Hormones and Cancer

, Volume 3, Issue 3, pp 65–78 | Cite as

Effects of Oestrogen on MicroRNA Expression in Hormone-Responsive Breast Cancer Cells

  • Lorenzo Ferraro
  • Maria Ravo
  • Giovanni Nassa
  • Roberta Tarallo
  • Maria Rosaria De Filippo
  • Giorgio Giurato
  • Francesca Cirillo
  • Claudia Stellato
  • Silvana Silvestro
  • Concita Cantarella
  • Francesca Rizzo
  • Daniela Cimino
  • Olivier Friard
  • Nicoletta Biglia
  • Michele De Bortoli
  • Luigi Cicatiello
  • Ernesto Nola
  • Alessandro WeiszEmail author


Oestrogen receptor alpha (ERα) is a ligand-dependent transcription factor that mediates oestrogen effects in hormone-responsive cells. Following oestrogenic activation, ERα directly regulates the transcription of target genes via DNA binding. MicroRNAs (miRNAs) represent a class of small noncoding RNAs that function as negative regulators of protein-coding gene expression. They are found aberrantly expressed or mutated in cancer, suggesting their crucial role as either oncogenes or tumour suppressor genes. Here, we analysed changes in miRNA expression in response to oestrogen in hormone-responsive breast cancer MCF-7 and ZR-75.1 cells by microarray-mediated expression profiling. This led to the identification of 172 miRNAs up- or down-regulated by ERα in response to 17β-oestradiol, of which 52 are similarly regulated by the hormone in the two cell models investigated. To identify mechanisms by which ERα exerts its effects on oestrogen-responsive miRNA genes, the oestrogen-dependent miRNA expression profiles were integrated with global in vivo ERα binding site mapping in the genome by ChIP-Seq. In addition, data from miRNA and messenger RNA (mRNA) expression profiles obtained under identical experimental conditions were compared to identify relevant miRNA target transcripts. Results show that miRNAs modulated by ERα represent a novel genomic pathway to impact oestrogen-dependent processes that affect hormone-responsive breast cancer cell behaviour. MiRNome analysis in tumour tissues from breast cancer patients confirmed a strong association between expression of these small RNAs and clinical outcome of the disease, although this appears to involve only marginally the oestrogen-regulated miRNAs identified in this study.


Oestrogen receptor Breast cancer MicroRNA Cell cycle Gene expression 



The authors thank Rosario Casale and Maria Francesca Papa for technical assistance and Claudio Scafoglio for critically reading the revised manuscript. Work supported by: European Union (CRESCENDO I.P., contract number LSHM-CT2005-018652), Italian Association for Cancer Research (grant IG-8586), Ministry for Education, University and Research (grants PRIN 2008CJ4SYW_004) and University of Salerno (Fondi FARB 2011). CC, FR, GN and RT are fellows of Fondazione con il Sud, MR is supported by a ‘Vladimir Ashkenazy’ fellowship of Italian Association for Cancer Research, MRDF is PhD student of the Research Doctorate ‘Computational Biology and Bioinformatics’ of the University of Napoli ‘Federico II’ supported by Fondazione IRCCS SDN, FC is PhD student of the Research Doctorate ‘Molecular Pathology and Physiopathology’ of the University of Napoli ‘Federico II’, GG and SS are PhD students of the Research Doctorate ‘Experimental Physiopathology and Neurosciences’ of the Second University of Napoli and CS is PhD student of the Research Doctorate ‘Molecular Oncology, Experimental Immunology and Innovative Therapy Development’ of the ‘Magna Graecia’ University of Catanzaro.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

12672_2012_102_MOESM1_ESM.doc (5.8 mb)
ESM 1 (DOC 1.77 mb)


  1. 1.
    Heldring N, Pike A, Andersson S, Matthews J, Cheng G, Hartman J, Tujague M et al (2007) Estrogen receptors: how do they signal and what are their targets. Physiol Rev 87:905–931PubMedCrossRefGoogle Scholar
  2. 2.
    Hall JM, Couse JF, Korach KS (2001) The multifaceted mechanisms of estradiol and estrogen receptor signaling. J Biol Chem 276:36869–36872PubMedCrossRefGoogle Scholar
  3. 3.
    Bai Z, Gust R (2009) Breast cancer, estrogen receptor and ligands. Arch Pharm (Weinheim) 342:133–149CrossRefGoogle Scholar
  4. 4.
    Kushner PJ, Agard DA, Greene GL, Scanlan TS, Shiau AK, Uht RM, Webb P (2000) Estrogen receptor pathways to AP-1. J Steroid Biochem Mol Biol 74:311–317PubMedCrossRefGoogle Scholar
  5. 5.
    Saville B, Wormke M, Wang F, Nguyen T, Enmark E, Kuiper G, Gustafsson JA et al (2000) Ligand-, cell-, estrogen receptor subtype (alpha/beta)-dependent activation at GC-rich (Sp1) promoter elements. J Biol Chem 275:5379–5387PubMedCrossRefGoogle Scholar
  6. 6.
    McDonnell DP, Norris JD (2002) Connections and regulation of the human estrogen receptor. Science 296:1642–1644PubMedCrossRefGoogle Scholar
  7. 7.
    Klinge CM, Jernigan SC, Mattingly KA, Risinger KE, Zhang J (2004) Estrogen response element-dependent regulation of transcriptional activation of estrogen receptors alpha and beta by coactivators and corepressors. J Mol Endocrinol 33:387–410PubMedCrossRefGoogle Scholar
  8. 8.
    Chen GG, Zeng Q, Tse GM (2008) Estrogen and its receptors in cancer. Med Res Rev 28:954–974PubMedCrossRefGoogle Scholar
  9. 9.
    Manavathi B, Kumar R (2006) Steering estrogen signals from the plasma membrane to the nucleus: two sides of the coin. J Cell Physiol 207:594–604PubMedCrossRefGoogle Scholar
  10. 10.
    Safe S, Kim KJ (2008) Non-classical genomic estrogen receptor (ER)/specificity protein and ER/activating protein-1 signaling pathways. J Mol Endocrinol 41:263–275PubMedCrossRefGoogle Scholar
  11. 11.
    Gronemeyer H, Gustafsson JA, Laudet V (2004) Principles for modulation of the nuclear receptor superfamily. Nat Rev Drug Discov 3:950–964PubMedCrossRefGoogle Scholar
  12. 12.
    Kim VN (2005) MicroRNA biogenesis: coordinated cropping and dicing. Nat Rev Mol Cell Biol 6:376–385PubMedCrossRefGoogle Scholar
  13. 13.
    Xiong H, Qian J, He T, Li F (2009) Indipendent transcription of miR-281 in the intron of ODA in Drosophila melanogaster. Biochem Biophys Res Commun 378:883–889PubMedCrossRefGoogle Scholar
  14. 14.
    Rodriguez A, Griffiths-Jones S, Ashurst JL, Bradley A (2004) Identification of mammalian microRNA host genes and transcription units. Genome Res 14:1902–1910PubMedCrossRefGoogle Scholar
  15. 15.
    Saini HK, Griffiths-Jones S, Enright AJ (2007) Genomic analysis of human microRNA transcripts. Proc Natl Acad Sci USA 104:17719–17724PubMedCrossRefGoogle Scholar
  16. 16.
    Lee Y, Kim M, Han JJ, Yeom KH, Lee S, Baek SH, Kim VN (2004) MicroRNA genes are transcribed by RNA polymerase II. EMBO J 23:4051–4060PubMedCrossRefGoogle Scholar
  17. 17.
    Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ (2004) Processing of primary microRNAs by the Microprocessor complex. Nature 432:231–235PubMedCrossRefGoogle Scholar
  18. 18.
    Lee Y, Jeon K, Lee JT, Kim S, Kim VN (2002) microRNA maturation: stepwise processing and subcellular localization. EMBO J 21:4663–4670PubMedCrossRefGoogle Scholar
  19. 19.
    Gregory RI, Chendrimada TP, Cooch N, Shiekhattar R (2005) Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell 123:631–640PubMedCrossRefGoogle Scholar
  20. 20.
    Engels BM, Hutvagner G (2006) Principles and effects of microRNA-mediated post-transcriptional gene regulation. Oncogene 25:6163–6169PubMedCrossRefGoogle Scholar
  21. 21.
    He L, Hannon GJ (2004) MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 5:522–531PubMedCrossRefGoogle Scholar
  22. 22.
    Linsen S, Tops B, Cuppen E (2008) miRNAs small changes, widespread effects. Cell Res 18:1157–1159PubMedCrossRefGoogle Scholar
  23. 23.
    Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120:15–20PubMedCrossRefGoogle Scholar
  24. 24.
    Zhao Y, Srivastava D (2007) A developmental view of microRNA function. Trends Biochem Sci 32:189–197PubMedCrossRefGoogle Scholar
  25. 25.
    Ventura A, Jacks T (2009) MicroRNAs and cancer: short RNAs go a long way. Cell 136:586–591PubMedCrossRefGoogle Scholar
  26. 26.
    Tessel MA, Krett NL, Rosen ST (2010) Steroid receptor and microRNA regulation in cancer. Curr Opin Oncol 22:592–597PubMedCrossRefGoogle Scholar
  27. 27.
    Klinge CM (2009) Estrogen regulation of miRNA expression. Curr Genomics 10:169–183PubMedCrossRefGoogle Scholar
  28. 28.
    Bhat-Nakshatri P, Wang G, Collins NR, Thomson MJ, Geistlinger TR, Carroll JS, Brown M et al (2009) Estradiol-regulated microRNAs control estradiol response in breast cancer cells. Nucleic Acids Res 37:4850–4861PubMedCrossRefGoogle Scholar
  29. 29.
    Yang Z, Wang L (2011) Regulation of microRNA expression and function by nuclear receptor signaling. Cell Biosci 1:31PubMedCrossRefGoogle Scholar
  30. 30.
    Paris O, Ferraro L, Grober OMV, Ravo M, De Filippo MR, Giurato G, Nassa G, Tarallo et al. (2012) Direct regulation of microRNA biogenesis and expression by estrogen receptor beta in hormone-responsive breast cancer. Oncogene. doi: 10.1038/onc.2011.583
  31. 31.
    Esquela-Kerscher A, Slack FJ (2006) Oncomirs—microRNAs with a role in cancer. Nat Rev Cancer 6:259–269PubMedCrossRefGoogle Scholar
  32. 32.
    Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, Sabbioni S, Magri E et al (2005) MicroRNA gene expression deregulation in human breast cancer. Cancer Res 65:7065–7070PubMedCrossRefGoogle Scholar
  33. 33.
    Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, Sweet-Cordero A et al (2005) MicroRNA expression profiles classify human cancers. Nature 435:834–838PubMedCrossRefGoogle Scholar
  34. 34.
    Blenkiron C, Goldstein LD, Thorne NP, Spiteri I, Chin SF, Dunning MJ, Barbosa-Morais NL et al (2007) MicroRNA expression profiling of human breast cancer identifies new markers of tumor subtype. Genome Biol 8:R214PubMedCrossRefGoogle Scholar
  35. 35.
    Gaur A, Jewell DA, Liang Y, Ridzon D, Moore JH, Chen C, Ambros VR et al (2007) Characterization of microRNA expression levels and their biological correlates in human cancer cell lines. Cancer Res 67:2456–2468PubMedCrossRefGoogle Scholar
  36. 36.
    O’Day E, Lal A (2010) MicroRNAs and their target gene networks in breast cancer. Breast Cancer Res 12:201PubMedCrossRefGoogle Scholar
  37. 37.
    Adams BD, Furneaux H, White BA (2007) The micro-ribonucleic acid (miRNA) miR-206 targets the human estrogen receptor alpha (ERalpha) and represses ERalpha messenger RNA and protein expression in breast cancer cell lines. Mol Endocrinol 21:1132–1147PubMedCrossRefGoogle Scholar
  38. 38.
    Hossain A, Kuo MT, Saunders GF (2006) Mir-17-5p regulates breast cancer cell proliferation by inhibiting translation of AIB1 mRNA. Mol Cell Biol 26:8191–8201PubMedCrossRefGoogle Scholar
  39. 39.
    Yu Z, Wang C, Wang M, Li Z, Casimiro MC, Liu M, Wu K et al (2008) A cyclin D1/microRNA 17/20 regulatory feedback loop in control of breast cancer cell proliferation. J Cell Biol 182:509–517PubMedCrossRefGoogle Scholar
  40. 40.
    Gaur A, Jewell DA, Liang Y, Ridzon D, Moore JH, Chen C, Ambros VR et al (2007) Characterization of microRNA expression levels and their biological correlates in human cancer cell lines. Cancer Res 67:2456–2468PubMedCrossRefGoogle Scholar
  41. 41.
    Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, Visone R et al (2006) A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci USA 103:2257–2261PubMedCrossRefGoogle Scholar
  42. 42.
    Gusev Y, Schmittgen TD, Lerner M, Postier R, Brackett D (2007) Computational analysis of biological functions and pathways collectively targeted by co-expressed microRNAs in cancer. BMC Bioinforma 8(Suppl 7):S16CrossRefGoogle Scholar
  43. 43.
    Cochrane DR, Cittelly DM, Howe EN, Spoelstra NS, McKinsey EL, LaPara K, Elias A et al (2010) MicroRNAs link estrogen receptor alpha status and dicer levels in breast cancer. Horm Cancer 1:306–319PubMedCrossRefGoogle Scholar
  44. 44.
    Ambrosino C, Tarallo R, Bamundo A, Cuomo D, Franci G, Nassa G, Paris O et al (2010) Identification of a hormone-regulated dynamic nuclear actin network associated with estrogen receptor alpha in human breast cancer cell nuclei. Mol Cell Proteomics 9:1352–1367PubMedCrossRefGoogle Scholar
  45. 45.
    Cimino D, Fuso L, Sfiligoi C, Biglia N, Ponzone R, Maggiorotto F, Russo G et al (2008) Identification of new genes associated with breast cancer progression by gene expression analysis of predefined set of neoplastic tissues. Int J Cancer 123:1327–1338PubMedCrossRefGoogle Scholar
  46. 46.
    Scafoglio C, Ambrosino C, Cicatiello L, Altucci L, Ardovino M, Bontempo P, Medici N et al (2006) Comparative gene expression profiling reveals partially overlapping but distinct genomic actions of different antiestrogens in human breast cancer cells. J Cell Biochem 98:1163–1184PubMedCrossRefGoogle Scholar
  47. 47.
    Cicatiello L, Scafoglio C, Altucci L, Cancemi M, Natoli G, Facchiano A, Iazzetti G et al (2004) A genomic view of estrogen actions in human breast cancer cells by expression profiling of the hormone-responsive transcriptome. J Mol Endocrinol 32:719–775PubMedCrossRefGoogle Scholar
  48. 48.
    Cicatiello L, Mutarelli M, Grober OMV, Paris O, Ferraro L, Ravo M, Tarallo R et al (2010) A gene network controlled by estrogen receptor α in luminal-like breast cancer cells comprising multiple transcription factors and microRNAs. Am J Pathol 176:2113–2130PubMedCrossRefGoogle Scholar
  49. 49.
    Ravo M, Mutarelli M, Ferraro L, Grober OMV, Paris O, Tarallo R, Vigilante A et al (2008) Quantitative expression profilino of highly degraded RNA from formalin-fixed, paraffin-embedded breast tumor biopsies by oligonucleotide microarrays. Lab Inv 88:430–440CrossRefGoogle Scholar
  50. 50.
    Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci 98:5116–5121PubMedCrossRefGoogle Scholar
  51. 51.
    Grober OMV, Mutarelli M, Giurato G, Ravo M, Cicatiello L, De Filippo MR, Ferraro L et al (2011) Global analysis of estrogen receptor beta binding to breast cancer cell genome reveals an extensive interplay with estrogen receptor alpha for target gene regulation. BMC Genomics 12:36PubMedCrossRefGoogle Scholar
  52. 52.
    Fejes AP, Robertson G, Bilenky M, Varhol R, Bainbridge M, Jones SJ (2008) FindPeaks 3.1: a tool for identifying areas of enrichment from massively parallel short-read sequencing technology. Bioinformatics 24:1729–1730PubMedCrossRefGoogle Scholar
  53. 53.
    Qiunian AR, Hall IN (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26:841–842CrossRefGoogle Scholar
  54. 54.
    Dennis G Jr, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA (2003) DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol 4:P3PubMedCrossRefGoogle Scholar
  55. 55.
    da Huang W, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. Nature Protoc 4:44–57CrossRefGoogle Scholar
  56. 56.
    Weigelt K, Moehle C, Stempfl T, Weber B, Langmann T (2008) An integrated workflow for analysis of ChIP-chip data. Biotechniques 45:131–132PubMedCrossRefGoogle Scholar
  57. 57.
    Cicatiello L, Scafoglio C, Altucci L, Cancemi M, Natoli G, Facchiano A, Iazzetti G et al (2004) A genomic view of estrogen actions in human breast cancer cells by expression profiling of the hormone-responsive transcriptome. J Mol Endocrinol 32:719–775PubMedCrossRefGoogle Scholar
  58. 58.
    Cicatiello L, Addeo R, Altucci L, Belsito Petrizzi V, Boccia V, Cancemi M, Germano D et al (2000) The antiestrogen ICI 182,780 inhibits proliferation of human breast cancer cells by interfering with multiple, sequential estrogen-regulated processes required for cell cycle completion. Mol Cell Endocrinol 165:199–209PubMedCrossRefGoogle Scholar
  59. 59.
    Castellano L, Giamas G, Jacob J, Coombes RC, Lucchesi W, Thiruchelvam P, Barton G et al (2009) The estrogen receptor-alpha-induced microRNA signature regulates itself and its transcriptional response. Proc Natl Acad Sci U S A 106:15732–15737PubMedCrossRefGoogle Scholar
  60. 60.
    Maillot G, Lacroix-Triki M, Pierredon S, Gratadou L, Schmidt S, Bénès V, Roché H et al (2009) Widespread estrogen-dependent repression of micrornas involved in breast tumor cell growth. Cancer Res 69:8332–8340PubMedCrossRefGoogle Scholar
  61. 61.
    Fullwood MJ, Liu MH, Pan YF, Liu J, Xu H, Mohamed YB, Orlov YL et al (2009) An oestrogen-receptor-alpha bound human chromatin interactome. Nature 462:58–64PubMedCrossRefGoogle Scholar
  62. 62.
    Kim VN, Han J, Siomi MC (2009) Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol 10:126–139PubMedCrossRefGoogle Scholar
  63. 63.
    Li X, Mertens-Talcott SU, Shu Z, KyoungHyun K, Judith B, Stephen S (2010) MicroRNA-27a indirectly regulates estrogen receptor alpha expression and hormone responsiveness in MCF-7 breast cancer cells. Endocrinology 151:2462–2473PubMedCrossRefGoogle Scholar
  64. 64.
    Nagel R, le Sage C, Diosdado B, van der Waal M, Oude Vrielink JA, Bolijn A, Meijer GA et al (2008) Regulation of the adenomatous polyposis coli gene by the miR-135 family in colorectal cancer. Cancer Res 68:5795–5802PubMedCrossRefGoogle Scholar
  65. 65.
    Navarro A, Diaz T, Martinez A, Gaya A, Pons A, Gel B, Codony C et al (2009) Regulation of JAK2 by miR-135a: prognostic impact in classic Hodgkin lymphoma. Blood 114:2945–2951PubMedCrossRefGoogle Scholar
  66. 66.
    Suzuki HI, Yamagata K, Sugimoto K, Iwamoto T, Kato S, Miyazono K (2009) Modulation of microRNA processing by p53. Nature 460:529–533PubMedCrossRefGoogle Scholar
  67. 67.
    Davis BN, Hilyard AC, Lagna G, Hata A (2008) SMAD proteins control DROSHA-mediated microRNA maturation. Nature 454:56–61PubMedCrossRefGoogle Scholar
  68. 68.
    Yamagata K, Fujiyama S, Ito S, Ueda T, Murata T, Naitou M, Takeyama K et al (2009) Maturation of microRNA is hormonally regulated by a nuclear receptor. Mol Cell 36:340–347PubMedCrossRefGoogle Scholar
  69. 69.
    Buffa FM, Camps C, Winchester L, Snell CE, Gee HE, Sheldon H, Taylor M et al (2011) microRNA-associated progression pathways and potential therapeutic targets identified by integrated mRNA and microRNA expression profiling in breast cancer. Cancer Res 71:5635–5645PubMedCrossRefGoogle Scholar
  70. 70.
    Qian B, Katsaros D, Lu L, Preti M, Durando A, Arisio R, Mu L et al (2009) High miR-21 expression in breast cancer associated with poor disease-free survival in early stage disease and highTGF-beta1. Breast Cancer Rea Treat 117:131–140CrossRefGoogle Scholar
  71. 71.
    Weisz A, Basile W, Scafoglio C, Altucci L, Bresciani F, Facchiano A, Sismondi P (2004) Molecular identification of ERalpha-positive breast cancer cells by the expression profile of an intrinsic set of estrogen regulated genes. J Cell Physiol 200:440–450PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Lorenzo Ferraro
    • 1
    • 2
  • Maria Ravo
    • 1
  • Giovanni Nassa
    • 1
  • Roberta Tarallo
    • 1
  • Maria Rosaria De Filippo
    • 3
  • Giorgio Giurato
    • 1
  • Francesca Cirillo
    • 2
  • Claudia Stellato
    • 1
  • Silvana Silvestro
    • 1
  • Concita Cantarella
    • 1
  • Francesca Rizzo
    • 1
  • Daniela Cimino
    • 4
  • Olivier Friard
    • 5
  • Nicoletta Biglia
    • 6
  • Michele De Bortoli
    • 5
  • Luigi Cicatiello
    • 2
  • Ernesto Nola
    • 2
  • Alessandro Weisz
    • 1
    • 2
    • 7
    Email author
  1. 1.Laboratory of Molecular Medicine and Genomics, Faculty of Medicine and SurgeryUniversity of SalernoBaronissiItaly
  2. 2.Department of General PathologySecond University of NaplesNapoliItaly
  3. 3.Fondazione IRCCS SDNNaplesItaly
  4. 4.Molecular Biotechnology Center and Department of Oncological SciencesUniversity of TurinTurinItaly
  5. 5.Center for Molecular Systems BiologyUniversity of TurinTurinItaly
  6. 6.Department of Obstetrics and Gynecology, Mauriziano ‘Umberto I’ HospitalUniversity of TurinTurinItaly
  7. 7.Division of Molecular Pathology and Medical Genomics, ‘SS. Giovanni di Dio e Ruggi d’Aragona’ HospitalUniversity of SalernoSalernoItaly

Personalised recommendations