Journal of Molecular Neuroscience

, Volume 56, Issue 4, pp 949–955 | Cite as

Pygo2 siRNA Inhibit the Growth and Increase Apoptosis of U251 Cell by Suppressing Histone H3K4 Trimethylation

  • Mingcong Li
  • Linlin Chao
  • Jian Wu
  • Hao Xu
  • Shanghan Shen
  • Sifang Chen
  • Xin Gao
  • Ning Yu
  • Zhanxiang WangEmail author


The development of novel therapeutic strategies for glioma requires the identification of molecular targets involved in malignancy. Pygopus (Pygo) is a new discovered and specific downstream component of canonical Wnt signaling. Our previous study has demonstrated that Pygo2 is highly expressed in and promotes the growth of glioma cells. However, the role of Pygo2 in glioma remains to be elucidated. In the current study, we investigated the role of Pygo2 in human glioma U251 cells and showed that knocking down of the expression of Pygo2 in U251 cells using lentivirally expressed siRNA have inhibited cell proliferation and increased apoptosis through decreasing H3K4me3 expression. Moreover, we found Pygo2 was enriched in U251 glioma cancer stem-like cells and Pygo2 siRNA resulted in a reduced number as well as size of tumor spheres. According to our result, this paper now links mechanistically Pygo2’s role in histone modification to its enhancement/reduction of proliferation/apoptosis in glioma cells and indicate that Pygo2 may play an important role in self-renew and proliferation in U251 glioma cancer stem-like cells.


Pygo2 Growth Apoptosis H3K4me3 



Central nervous system


Lymphoid-enhancing factor 1/T-cell factor


Trimethylation of histone H3 at lysine 4


Epidermal growth factor


Basic fibroblast growth factor


Green fluorescent protein


Glyceraldehyde-3-phosphate dehydrogenase


Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling





This work was supported by the National Natural Science Foundation of China (no. 81271332) and Fujiani Natural Science Foundation (2009D002).

Conflict of Interest

All the authors of this study declare no conflict of interest.


  1. Andrews PG, Lake BB, Popadiuk C, Kao KR (2007) Requirement of Pygopus 2 in breast cancer. Int J Oncol 30:357–363PubMedGoogle Scholar
  2. Andrews PG, He Z, Popadiuk C, Kao KR (2009) The transcriptional activity of Pygopus is enhanced by its interaction with cAMP-response-element-binding protein (CREB)-binding protein. Biochem J 422:493–501. doi: 10.1042/BJ20090134 PubMedCrossRefGoogle Scholar
  3. Belenkaya TY, Han C, Standley HJ, Lin X, Houston DW, Heasman J (2002) Pygopus encodes a nuclear protein essential for wingless/Wnt signaling. Development 129:4089–4101PubMedGoogle Scholar
  4. Bienz M (2006) The PHD finger, a nuclear protein-interaction domain. Trends Biochem Sci 31:35–40. doi: 10.1016/j.tibs.2005.11.001 PubMedCrossRefGoogle Scholar
  5. Chen J, Luo Q, Yuan Y, Huang X, Cai W, Li C, Wei T, Zhang L, Yang M, Liu Q, Ye G, Dai X, Li B (2010) Pygo2 associates with MLL2 histone methyltransferase and GCN5 histone acetyltransferase complexes to augment Wnt target gene expression and breast cancer stem-like cell expansion. Mol Cell Biol 30:5621–5635. doi: 10.1128/MCB.00465-10 PubMedCentralPubMedCrossRefGoogle Scholar
  6. Cruceru ML, Neagu M, Demoulin JB, Constantinescu SN (2013) Therapy targets in glioblastoma and cancer stem cells: lessons from haematopoietic neoplasms. J Cell Mol Med 17:1218–1235. doi: 10.1111/jcmm.12122 PubMedCentralPubMedCrossRefGoogle Scholar
  7. Fiedler M, Sanchez-Barrena MJ, Nekrasov M, Mieszczanek J, Rybin V, Muller J, Evans P, Bienz M (2008) Decoding of methylated histone H3 tail by the Pygo-BCL9 Wnt signaling complex. Mol Cell 30:507–518. doi: 10.1016/j.molcel.2008.03.011 PubMedCentralPubMedCrossRefGoogle Scholar
  8. Gu B, Sun P, Yuan Y, Moraes RC, Li A, Teng A, Agrawal A, Rheaume C, Bilanchone V, Veltmaat JM, Takemaru K, Millar S, Lee EY, Lewis MT, Li B, Dai X (2009) Pygo2 expands mammary progenitor cells by facilitating histone H3 K4 methylation. J Cell Biol 185:811–826. doi: 10.1083/jcb.200810133 PubMedCentralPubMedCrossRefGoogle Scholar
  9. Gu B, Watanabe K, Dai X (2012) Pygo2 regulates histone gene expression and H3 K56 acetylation in human mammary epithelial cells. Cell Cycle 11:79–87. doi: 10.4161/cc.11.1.18402 PubMedCentralPubMedCrossRefGoogle Scholar
  10. He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, Morin PJ, Vogelstein B, Kinzler KW (1998) Identification of c-MYC as a target of the APC pathway. Science 281:1509–1512PubMedCrossRefGoogle Scholar
  11. Jessen S, Gu B, Dai X (2008) Pygopus and the Wnt signaling pathway: a diverse set of connections. Bioessays 30:448–456. doi: 10.1002/bies.20757 PubMedCrossRefGoogle Scholar
  12. Kessler R, Hausmann G, Basler K (2009) The PHD domain is required to link Drosophila Pygopus to legless/beta-catenin and not to histone H3. Mech Dev 126:752–759. doi: 10.1016/j.mod.2009.04.003 PubMedCrossRefGoogle Scholar
  13. Kramps T, Peter O, Brunner E, Nellen D, Froesch B, Chatterjee S, Murone M, Zullig S, Basler K (2002) Wnt/wingless signaling requires BCL9/legless-mediated recruitment of pygopus to the nuclear beta-catenin-TCF complex. Cell 109:47–60PubMedCrossRefGoogle Scholar
  14. Li B, Mackay DR, Ma J, Dai X (2004) Cloning and developmental expression of mouse pygopus 2, a putative Wnt signaling component. Genomics 84:398–405. doi: 10.1016/j.ygeno.2004.04.007 PubMedCentralPubMedCrossRefGoogle Scholar
  15. Li H, Ilin S, Wang W, Duncan EM, Wysocka J, Allis CD, Patel DJ (2006) Molecular basis for site-specific read-out of histone H3K4me3 by the BPTF PHD finger of NURF. Nature 442:91–95. doi: 10.1038/nature04802 PubMedCrossRefGoogle Scholar
  16. Liu C, Li Y, Semenov M, Han C, Baeg GH, Tan Y, Zhang Z, Lin X, He X (2002) Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell 108:837–847PubMedCrossRefGoogle Scholar
  17. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 25:402–408. doi: 10.1006/meth.2001.1262 PubMedCrossRefGoogle Scholar
  18. Logan CY, Nusse R (2004) The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol 20:781–810. doi: 10.1146/annurev.cellbio.20.010403.113126 PubMedCrossRefGoogle Scholar
  19. Modrek AS, Bayin NS, Placantonakis DG (2014) Brain stem cells as the cell of origin in glioma. World J Stem Cells 6:43–52. doi: 10.4252/wjsc.v6.i1.43 PubMedCentralPubMedCrossRefGoogle Scholar
  20. Nair M, Nagamori I, Sun P, Mishra DP, Rheaume C, Li B, Sassone-Corsi P, Dai X (2008) Nuclear regulator Pygo2 controls spermiogenesis and histone H3 acetylation. Dev Biol 320:446–455. doi: 10.1016/j.ydbio.2008.05.553 PubMedCentralPubMedCrossRefGoogle Scholar
  21. Niwa H (2007) Open conformation chromatin and pluripotency. Genes Dev 21:2671–2676. doi: 10.1101/gad.1615707 PubMedCrossRefGoogle Scholar
  22. Parker DS, Jemison J, Cadigan KM (2002) Pygopus, a nuclear PHD-finger protein required for Wingless signaling in Drosophila. Development 129:2565–2576PubMedGoogle Scholar
  23. Polakis P (2000) Wnt signaling and cancer. Genes Dev 14:1837–1851PubMedGoogle Scholar
  24. Popadiuk CM, Xiong J, Wells MG, Andrews PG, Dankwa K, Hirasawa K, Lake BB, Kao KR (2006) Antisense suppression of pygopus2 results in growth arrest of epithelial ovarian cancer. Clin Cancer Res 12:2216–2223. doi: 10.1158/1078-0432.CCR-05-2433 PubMedCrossRefGoogle Scholar
  25. Qiu B, Zhang D, Tao J, Wu A, Wang Y (2012) A simplified and modified procedure to culture brain glioma stem cells from clinical specimens. Oncol Lett 3:50–54. doi: 10.3892/ol.2011.433 PubMedCentralPubMedGoogle Scholar
  26. Shtutman M, Zhurinsky J, Simcha I, Albanese C, D’Amico M, Pestell R, Ben-Ze’ev A (1999) The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway. Proc Natl Acad Sci U S A 96:5522–5527PubMedCentralPubMedCrossRefGoogle Scholar
  27. Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, Dirks PB (2003) Identification of a cancer stem cell in human brain tumors. Cancer Res 63:5821–5828PubMedGoogle Scholar
  28. Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, Ludwin SK, Allgeier A, Fisher B, Belanger K, Hau P, Brandes AA, Gijtenbeek J, Marosi C, Vecht CJ, Mokhtari K, Wesseling P, Villa S, Eisenhauer E, Gorlia T, Weller M, Lacombe D, Cairncross JG, Mirimanoff RO (2009) Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 10:459–466. doi: 10.1016/S1470-2045(09)70025-7 PubMedCrossRefGoogle Scholar
  29. Thompson B, Townsley F, Rosin-Arbesfeld R, Musisi H, Bienz M (2002) A new nuclear component of the Wnt signalling pathway. Nat Cell Biol 4:367–373. doi: 10.1038/ncb786 PubMedCrossRefGoogle Scholar
  30. van Meir EG, Hadjipanayis CG, Norden AD, Shu HK, Wen PY, Olson JJ (2010) Exciting new advances in neuro-oncology: the avenue to a cure for malignant glioma. CA Cancer J Clin 60:166–193. doi: 10.3322/caac.20069 PubMedCentralPubMedCrossRefGoogle Scholar
  31. Vescovi AL, Galli R, Reynolds BA (2006) Brain tumour stem cells. Nat Rev Cancer 6:425–436. doi: 10.1038/nrc1889 PubMedCrossRefGoogle Scholar
  32. Wang ZX, Chen YY, Li BA, Tan GW, Liu XY, Shen SH, Zhu HW, Wang HD (2010) Decreased pygopus 2 expression suppresses glioblastoma U251 cell growth. J Neurooncol 100:31–41. doi: 10.1007/s11060-010-0144-6 PubMedCrossRefGoogle Scholar
  33. Watanabe K, Fallahi M, Dai X (2014) Chromatin effector Pygo2 regulates mammary tumor initiation and heterogeneity in MMTV-Wnt1 mice. Oncogene 33:632–642. doi: 10.1038/onc.2012.620 PubMedCentralPubMedCrossRefGoogle Scholar
  34. Wodarz A, Nusse R (1998) Mechanisms of Wnt signaling in development. Annu Rev Cell Dev Biol 14:59–88. doi: 10.1146/annurev.cellbio.14.1.59 PubMedCrossRefGoogle Scholar
  35. Zhou SY, Xu ML, Wang SQ, Zhang F, Wang L, Wang HQ (2014) Overexpression of Pygopus-2 is required for canonical Wnt activation in human lung cancer. Oncol Lett 7:233–238. doi: 10.3892/ol.2013.1691 PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Mingcong Li
    • 1
  • Linlin Chao
    • 1
  • Jian Wu
    • 3
  • Hao Xu
    • 2
  • Shanghan Shen
    • 3
  • Sifang Chen
    • 3
  • Xin Gao
    • 1
  • Ning Yu
    • 3
  • Zhanxiang Wang
    • 3
    Email author
  1. 1.The First Clinical Medical CollegeFujian Medical UniversityFuzhouThe People’s Republic of China
  2. 2.Department of NeurosurgeryAnhui Provincial HospitalHefeiPeople’s Republic of China
  3. 3.Department of NeurosurgeryThe First Affiliated Hospital of Xiamen UniversityXiamenThe People’s Republic of China

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