, Volume 18, Issue 5, pp 674–684 | Cite as

Epidrug mediated re-expression of miRNA targeting the HMGA transcripts in pituitary cells

  • Mark O. Kitchen
  • Kiren Yacqub-Usman
  • Richard D. Emes
  • Alan Richardson
  • Richard N. Clayton
  • William E. FarrellEmail author



Transgenic mice overexpressing the high mobility group A (HMGA) genes, Hmga1 or Hmga2 develop pituitary tumours and their overexpression is also a frequent finding in human pituitary adenomas. In some cases, increased expression of HMGA2 but not that of HMGA1 is consequent to genetic perturbations. However, recent studies show that down-regulation of microRNA (miRNA), that contemporaneously target the HMGA1 and HMGA2 transcripts, are associated with their overexpression.


In a cohort of primary pituitary adenoma we determine the impact of epigenetic modifications on the expression of HMGA-targeting miRNA. For these miRNAs, chromatin immunoprecipitations showed that transcript down-regulation is correlated with histone tail modifications associated with condensed silenced genes. The functional impact of epigenetic modification on miRNA expression was determined in the rodent pituitary cell line, GH3. In these cells, histone tail, miRNA-associated, modifications were similar to those apparent in human adenoma and likely account for their repression. Indeed, challenge of GH3 cells with the epidrugs, zebularine and TSA, led to enrichment of the histone modification, H3K9Ac, associated with active genes, and depletion of the modification, H3K27me3, associated with silent genes and re-expression of HMGA-targeting miRNA. Moreover, epidrugs challenges were also associated with a concomitant decrease in hmga1 transcript and protein levels and concurrent increase in bmp-4 expression.


These findings show that the inverse relationship between HMGA expression and targeting miRNA is reversible through epidrug interventions. In addition to showing a mechanistic link between epigenetic modifications and miRNA expression these findings underscore their potential as therapeutic targets in this and other diseases.


Pituitary microRNA Chromatin Epidrugs HMGA1 



We are grateful and wish to extend our thanks to Dr Kim Haworth for assistance with pyrosequencing. This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

Conflict of interest

We hereby confirm that there is no financial or personal relationship between the authors and other people or organizations that can inappropriately influence the work and there is thereby no conflict of interest.

Supplementary material

11102_2014_630_MOESM1_ESM.pdf (57 kb)
Supplementary material 1 (PDF 56 kb)
11102_2014_630_MOESM2_ESM.docx (24 kb)
Supplementary material 2 (DOCX 24 kb)
11102_2014_630_MOESM3_ESM.pdf (506 kb)
Expression and histone tail modifications associated with HMGA-targeting miRNA in human pituitary adenomas. (A) From left to right, quantitative RT-PCR of miR-16, miR34b and miR-320 in non-functioning (NF), prolactinoma (P), corticotrophinoma (A) and growth hormone secreting adenomas (GH). Expression is reported relative to the mean of three post-mortem pituitaries, where the mean value was expressed as equal to 1. Each bar represents the mean value ± SEM from three independent experiments performed in triplicate. (B) Chromatin immunoprecipitation analysis (ChIP) for the histone tail modification associated with active genes, H3K9Ac. Adenomas, and three post-mortem normal pituitaries, are those shown in panel A and enrichment in each case is relative to input chromatin. (C) ChIP analysis of adenomas and post-mortem normal pituitaries shown in panels A and B, for the histone tail modification associated with silenced gene, H3K27Me3. In panels B and C each bar represents mean value ± SEM from three independent experiments performed in triplicate
11102_2014_630_MOESM4_ESM.pdf (251 kb)
Epidrug mediated effects on hmga1 targeting miRNA (A) From left to right, expression of miR-16, miR-34b and miR-320 in GH3 cells as determined by RT-qPCR and relative to normal rat pituitaries (NRP). Expression was determined in the absence or presence of the epidrugs, zebularine (Zeb) and trichostatin A (TSA) either alone or in combination. Doses of drugs are shown on the x axis. Expression is reported relative to the mean of three normal rat pituitaries (NRP), where the mean value was expressed as equal to 100 % and where each bar represents the mean value ± SEM from three independent experiments performed in triplicate. Panels B and C show the ChIP analysis of the cells shown in panel A, where panel B is the modification associated with active gene, H3K9Ac and panel C is the modification associated with silencing, H3K27me3. Each bar represents the mean value ± SEM from three independent experiments performed in triplicate. Data was analyzed for significance by one-way ANOVA with Dunnett’s multiple comparison post-test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 versus NRP


  1. 1.
    Melmed S (2003) Mechanisms for pituitary tumorigenesis: the plastic pituitary. J Clin Invest 112(11):1603–1618. doi: 10.1172/jci20401 PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Melmed S (2011) Pathogenesis of pituitary tumors. Nat Rev Endocrinol 7(5):257–266. doi: 10.1038/nrendo.2011.40 CrossRefPubMedGoogle Scholar
  3. 3.
    Dudley KJ, Revill K, Clayton RN, Farrell WE (2009) Pituitary tumours: all silent on the epigenetics front. J Mol Endocrinol 42(6):461–468. doi: 10.1677/jme-09-0009 CrossRefPubMedGoogle Scholar
  4. 4.
    Vandeva S, Jaffrain-Rea ML, Daly AF, Tichomirowa M, Zacharieva S, Beckers A (2010) The genetics of pituitary adenomas. Best practice & research. Clin Endocrinol Metab 24(3):461–476. doi: 10.1016/j.beem.2010.03.001 Google Scholar
  5. 5.
    Zhou Y, Zhang X, Klibanski A (2013) Genetic and epigenetic mutations of tumor suppressive genes in sporadic pituitary adenoma. Mol Cell Endocrinol. doi: 10.1016/j.mce.2013.09.006 PubMedCentralGoogle Scholar
  6. 6.
    Duong CV, Emes RD, Wessely F, Yacqub-Usman K, Clayton RN, Farrell WE (2012) Quantitative, genome-wide analysis of the DNA methylome in sporadic pituitary adenomas. Endocr Relat Cancer 19(6):805–816. doi: 10.1530/ERC-12-0251 CrossRefPubMedGoogle Scholar
  7. 7.
    Pease M, Ling C, Mack WJ, Wang K, Zada G (2013) The role of epigenetic modification in tumorigenesis and progression of pituitary adenomas: a systematic review of the literature. PLoS One 8(12):e82619. doi: 10.1371/journal.pone.0082619 PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Hayward BE, Barlier A, Korbonits M, Grossman AB, Jacquet P, Enjalbert A, Bonthron DT (2001) Imprinting of the G(s)alpha gene GNAS1 in the pathogenesis of acromegaly. J Clin Invest 107(6):R31–R36. doi: 10.1172/jci11887 PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Fedele M, Pierantoni GM, Visone R, Fusco A (2006) Critical role of the HMGA2 gene in pituitary adenomas. Cell Cycle 5(18):2045–2048CrossRefPubMedGoogle Scholar
  10. 10.
    Finelli P, Pierantoni GM, Giardino D, Losa M, Rodeschini O, Fedele M, Valtorta E, Mortini P, Croce CM, Larizza L, Fusco A (2002) The high mobility group A2 gene is amplified and overexpressed in human prolactinomas. Cancer Res 62(8):2398–2405PubMedGoogle Scholar
  11. 11.
    Palmieri D, D’Angelo D, Valentino T, De Martino I, Ferraro A, Wierinckx A, Fedele M, Trouillas J, Fusco A (2012) Downregulation of HMGA-targeting microRNAs has a critical role in human pituitary tumorigenesis. Oncogene 31(34):3857–3865. doi: 10.1038/onc.2011.557 CrossRefPubMedGoogle Scholar
  12. 12.
    Zamore PD, Haley B (2005) Ribo-gnome: the big world of small RNAs. Science 309(5740):1519–1524. doi: 10.1126/science.1111444 CrossRefPubMedGoogle Scholar
  13. 13.
    Huntzinger E, Izaurralde E (2011) Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nat Rev Genet 12(2):99–110. doi: 10.1038/nrg2936 CrossRefPubMedGoogle Scholar
  14. 14.
    D’Angelo D, Palmieri D, Mussnich P, Roche M, Wierinckx A, Raverot G, Fedele M, Croce CM, Trouillas J, Fusco A (2012) Altered microRNA expression profile in human pituitary GH adenomas: down-regulation of miRNA targeting HMGA1, HMGA2, and E2F1. J Clin Endocrinol Metab 97(7):E1128–E1138. doi: 10.1210/jc.2011-3482 CrossRefPubMedGoogle Scholar
  15. 15.
    Palumbo T, Faucz FR, Azevedo M, Xekouki P, Iliopoulos D, Stratakis CA (2013) Functional screen analysis reveals miR-26b and miR-128 as central regulators of pituitary somatomammotrophic tumor growth through activation of the PTEN–AKT pathway. Oncogene 32(13):1651–1659. doi: 10.1038/onc.2012.190 PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Gentilin E, Tagliati F, Filieri C, Mole D, Minoia M, Rosaria Ambrosio M, Degli Uberti EC, Zatelli MC (2013) miR-26a plays an important role in cell cycle regulation in ACTH-secreting pituitary adenomas by modulating protein kinase Cdelta. Endocrinology 154(5):1690–1700. doi: 10.1210/en.2012-2070 PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Fabbri M, Calin GA (2010) Epigenetics and miRNAs in human cancer. Adv Genet 70:87–99. doi: 10.1016/B978-0-12-380866-0.60004-6 CrossRefPubMedGoogle Scholar
  18. 18.
    Saito Y, Liang G, Egger G, Friedman JM, Chuang JC, Coetzee GA, Jones PA (2006) Specific activation of microRNA-127 with downregulation of the proto-oncogene BCL6 by chromatin-modifying drugs in human cancer cells. Cancer Cell 9(6):435–443. doi: 10.1016/j.ccr.2006.04.020 CrossRefPubMedGoogle Scholar
  19. 19.
    Al-Azzawi H, Yacqub-Usman K, Richardson A, Hofland LJ, Clayton RN, Farrell WE (2011) Reversal of endogenous dopamine receptor silencing in pituitary cells augments receptor-mediated apoptosis. Endocrinology 152(2):364–373. doi: 10.1210/en.2010-0886 CrossRefPubMedGoogle Scholar
  20. 20.
    Yacqub-Usman K, Duong CV, Clayton RN, Farrell WE (2012) Epigenomic silencing of the BMP-4 gene in pituitary adenomas: a potential target for epidrug-induced re-expression. Endocrinology. doi: 10.1210/en.2012-1231 Google Scholar
  21. 21.
    Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ (2008) miRBase: tools for microRNA genomics. Nucleic Acids Res 36:D154–D158. doi: 10.1093/nar/gkm952 PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Bhattacharyya M, Das M, Bandyopadhyay S (2012) miRT: a database of validated transcription start sites of human microRNAs. Genomics Proteomics Bioinformatics 10(5):310–316. doi: 10.1016/j.gpb.2012.08.005 CrossRefPubMedGoogle Scholar
  23. 23.
    Chien CH, Sun YM, Chang WC, Chiang-Hsieh PY, Lee TY, Tsai WC, Horng JT, Tsou AP, Huang HD (2011) Identifying transcriptional start sites of human microRNAs based on high-throughput sequencing data. Nucleic Acids Res 39(21):9345–9356. doi: 10.1093/nar/gkr604 PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Kramer MF (2011) Stem-loop RT-qPCR for miRNAs. In: Frederick M. Ausubel et al. (ed) Current protocols in molecular biology 15(15–10). doi: 10.1002/0471142727.mb1510s95
  25. 25.
    Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33(20):e179. doi: 10.1093/nar/gni178 PubMedCentralCrossRefPubMedGoogle Scholar
  26. 26.
    Bahar A, Simpson DJ, Cutty SJ, Bicknell JE, Hoban PR, Holley S, Mourtada-Maarabouni M, Williams GT, Clayton RN, Farrell WE (2004) Isolation and characterization of a novel pituitary tumor apoptosis gene. Mol Endocrinol 18(7):1827–1839. doi: 10.1210/me.2004-0087 CrossRefPubMedGoogle Scholar
  27. 27.
    Peltier HJ, Latham GJ (2008) Normalization of microRNA expression levels in quantitative RT-PCR assays: identification of suitable reference RNA targets in normal and cancerous human solid tissues. RNA 14(5):844–852. doi: 10.1261/rna.939908 PubMedCentralCrossRefPubMedGoogle Scholar
  28. 28.
    Revill K, Dudley KJ, Clayton RN, McNicol AM, Farrell WE (2009) Loss of neuronatin expression is associated with promoter hypermethylation in pituitary adenoma. Endocr Relat Cancer 16(2):537–548. doi: 10.1677/ERC-09-0008 CrossRefPubMedGoogle Scholar
  29. 29.
    Dudley KJ, Revill K, Whitby P, Clayton RN, Farrell WE (2008) Genome-wide analysis in a murine Dnmt1 knockdown model identifies epigenetically silenced genes in primary human pituitary tumors. MCR 6(10):1567–1574. doi: 10.1158/1541-7786.mcr-08-0234 CrossRefPubMedGoogle Scholar
  30. 30.
    Jenuwein T, Allis CD (2001) Translating the histone code. Science (New York, N.Y.) 293(5532):1074–1080. doi: 10.1126/science.1063127 CrossRefGoogle Scholar
  31. 31.
    Cao R, Wang L, Wang H, Xia L, Erdjument-Bromage H, Tempst P, Jones RS, Zhang Y (2002) Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science (New York, N.Y.) 298(5595):1039–1043. doi: 10.1126/science.1076997 CrossRefGoogle Scholar
  32. 32.
    Walker JM (1994) The bicinchoninic acid (BCA) assay for protein quantitation. Methods Mol Biol 32:5–8. doi: 10.1385/0-89603-268-x:5 PubMedGoogle Scholar
  33. 33.
    Pierantoni GM, Finelli P, Valtorta E, Giardino D, Rodeschini O, Esposito F, Losa M, Fusco A, Larizza L (2005) High-mobility group A2 gene expression is frequently induced in non-functioning pituitary adenomas (NFPAs), even in the absence of chromosome 12 polysomy. Endocr Relat Cancer 12(4):867–874. doi: 10.1677/erc.1.01049 CrossRefPubMedGoogle Scholar
  34. 34.
    Wang EL, Qian ZR, Yamada S, Rahman MM, Inosita N, Kageji T, Endo H, Kudo E, Sano T (2009) Clinicopathological characterization of TSH-producing adenomas: special reference to TSH-immunoreactive but clinically non-functioning adenomas. Endocr Pathol 20(4):209–220. doi: 10.1007/s12022-009-9094-y CrossRefPubMedGoogle Scholar
  35. 35.
    De Martino I, Visone R, Fedele M, Petrocca F, Palmieri D, Martinez Hoyos J, Forzati F, Croce CM, Fusco A (2009) Regulation of microRNA expression by HMGA1 proteins. Oncogene 28(11):1432–1442. doi: 10.1038/onc.2008.495 CrossRefPubMedGoogle Scholar
  36. 36.
    Baldassarre G, Fedele M, Battista S, Vecchione A, Klein-Szanto AJ, Santoro M, Waldmann TA, Azimi N, Croce CM, Fusco A (2001) Onset of natural killer cell lymphomas in transgenic mice carrying a truncated HMGI-C gene by the chronic stimulation of the IL-2 and IL-15 pathway. Proc Natl Acad Sci USA 98(14):7970–7975. doi: 10.1073/pnas.141224998 PubMedCentralCrossRefPubMedGoogle Scholar
  37. 37.
    Fedele M, Battista S, Kenyon L, Baldassarre G, Fidanza V, Klein-Szanto AJ, Parlow AF, Visone R, Pierantoni GM, Outwater E, Santoro M, Croce CM, Fusco A (2002) Overexpression of the HMGA2 gene in transgenic mice leads to the onset of pituitary adenomas. Oncogene 21(20):3190–3198. doi: 10.1038/sj.onc.1205428 CrossRefPubMedGoogle Scholar
  38. 38.
    Fedele M, Pentimalli F, Baldassarre G, Battista S, Klein-Szanto AJ, Kenyon L, Visone R, De Martino I, Ciarmiello A, Arra C, Viglietto G, Croce CM, Fusco A (2005) Transgenic mice overexpressing the wild-type form of the HMGA1 gene develop mixed growth hormone/prolactin cell pituitary adenomas and natural killer cell lymphomas. Oncogene 24(21):3427–3435. doi: 10.1038/sj.onc.1208501 CrossRefPubMedGoogle Scholar
  39. 39.
    Fedele M, Palmieri D, Fusco A (2010) HMGA2: A pituitary tumour subtype-specific oncogene? Mol Cell Endocrinol 326(1–2):19–24. doi: 10.1016/j.mce.2010.03.019 CrossRefPubMedGoogle Scholar
  40. 40.
    Butz H, Liko I, Czirjak S, Igaz P, Korbonits M, Racz K, Patocs A (2011) MicroRNA profile indicates downregulation of the TGFbeta pathway in sporadic non-functioning pituitary adenomas. Pituitary 14(2):112–124. doi: 10.1007/s11102-010-0268-x CrossRefPubMedGoogle Scholar
  41. 41.
    Yacqub-Usman K, Duong CV, Clayton RN, Farrell WE (2013) Preincubation of pituitary tumor cells with the epidrugs zebularine and trichostatin A are permissive for retinoic acid-augmented expression of the BMP-4 and D2R genes. Endocrinology 154(5):1711–1721. doi: 10.1210/en.2013-1061 CrossRefPubMedGoogle Scholar
  42. 42.
    Lujambio A, Ropero S, Ballestar E, Fraga MF, Cerrato C, Setien F, Casado S, Suarez-Gauthier A, Sanchez-Cespedes M, Git A, Spiteri I, Das PP, Caldas C, Miska E, Esteller M (2007) Genetic unmasking of an epigenetically silenced microRNA in human cancer cells. Cancer Res 67(4):1424–1429. doi: 10.1158/0008-5472.CAN-06-4218 CrossRefPubMedGoogle Scholar
  43. 43.
    Rhodes LV, Nitschke AM, Segar HC, Martin EC, Driver JL, Elliott S, Nam SY, Li M, Nephew KP, Burow ME, Collins-Burow BM (2012) The histone deacetylase inhibitor trichostatin A alters microRNA expression profiles in apoptosis-resistant breast cancer cells. Oncol Rep 27(1):10–16. doi: 10.3892/or.2011.1488 PubMedCentralPubMedGoogle Scholar
  44. 44.
    Sampath D, Liu C, Vasan K, Sulda M, Puduvalli VK, Wierda WG, Keating MJ (2012) Histone deacetylases mediate the silencing of miR-15a, miR-16, and miR-29b in chronic lymphocytic leukemia. Blood 119(5):1162–1172. doi: 10.1182/blood-2011-05-351510 PubMedCentralCrossRefPubMedGoogle Scholar
  45. 45.
    Leone V, Langella C, D’Angelo D, Mussnich P, Wierinckx A, Terracciano L, Raverot G, Lachuer J, Rotondi S, Jaffrain-Rea ML, Trouillas J, Fusco A (2014) miR-23b and miR-130b expression is downregulated in pituitary adenomas. Mol Cell Endocrinol 390(1–2):1–7. doi: 10.1016/j.mce.2014.03.002 CrossRefPubMedGoogle Scholar
  46. 46.
    Bottoni A, Zatelli MC, Ferracin M, Tagliati F, Piccin D, Vignali C, Calin GA, Negrini M, Croce CM, Degli Uberti EC (2007) Identification of differentially expressed microRNAs by microarray: a possible role for microRNA genes in pituitary adenomas. J Cell Physiol 210(2):370–377. doi: 10.1002/jcp.20832 CrossRefPubMedGoogle Scholar
  47. 47.
    Mao ZG, He DS, Zhou J, Yao B, Xiao WW, Chen CH, Zhu YH, Wang HJ (2010) Differential expression of microRNAs in GH-secreting pituitary adenomas. Diagn Pathol 5:79. doi: 10.1186/1746-1596-5-79 PubMedCentralCrossRefPubMedGoogle Scholar
  48. 48.
    Gadelha MR, Kasuki L, Denes J, Trivellin G, Korbonits M (2013) MicroRNAs: suggested role in pituitary adenoma pathogenesis. J Endocrinol Invest 36(10):889–895CrossRefPubMedGoogle Scholar
  49. 49.
    Jiang X, Zhang X (2013) The molecular pathogenesis of pituitary adenomas: an update. Endocrinol Metab 28(4):245–254. doi: 10.3803/EnM.2013.28.4.245 CrossRefGoogle Scholar
  50. 50.
    Shi X, Tao B, He H, Sun Q, Fan C, Bian L, Zhao W, Lu YC (2012) MicroRNAs-based network: a novel therapeutic agent in pituitary adenoma. Med Hypotheses 78(3):380–384. doi: 10.1016/j.mehy.2011.12.001 CrossRefPubMedGoogle Scholar
  51. 51.
    Rostad S (2012) Pituitary adenoma pathogenesis: an update. Curr Opin Endocrinol Diabetes Obes 19(4):322–327. doi: 10.1097/MED.0b013e328354b2e2 CrossRefPubMedGoogle Scholar
  52. 52.
    Yacqub-Usman K, Richardson A, Duong CV, Clayton RN, Farrell WE (2012) The pituitary tumour epigenome: aberrations and prospects for targeted therapy. Nat Rev Endocrinol 8(8):486–494. doi: 10.1038/nrendo.2012.54 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Mark O. Kitchen
    • 1
  • Kiren Yacqub-Usman
    • 1
  • Richard D. Emes
    • 2
  • Alan Richardson
    • 1
  • Richard N. Clayton
    • 1
  • William E. Farrell
    • 1
    Email author
  1. 1.Human Disease and Genomics Group, Institute of Science and Technology in Medicine, School of MedicineKeele UniversityStoke-on-TrentUK
  2. 2.School of Veterinary Medicine and ScienceUniversity of NottinghamSutton BoningtonUK

Personalised recommendations