progenitors. Dev Cell 14(6):843–853PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Johnnidis JB et al (2008) Regulation of progenitor cell proliferation and granulocyte function by microRNA-223. Nature 451(7182): 1125–1129PubMedCrossRefGoogle Scholar
  32. 32.
    Cobb BS et al (2005) T cell lineage choice and differentiation in the absence of the RNase III enzyme Dicer. J Exp Med 201(9): 1367–1373PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Li QJ et al (2007) miR-181a is an intrinsic modulator of T cell sensitivity and selection. Cell 129(1):147–161PubMedCrossRefGoogle Scholar
  34. 34.
    Koralov SB et al (2008) Dicer ablation affects antibody diversity and cell survival in the B lymphocyte lineage. Cell 132(5):860–874PubMedCrossRefGoogle Scholar
  35. 35.
    O'Carroll D et al (2007) A Slicer-independent role for Argonaute 2 in hematopoiesis and the microRNA pathway. Genes Dev 21(16): 1999–2004PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Xiao C et al (2007) MiR-150 controls B cell differentiation by targeting the transcription factor c-Myb. Cell 131(1):146–159PubMedCrossRefGoogle Scholar
  37. 37.
    Zhou B et al (2007) miR-150, a microRNA expressed in mature B and T cells, blocks early B cell development when expressed prematurely. Proc Natl Acad Sci U S A 104(17): 7080–7085PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Rao DS et al (2010) MicroRNA-34a perturbs B lymphocyte development by repressing the forkhead box transcription factor Foxp1. Immunity 33(1):48–59PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Fontana L et al (2007) MicroRNAs 17-5p-20a-106a control monocytopoiesis through AML1 targeting and M-CSF receptor upregulation. Nat Cell Biol 9(7):775–787PubMedCrossRefGoogle Scholar
  40. 40.
    Fukao T et al (2007) An evolutionarily conserved mechanism for microRNA-223 expression revealed by microRNA gene profiling. Cell 129(3):617–631PubMedCrossRefGoogle Scholar
  41. 41.
    Fazi F et al (2005) A minicircuitry comprised of microRNA-223 and transcription factors NFI-A and C/EBPalpha regulates human granulopoiesis. Cell 123(5):819–831PubMedCrossRefGoogle Scholar
  42. 42.
    Costinean S et al (2006) Pre-B cell proliferation and lymphoblastic leukemia/high-grade lymphoma in E(mu)-miR155 transgenic mice. Proc Natl Acad Sci U S A 103(18): 7024–7029PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    O'Connell RM et al (2008) Sustained expression of microRNA-155 in hematopoietic stem cells causes a myeloproliferative disorder. J Exp Med 205(3):585–594PubMedCentralPubMedCrossRefGoogle Scholar
  44. 44.
    Han YC et al (2010) microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors, biased myeloid development, and acute myeloid leukemia. J Exp Med 207(3): 475–489PubMedCentralPubMedCrossRefGoogle Scholar
  45. 45.
    Calin GA et al (2002) Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A 99(24): 15524–15529PubMedCentralPubMedCrossRefGoogle Scholar
  46. 46.
    Klein U et al (2010) The DLEU2/miR-15a/ 16-1 cluster controls B cell proliferation and its deletion leads to chronic lymphocytic leukemia. Cancer Cell 17(1):28–40PubMedCrossRefGoogle Scholar
  47. 47.
    Chen XM et al (2007) A cellular micro-RNA, let-7i, regulates Toll-like receptor 4 expression and contributes to cholangiocyte immune responses against Cryptosporidium parvum infection. J Biol Chem 282(39):28929–28938PubMedCentralPubMedCrossRefGoogle Scholar
  48. 48.
    Bazzoni F et al (2009) Induction and regulatory function of miR-9 in human monocytes and neutrophils exposed to proinflammatory signals. Proc Natl Acad Sci U S A 106(13): 5282–5287PubMedCentralPubMedCrossRefGoogle Scholar
  49. 49.
    O’Connell RM et al (2007) MicroRNA-155 is induced during the macrophage inflammatory response. Proc Natl Acad Sci U S A 104(5): 1604–1609PubMedCentralPubMedCrossRefGoogle Scholar
  50. 50.
    Liu G et al (2009) miR-147, a microRNA that is induced upon Toll-like receptor stimulation, regulates murine macrophage inflammatory responses. Proc Natl Acad Sci U S A 106(37): 15819–15824PubMedCentralPubMedCrossRefGoogle Scholar
  51. 51.
    Sheedy FJ et al (2010) Negative regulation of TLR4 via targeting of the proinflammatory tumor suppressor PDCD4 by the microRNA miR-21. Nat Immunol 11(2):141–147PubMedCrossRefGoogle Scholar
  52. 52.
    Taganov KD et al (2006) NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc Natl Acad Sci U S A 103(33): 12481–12486PubMedCentralPubMedCrossRefGoogle Scholar
  53. 53.
    Rodriguez A et al (2007) Requirement of bic/microRNA-155 for normal immune function. Science 316(5824):608–611PubMedCentralPubMedCrossRefGoogle Scholar
  54. 54.
    Thai TH et al (2007) Regulation of the germinal center response by microRNA-155. Science 316(5824):604–608PubMedCrossRefGoogle Scholar
  55. 55.
    Du C et al (2009) MicroRNA miR-326 regulates TH-17 differentiation and is associated with the pathogenesis of multiple sclerosis. Nat Immunol 10(12):1252–1259PubMedCrossRefGoogle Scholar
  56. 56.
    Mattes J et al (2009) Antagonism of micro RNA-126 suppresses the effector function of TH2 cells and the development of allergic airways disease. Proc Natl Acad Sci U S A 106(44):18704–18709PubMedCentralPubMedCrossRefGoogle Scholar
  57. 57.
    Lu TX, Munitz A, Rothenberg ME (2009) MicroRNA-21 is up-regulated in allergic airway inflammation and regulates IL-12p35 expression. J Immunol 182(8):4994–5002PubMedCrossRefGoogle Scholar
  58. 58.
    Liu G et al (2010) miR-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis. J Exp Med 207(8):1589–1597PubMedCentralPubMedCrossRefGoogle Scholar
  59. 59.
    Monticelli S et al (2005) MicroRNA profiling of the murine hematopoietic system. Genome Biol 6(8):R71PubMedCentralPubMedCrossRefGoogle Scholar
  60. 60.
    Kuchen S et al (2010) Regulation of microRNA expression and abundance during lymphopoiesis. Immunity 32(6):828–839PubMedCentralPubMedCrossRefGoogle Scholar
  61. 61.
    Mayoral RJ et al (2009) MicroRNA-221-222 regulate the cell cycle in mast cells. J Immunol 182(1):433–445PubMedCentralPubMedCrossRefGoogle Scholar
  62. 62.
    Lee YN et al (2011) KIT signaling regulates MITF expression through miRNAs in normal and malignant mast cell proliferation. Blood 117(13):3629–3640PubMedCentralPubMedCrossRefGoogle Scholar
  63. 63.
    Ishizaki T et al (2011) miR126 positively regulates mast cell proliferation and cytokine production through suppressing Spred1. Genes Cells 16(7):803–814PubMedCrossRefGoogle Scholar
  64. 64.
    Mayoral RJ et al (2011) MiR-221 influences effector functions and actin cytoskeleton in mast cells. PLoS One 6(10):e26133PubMedCentralPubMedCrossRefGoogle Scholar
  65. 65.
    Molnar V et al (2012) MicroRNA-132 targets HB-EGF upon IgE-mediated activation in murine and human mast cells. Cell Mol Life Sci 69(5):793–808Google Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Priority Research Centre for Asthma and Respiratory Diseases, School of Biomedical Sciences and Pharmacy, Faculty of HealthUniversity of NewcastleNew Lambton HeightsAustralia
  2. 2.Hunter Medical Research InstituteNew Lambton HeightsAustralia
  3. 3.Department of PathologyUniversity of MichiganAnn ArborUSA

Personalised recommendations