Expression Profiles of Estrogen-Regulated MicroRNAs in Breast Cancer Cells

  • Anne Katchy
  • Cecilia Williams
Part of the Methods in Molecular Biology book series (MIMB, volume 1366)


Molecular signaling through both estrogen and microRNAs are critical for breast cancer development and growth. The activity of estrogen is mediated by transcription factors, the estrogen receptors. Here we describe a method for robust characterization of estrogen-regulated microRNA profiles. The method details how to prepare cells for optimal estrogen response, directions for estrogen treatment, RNA extraction, microRNA large-scale profiling, and subsequent confirmations.

Key words

Breast cancer MicroRNA Estrogen Estrogenreceptor Microarray qPCR 



This work was supported by NIH grant CA172437 and by co-funding from Marie Curie Actions FP7-PEOPLE-2011-COFUND (GROWTH 291795) via the VINNOVA programme Mobility for Growth.


  1. 1.
    Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297CrossRefGoogle Scholar
  2. 2.
    Williams C, Lin CY (2013) Oestrogen receptors in breast cancer: basic mechanisms and clinical implications. Ecancermedicalscience 7:370PubMedPubMedCentralGoogle Scholar
  3. 3.
    Klinge CM (2012) miRNAs and estrogen action. Trends Endocrinol Metab 23:223–233CrossRefGoogle Scholar
  4. 4.
    Wickramasinghe NS, Manavalan TT, Dougherty SM et al (2009) Estradiol downregulates miR-21 expression and increases miR-21 target gene expression in MCF-7 breast cancer cells. Nucleic Acids Res 37:2584–2595CrossRefGoogle Scholar
  5. 5.
    Bhat-Nakshatri P, Wang G, Collins NR et al (2009) Estradiol-regulated microRNAs control estradiol response in breast cancer cells. Nucleic Acids Res 37:4850–4861CrossRefGoogle Scholar
  6. 6.
    Gupta A, Caffrey E, Callagy G et al (2012) Oestrogen-dependent regulation of miRNA biogenesis: many ways to skin the cat. Biochem Soc Trans 40:752–758CrossRefGoogle Scholar
  7. 7.
    Katchy A, Edvardsson K, Aydogdu E et al (2012) Estradiol-activated estrogen receptor alpha does not regulate mature microRNAs in T47D breast cancer cells. J Steroid Biochem Mol Biol 128:145–153CrossRefGoogle Scholar
  8. 8.
    Katchy A, Williams C (2014) Profiling of estrogen-regulated microRNAs in breast cancer cells. J Vis Exp 84:e51285Google Scholar
  9. 9.
    Kao J, Salari K, Bocanegra M et al (2009) Molecular profiling of breast cancer cell lines defines relevant tumor models and provides a resource for cancer gene discovery. PLoS One 4:e6146CrossRefGoogle Scholar
  10. 10.
    Lacroix M, Leclercq G (2004) Relevance of breast cancer cell lines as models for breast tumours: an update. Breast Cancer Res Treat 83:249–289CrossRefGoogle Scholar
  11. 11.
    Ross DT, Perou CM (2001) A comparison of gene expression signatures from breast tumors and breast tissue derived cell lines. Dis Markers 17:99–109CrossRefGoogle Scholar
  12. 12.
    Neve RM, Chin K, Fridlyand J et al (2006) A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 10:515–527CrossRefGoogle Scholar
  13. 13.
    Frasor J, Danes JM, Komm B et al (2003) Profiling of estrogen up- and down-regulated gene expression in human breast cancer cells: insights into gene networks and pathways underlying estrogenic control of proliferation and cell phenotype. Endocrinology 144:4562–4574CrossRefGoogle Scholar
  14. 14.
    Williams C, Edvardsson K, Lewandowski SA et al (2008) A genome-wide study of the repressive effects of estrogen receptor beta on estrogen receptor alpha signaling in breast cancer cells. Oncogene 27:1019–1032CrossRefGoogle Scholar
  15. 15.
    Zhou X, Zhu Q, Eicken C et al (2012) MicroRNA profiling using microParaflo microfluidic array technology. Methods Mol Biol 822:153–182CrossRefGoogle Scholar
  16. 16.
    Griffiths-Jones S, Grocock RJ, van Dongen S et al (2006) miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res 34:D140–D144CrossRefGoogle Scholar
  17. 17.
    Chen C, Ridzon DA, Broomer AJ et al (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33:e179CrossRefGoogle Scholar
  18. 18.
    Benes V, Castoldi M (2010) Expression profiling of microRNA using real-time quantitative PCR, how to use it and what is available. Methods 50:244–249CrossRefGoogle Scholar
  19. 19.
    Baker M (2010) MicroRNA profiling: separating signal from noise. Nat Methods 7:687–692CrossRefGoogle Scholar
  20. 20.
    Schmittgen TD, Jiang J, Liu Q et al (2004) A high-throughput method to monitor the expression of microRNA precursors. Nucleic Acids Res 32:e43CrossRefGoogle Scholar
  21. 21.
    Wesierska-Gadek J, Schreiner T, Maurer M et al (2007) Phenol red in the culture medium strongly affects the susceptibility of human MCF-7 cells to roscovitine. Cell Mol Biol Lett 12:280–293CrossRefGoogle Scholar
  22. 22.
    Welshons WV, Wolf MF, Murphy CS et al (1988) Estrogenic activity of phenol red. Mol Cell Endocrinol 57:169–178CrossRefGoogle Scholar
  23. 23.
    Katchy A, Pinto C, Jonsson J et al (2014) Co-exposure to phytoestrogens and bisphenol A mimics estrogenic effects in an additive manner. Toxicol Sci 138:21–35CrossRefGoogle Scholar
  24. 24.
    Kim YK, Yeo J, Kim B et al (2012) Short structured RNAs with low GC content are selectively lost during extraction from a small number of cells. Mol Cell 46:893–895CrossRefGoogle Scholar
  25. 25.
    Setiawan AN, Lokman PM (2010) The use of reference gene selection programs to study the silvering transformation in a freshwater eel Anguilla australis: a cautionary tale. BMC Mol Biol 11:75CrossRefGoogle Scholar
  26. 26.
    Yu YT, Maroney PA, Darzynkiwicz E et al (1995) U6 snRNA function in nuclear pre-mRNA splicing: a phosphorothioate interference analysis of the U6 phosphate backbone. RNA 1:46–54PubMedPubMedCentralGoogle Scholar
  27. 27.
    Yuan JS, Reed A, Chen F et al (2006) Statistical analysis of real-time PCR data. BMC Bioinformatics 7:85CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Center for Nuclear Receptors and Cell Signaling, Department of Biology and BiochemistryUniversity of HoustonHoustonUSA
  2. 2.Department of Biology and BiochemistryCenter for Nuclear Receptors and Cell Signaling, University of HoustonHoustonUSA
  3. 3.SciLifeLab, Department of Proteomics and NanobiotechnologyKTH—Royal Institute of TechnologySolnaSweden

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