Methods for MicroRNA Profiling in Cancer

  • Sushuma Yarlagadda
  • Anusha Thota
  • Ruchi Bansal
  • Jason Kwon
  • Murray Korc
  • Janaiah KotaEmail author
Part of the Methods in Pharmacology and Toxicology book series (MIPT)


MicroRNAs (miRNA) are small non-coding RNAs that negatively regulate post-transcriptional gene expression. Almost all human cancers are characterized by abnormal microRNA expression patterns, which are unique to tumor types. A large body of experimental evidence documents the role of miRNAs in cancer pathogenesis, and specific miRNAs function as oncogenes or tumor suppressors. Due to unique expression profiles and anti/pro-tumorigenic properties of miRNAs, efforts are underway to explore their therapeutic and diagnostic potential. Many miRNA profiling methods have been developed, ranging from Northern blotting and qRT-PCR to the more recent microarray and RNA-Seq platforms. The following chapter details an imaging technique for cellular-specific miRNA expression profiling called in situ hybridization (ISH).

Key words

MicroRNA Expression profiling Molecular imaging ISH (in situ hybridization) Cancer 


  1. 1.
    Lee RC, Feinbaum RL, Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75(5):843–854CrossRefPubMedGoogle Scholar
  2. 2.
    Reinhart BJ et al (2000) The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403(6772):901–906CrossRefPubMedGoogle Scholar
  3. 3.
    Lagos-Quintana M et al (2001) Identification of novel genes coding for small expressed RNAs. Science 294(5543):853–858CrossRefPubMedGoogle Scholar
  4. 4.
    Lau NC et al (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294(5543):858–862CrossRefPubMedGoogle Scholar
  5. 5.
    Lee RC, Ambros V (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science 294(5543):862–864CrossRefPubMedGoogle Scholar
  6. 6.
    Londin E et al (2015) Analysis of 13 cell types reveals evidence for the expression of numerous novel primate- and tissue-specific microRNAs. Proc Natl Acad Sci U S A 112(10):E1106–E1115CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Berezikov E (2011) Evolution of microRNA diversity and regulation in animals. Nat Rev Genet 12(12):846–860CrossRefPubMedGoogle Scholar
  8. 8.
    Lee Y et al (2004) MicroRNA genes are transcribed by RNA polymerase II. EMBO J 23(20):4051–4060CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Lee Y et al (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature 425(6956):415–419CrossRefPubMedGoogle Scholar
  10. 10.
    Yi R et al (2003) Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev 17(24):3011–3016CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Lee Y et al (2002) MicroRNA maturation: stepwise processing and subcellular localization. EMBO J 21(17):4663–4670CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Hutvagner G, Zamore PD (2002) A microRNA in a multiple-turnover RNAi enzyme complex. Science 297(5589):2056–2060CrossRefPubMedGoogle Scholar
  13. 13.
    Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136(2):215–233CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Wang X (2014) Composition of seed sequence is a major determinant of microRNA targeting patterns. Bioinformatics 30(10):1377–1383CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Concepcion CP, Bonetti C, Ventura A (2012) The microRNA-17-92 family of microRNA clusters in development and disease. Cancer J 18(3):262–267CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Lim LP et al (2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433(7027):769–773CrossRefPubMedGoogle Scholar
  17. 17.
    Krek A et al (2005) Combinatorial microRNA target predictions. Nat Genet 37(5):495–500CrossRefPubMedGoogle Scholar
  18. 18.
    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):15–20CrossRefPubMedGoogle Scholar
  19. 19.
    Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2):281–297CrossRefPubMedGoogle Scholar
  20. 20.
    Rodriguez A et al (2007) Requirement of bic/microRNA-155 for normal immune function. Science 316(5824):608–611CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Tijsen AJ, Pinto YM, Creemers EE (2012) Circulating microRNAs as diagnostic biomarkers for cardiovascular diseases. Am J Physiol Heart Circ Physiol 303(9):H1085–H1095CrossRefPubMedGoogle Scholar
  22. 22.
    Karolina DS et al (2011) MicroRNA 144 impairs insulin signaling by inhibiting the expression of insulin receptor substrate 1 in type 2 diabetes mellitus. PLoS One 6(8), e22839CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Munker R, Calin GA (2011) MicroRNA profiling in cancer. Clin Sci 121(4):141–158CrossRefPubMedGoogle Scholar
  24. 24.
    Croce CM (2009) Causes and consequences of microRNA dysregulation in cancer. Nat Rev Genet 10(10):704–714CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Calin GA, Croce CM (2006) MicroRNA signatures in human cancers. Nat Rev Cancer 6(11):857–866CrossRefPubMedGoogle Scholar
  26. 26.
    Cimmino A et al (2005) miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci U S A 102(39):13944–13949CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Zhang BG et al (2012) microRNA-21 promotes tumor proliferation and invasion in gastric cancer by targeting PTEN. Oncol Rep 27(4):1019–1026PubMedPubMedCentralGoogle Scholar
  28. 28.
    O'Donnell KA et al (2005) c-Myc-regulated microRNAs modulate E2F1 expression. Nature 435(7043):839–843CrossRefPubMedGoogle Scholar
  29. 29.
    Chang TC et al (2008) Widespread microRNA repression by Myc contributes to tumorigenesis. Nat Genet 40(1):43–50CrossRefPubMedGoogle Scholar
  30. 30.
    He L et al (2007) microRNAs join the p53 network—another piece in the tumour-suppression puzzle. Nat Rev Cancer 7(11):819–822CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Kota J et al (2009) Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model. Cell 137(6):1005–1017CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Orellana EA, Kasinski AL (2015) MicroRNAs in cancer: a historical perspective on the path from discovery to therapy. Cancers 7(3):1388–1405CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Mirna T (2013) Mirna therapuetics is first to advance microRNA into the clinic for cancer. Accessed 13 May 2013
  34. 34.
    Daige CL et al (2014) Systemic delivery of a miR-34a mimic as a potential therapeutic for liver cancer. Mol Cancer Ther 13(10):2352–2360CrossRefPubMedGoogle Scholar
  35. 35.
    Kasinski AL et al (2014) A combinatorial microRNA therapeutics approach to suppressing non-small cell lung cancer. Oncogene 34(27):3547–3555CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Xue W et al (2014) Small RNA combination therapy for lung cancer. Proc Natl Acad Sci U S A 111(34):E3553–E3561CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Iorio MV, Croce CM (2012) MicroRNA dysregulation in cancer: diagnostics, monitoring and therapeutics. A comprehensive review. EMBO Mol Med 4(3):143–159CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Cortez MA et al (2011) MicroRNAs in body fluids—the mix of hormones and biomarkers. Nat Rev Clin Oncol 8(8):467–477CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Boeri M et al (2011) MicroRNA signatures in tissues and plasma predict development and prognosis of computed tomography detected lung cancer. Proc Natl Acad Sci U S A 108(9):3713–3718CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Allegra A et al (2012) Circulating microRNAs: new biomarkers in diagnosis, prognosis and treatment of cancer (review). Int J Oncol 41(6):1897–1912PubMedGoogle Scholar
  41. 41.
    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):15524–15529CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Iram S (2014) Northern hybridization: a proficient method for detection of small RNAs and microRNAs. Methods Mol Biol 1099:179–188CrossRefPubMedGoogle Scholar
  43. 43.
    Babak T et al (2004) Probing microRNAs with microarrays: tissue specificity and functional inference. RNA 10(11):1813–1819CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Nelson PT et al (2004) Microarray-based, high-throughput gene expression profiling of microRNAs. Nat Methods 1(2):155–161CrossRefPubMedGoogle Scholar
  45. 45.
    Baskerville S, Bartel DP (2005) Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes. RNA 11(3):241–247CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Landgraf P et al (2007) A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 129(7):1401–1414CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Hu G et al (2012) Identification of miRNA signatures during the differentiation of hESCs into retinal pigment epithelial cells. PLoS One 7(7), e37224CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Meacham CE, Morrison SJ (2013) Tumour heterogeneity and cancer cell plasticity. Nature 501(7467):328–337CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Marte B (2013) Tumour heterogeneity. Nature 501(7467):327CrossRefPubMedGoogle Scholar
  50. 50.
    Junttila MR, de Sauvage FJ (2013) Influence of tumour micro-environment heterogeneity on therapeutic response. Nature 501(7467):346–354CrossRefPubMedGoogle Scholar
  51. 51.
    Kwon JJ et al (2015) Pathophysiological role of microRNA-29 in pancreatic cancer stroma. Sci Rep 5:11450CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Sempere LF et al (2010) Fluorescence-based codetection with protein markers reveals distinct cellular compartments for altered MicroRNA expression in solid tumors. Clin Cancer Res 16(16):4246–4255CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Burgess A et al (2010) Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance. Proc Natl Acad Sci U S A 107(28):12564–12569CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Sushuma Yarlagadda
    • 1
  • Anusha Thota
    • 1
  • Ruchi Bansal
    • 1
  • Jason Kwon
    • 1
  • Murray Korc
    • 1
  • Janaiah Kota
    • 1
    Email author
  1. 1.Department of Medical and Molecular Genetics, 2 Simon Cancer CenterIndiana University School of MedicineIndianapolisUSA

Personalised recommendations