Estradiol-Induced Transcriptional Regulation of Long Non-Coding RNA, HOTAIR

  • Arunoday Bhan
  • Subhrangsu S. Mandal
Part of the Methods in Molecular Biology book series (MIMB, volume 1366)


HOTAIR (HOX antisense intergenic RNA) is a 2.2 kb long non-coding RNA (lncRNA), transcribed from the antisense strand of homeobox C (HOXC) gene locus in chromosome 12. HOTAIR acts as a scaffolding lncRNA. It interacts and guides various chromatin-modifying complexes such as PRC2 (polycomb-repressive complex 2) and LSD1 (lysine-specific demethylase 1) to the target gene promoters leading to their gene silencing. Various studies have demonstrated that HOTAIR overexpression is associated with breast cancer. Recent studies from our laboratory demonstrate that HOTAIR is required for viability of breast cancer cells and is transcriptionally regulated by estradiol (E2) in vitro and in vivo. This chapter describes protocols for analysis of the HOTAIR promoter, cloning, transfection and dual luciferase assays, knockdown of protein synthesis by antisense oligonucleotides, and chromatin immunoprecipitation (ChIP) assay. These protocols are useful for studying the estrogen-mediated transcriptional regulation of lncRNA HOTAIR, as well as other protein coding genes and non-coding RNAs.

Key words

Long non-coding RNA HOTAIR Estradiol Estrogenreceptors Gene expression Epigenetics Mixed lineage leukemia Polycomb-repressive complex 2 



We thank all the Mandal lab members for helpful discussions. Research in Mandal laboratory is supported by a grant from NIH (1R15 ES019129-01).


  1. 1.
    Wahlestedt C (2013) Targeting long non-coding RNA to therapeutically upregulate gene expression. Nat Rev Drug Discov 12:433–446CrossRefGoogle Scholar
  2. 2.
    Bhan A, Mandal SS (2014) Long noncoding RNAs: emerging stars in gene regulation, epigenetics and human disease. ChemMedChem 9:1932–1956CrossRefGoogle Scholar
  3. 3.
    Grammatikakis I, Panda AC, Abdelmohsen K, Gorospe M (2014) Long noncoding RNAs (lncRNAs) and the molecular hallmarks of aging. Aging (Albany, NY) 6:992–1009CrossRefGoogle Scholar
  4. 4.
    Hu CC, Gan P, Zhang RY, Xue JX, Ran LK (2014) Identification of prostate cancer LncRNAs by RNA-Seq. Asian Pac J Cancer Prev 15:9439–9444CrossRefGoogle Scholar
  5. 5.
    Cogill SB, Wang L (2014) Co-expression network analysis of human lncRNAs and cancer genes. Cancer Inform 13:49–59PubMedPubMedCentralGoogle Scholar
  6. 6.
    Rinn JL, Kertesz M, Wang JK et al (2007) Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129:1311–1323CrossRefGoogle Scholar
  7. 7.
    Tsai MC, Manor O, Wan Y et al (2010) Long noncoding RNA as modular scaffold of histone modification complexes. Science 329:689–693CrossRefGoogle Scholar
  8. 8.
    Gupta RA, Shah N, Wang KC et al (2010) Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature 464:1071–1076CrossRefGoogle Scholar
  9. 9.
    Shang D, Yang H, Xu Y et al (2015) A global view of network of lncRNAs and their binding proteins. Mol Biosyst 11:656–663CrossRefGoogle Scholar
  10. 10.
    Sorensen KP, Thomassen M, Tan Q et al (2013) Long non-coding RNA HOTAIR is an independent prognostic marker of metastasis in estrogen receptor-positive primary breast cancer. Breast Cancer Res Treat 142:529–536CrossRefGoogle Scholar
  11. 11.
    Bhan A, Hussain I, Ansari KI, Kasiri S, Bashyal A, Mandal SS (2013) Antisense transcript long noncoding RNA (lncRNA) HOTAIR is transcriptionally induced by estradiol. J Mol Biol 425:3707–3722CrossRefGoogle Scholar
  12. 12.
    Bhan A, Hussain I, Ansari KI, Bobzean SA, Perrotti LI, Mandal SS (2014) Bisphenol-A and diethylstilbestrol exposure induces the expression of breast cancer associated long noncoding RNA HOTAIR in vitro and in vivo. J Steroid Biochem Mol Biol 141:160–170CrossRefGoogle Scholar
  13. 13.
    Hussain I, Bhan A, Ansari KI et al (2015) Bisphenol-A induces expression of HOXC6, an estrogen-regulated homeobox-containing gene associated with breast cancer. Biochim Biophys Acta 1849(6):697–708CrossRefGoogle Scholar
  14. 14.
    Shrestha B, Ansari KI, Bhan A, Kasiri S, Hussain I, Mandal SS (2012) Homeodomain-containing protein HOXB9 regulates expression of growth and angiogenic factors, facilitates tumor growth in vitro and is overexpressed in breast cancer tissue. FEBS J 279:3715–3726CrossRefGoogle Scholar
  15. 15.
    Bhan A, Hussain I, Ansari KI, Bobzean SA, Perrotti LI, Mandal SS (2014) Histone methyltransferase EZH2 is transcriptionally induced by estradiol as well as estrogenic endocrine disruptors bisphenol-A and diethylstilbestrol. J Mol Biol 426:3426–3441CrossRefGoogle Scholar
  16. 16.
    Ansari KI, Kasiri S, Hussain I, Bobzean SA, Perrotti LI, Mandal SS (2013) MLL histone methylases regulate expression of HDLR-SR-B1 in presence of estrogen and control plasma cholesterol in vivo. Mol Endocrinol 27:92–105CrossRefGoogle Scholar
  17. 17.
    Ansari KI, Kasiri S, Mandal SS (2013) Histone methylase MLL1 has critical roles in tumor growth and angiogenesis and its knockdown suppresses tumor growth in vivo. Oncogene 32:3359–3370CrossRefGoogle Scholar
  18. 18.
    Ansari KI, Kasiri S, Mishra BP, Mandal SS (2012) Mixed lineage leukaemia-4 regulates cell-cycle progression and cell viability and its depletion suppresses growth of xenografted tumour in vivo. Br J Cancer 107:315–324CrossRefGoogle Scholar
  19. 19.
    Ansari KI, Hussain I, Kasiri S, Mandal SS (2012) HOXC10 is overexpressed in breast cancer and transcriptionally regulated by estrogen via involvement of histone methylases MLL3 and MLL4. J Mol Endocrinol 48:61–75CrossRefGoogle Scholar
  20. 20.
    Ansari KI, Hussain I, Shrestha B, Kasiri S, Mandal SS (2011) HOXC6 is transcriptionally regulated via coordination of MLL histone methylase and estrogen receptor in an estrogen environment. J Mol Biol 411:334–349CrossRefGoogle Scholar
  21. 21.
    Ansari KI, Shrestha B, Hussain I, Kasiri S, Mandal SS (2011) Histone methylases MLL1 and MLL3 coordinate with estrogen receptors in estrogen-mediated HOXB9 expression. Biochemistry 50:3517–3527CrossRefGoogle Scholar
  22. 22.
    Aartsma-Rus A, van Vliet L, Hirschi M et al (2009) Guidelines for antisense oligonucleotide design and insight into splice-modulating mechanisms. Mol Ther 17:548–553CrossRefGoogle Scholar
  23. 23.
    Malcolm AD (1992) Uses of antisense nucleic acids—an introduction. Biochem Soc Trans 20:745–746CrossRefGoogle Scholar
  24. 24.
    Kasiri S, Ansari KI, Hussain I, Bhan A, Mandal SS (2013) Antisense oligonucleotide mediated knockdown of HOXC13 affects cell growth and induces apoptosis in tumor cells and over expression of HOXC13 induces 3D-colony formation. RSC Adv 3:3260–3269CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryThe University of Texas at ArlingtonArlingtonUSA

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