Tumor Biology

, Volume 36, Issue 6, pp 4271–4278 | Cite as

Stable knockdown of LRG1 by RNA interference inhibits growth and promotes apoptosis of glioblastoma cells in vitro and in vivo

  • Di Zhong
  • Siren Zhao
  • Guangxu He
  • Jinku Li
  • Yanbin Lang
  • Wei Ye
  • Yongli Li
  • Chuanlu Jiang
  • Xianfeng Li
Research Article

Abstract

Leucine-rich α2 glycoprotein 1 (LRG1) has been shown to be aberrantly expressed in multiple human malignancies. However, the biological functions of LRG1 in human glioblastoma remain unknown. Here, we report for the first time the role of LRG1 in glioblastoma development based on the preliminary in vitro and in vivo data. We first confirmed the expression of LRG1 in human glioblastoma cell lines. Next, to investigate the role of LRG1 in the tumorigenesis and development of glioblastoma, a short hairpin RNA (shRNA) construct targeting LRG1 mRNA was transfected into U251 glioblastoma cells to generate a cell line with stably silenced LRG1 expression. The results showed that silencing of LRG1 significantly inhibited cell proliferation, induced cell cycle arrest at G0/G1 phase, and enhanced apoptosis in U251 cells in vitro. Consistently, LRG1 silencing resulted in the downregulation of key cell cycle factors including cyclin D1, B, and E and apoptotic gene Bcl-2 while elevated the levels of pro-apoptotic Bax and cleaved caspase-3, as determined by Western blot analysis. We further demonstrate that the silencing of LRG1 expression effectively reduced the tumorigenicity of U251 cells, delayed tumor formation, and promoted apoptosis in a xenograft tumor model in vivo. In conclusion, silencing the expression of LRG1 suppresses the growth of glioblastoma U251 cells in vitro and in vivo, suggesting that LRG1 may play a critical role in glioblastoma development, and it may have potential clinical implications in glioblastoma therapy.

Keywords

LRG1 Glioblastoma Knockdown Proliferation Cell cycle Apoptosis 

Notes

Acknowledgments

This study was supported by a grant from the National Natural Science Foundation of China (No.: 30640008).

References

  1. 1.
    Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005;352:987–96.CrossRefPubMedGoogle Scholar
  2. 2.
    Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, et al. The 2007 who classification of tumours of the central nervous system. Acta Neuropathol. 2007;114:97–109.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Cuddapah VA, Robel S, Watkins S, Sontheimer H. A neurocentric perspective on glioma invasion. Nat Rev Neurosci. 2014;15:455–65.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Arrillaga-Romany I, Reardon DA, Wen PY. Current status of antiangiogenic therapies for glioblastomas. Expert Opin Investig Drugs. 2014;23:199–210.CrossRefPubMedGoogle Scholar
  5. 5.
    Cloughesy TF, Cavenee WK, Mischel PS. Glioblastoma: from molecular pathology to targeted treatment. Annu Rev Pathol. 2014;9:1–25.CrossRefPubMedGoogle Scholar
  6. 6.
    Shirai R, Hirano F, Ohkura N, Ikeda K, Inoue S. Up-regulation of the expression of leucine-rich alpha(2)-glycoprotein in hepatocytes by the mediators of acute-phase response. Biochem Biophys Res Commun. 2009;382:776–9.CrossRefPubMedGoogle Scholar
  7. 7.
    Haupt H, Baudner S. Isolation and characterization of an unknown, leucine-rich 3.1-s-alpha2-glycoprotein from human serum (author's transl). Hoppe Seylers Z Physiol Chem. 1977;358:639–46.CrossRefPubMedGoogle Scholar
  8. 8.
    Weivoda S, Andersen JD, Skogen A, Schlievert PM, Fontana D, Schacker T, et al. Elisa for human serum leucine-rich alpha-2-glycoprotein-1 employing cytochrome c as the capturing ligand. J Immunol Methods. 2008;336:22–9.CrossRefPubMedGoogle Scholar
  9. 9.
    O'Donnell LC, Druhan LJ, Avalos BR. Molecular characterization and expression analysis of leucine-rich alpha2-glycoprotein, a novel marker of granulocytic differentiation. J Leukoc Biol. 2002;72:478–85.PubMedGoogle Scholar
  10. 10.
    Nakajima M, Miyajima M, Ogino I, Watanabe M, Miyata H, Karagiozov KL, et al. Leucine-rich alpha-2-glycoprotein is a marker for idiopathic normal pressure hydrocephalus. Acta Neurochir (Wien). 2011;153:1339–46. discussion 1346.CrossRefGoogle Scholar
  11. 11.
    Linden M, Segersten U, Runeson M, Wester K, Busch C, Pettersson U, et al. Tumour expression of bladder cancer-associated urinary proteins. BJU Int. 2013;112:407–15.CrossRefPubMedGoogle Scholar
  12. 12.
    Liu Y, Luo X, Hu H, Wang R, Sun Y, Zeng R, et al. Integrative proteomics and tissue microarray profiling indicate the association between overexpressed serum proteins and non-small cell lung cancer. PLoS One. 2012;7:e51748.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Wang X, Abraham S, McKenzie JA, Jeffs N, Swire M, Tripathi VB, et al. Lrg1 promotes angiogenesis by modulating endothelial tgf-beta signalling. Nature. 2013;499:306–11.CrossRefPubMedGoogle Scholar
  14. 14.
    Andersen JD, Boylan KL, Jemmerson R, Geller MA, Misemer B, Harrington KM, et al. Leucine-rich alpha-2-glycoprotein-1 is upregulated in sera and tumors of ovarian cancer patients. J Ovarian Res. 2010;3:21.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Li Y, Zhang Y, Qiu F, Qiu Z. Proteomic identification of exosomal lrg1: a potential urinary biomarker for detecting nsclc. Electrophoresis. 2011;32:1976–83.CrossRefPubMedGoogle Scholar
  16. 16.
    Ladd JJ, Busald T, Johnson MM, Zhang Q, Pitteri SJ, Wang H, et al. Increased plasma levels of the apc-interacting protein mapre1, lrg1, and igfbp2 preceding a diagnosis of colorectal cancer in women. Cancer Prev Res (Phila). 2012;5:655–64.CrossRefGoogle Scholar
  17. 17.
    Schonberg DL, Lubelski D, Miller TE, Rich JN. Brain tumor stem cells: molecular characteristics and their impact on therapy. Mol Asp Med. 2014;39:82–101.CrossRefGoogle Scholar
  18. 18.
    Sherr CJ. Cancer cell cycles. Science. 1996;274:1672–7.CrossRefPubMedGoogle Scholar
  19. 19.
    Schafer KA. The cell cycle: a review. Vet Pathol. 1998;35:461–78.CrossRefPubMedGoogle Scholar
  20. 20.
    Lee MH, Yang HY. Regulators of g1 cyclin-dependent kinases and cancers. Cancer Metastasis Rev. 2003;22:435–49.CrossRefPubMedGoogle Scholar
  21. 21.
    Hochegger H, Takeda S, Hunt T. Cyclin-dependent kinases and cell-cycle transitions: does one fit all? Nat Rev Mol Cell Biol. 2008;9:910–6.CrossRefPubMedGoogle Scholar
  22. 22.
    Gautschi O, Ratschiller D, Gugger M, Betticher DC, Heighway J. Cyclin d1 in non-small cell lung cancer: a key driver of malignant transformation. Lung Cancer. 2007;55:1–14.CrossRefPubMedGoogle Scholar
  23. 23.
    Chen X, Zhao T, Li L, Xu C, Zhang X, Tse V, et al. Ccnd1 g870a polymorphism with altered cyclin d1 transcripts expression is associated with the risk of glioma in a Chinese population. DNA Cell Biol. 2012;31:1107–13.CrossRefPubMedGoogle Scholar
  24. 24.
    Simpson DJ, Fryer AA, Grossman AB, Wass JA, Pfeifer M, Kros JM, et al. Cyclin d1 (ccnd1) genotype is associated with tumour grade in sporadic pituitary adenomas. Carcinogenesis. 2001;22:1801–7.CrossRefPubMedGoogle Scholar
  25. 25.
    Sun G, Shi L, Yan S, Wan Z, Jiang N, Fu L, et al. Mir-15b targets cyclin d1 to regulate proliferation and apoptosis in glioma cells. Biomed Res Int. 2014;2014:687826.PubMedPubMedCentralGoogle Scholar
  26. 26.
    Daniel P, Filiz G, Brown DV, Hollande F, Gonzales M, D'Abaco G, et al. Selective creb-dependent cyclin expression mediated by the pi3k and mapk pathways supports glioma cell proliferation. Oncogenesis. 2014;3:e108.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Qi S, Song Y, Peng Y, Wang H, Long H, Yu X, et al. Zeb2 mediates multiple pathways regulating cell proliferation, migration, invasion, and apoptosis in glioma. PLoS One. 2012;7:e38842.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Cory S, Adams JM. The bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer. 2002;2:647–56.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Di Zhong
    • 1
  • Siren Zhao
    • 2
  • Guangxu He
    • 2
  • Jinku Li
    • 2
  • Yanbin Lang
    • 2
  • Wei Ye
    • 2
  • Yongli Li
    • 2
  • Chuanlu Jiang
    • 2
  • Xianfeng Li
    • 2
  1. 1.Department of NeurologyThe First Affiliated Hospital of Harbin Medical UniversityHarbinChina
  2. 2.Department of NeurosurgeryThe Second Affiliated Hospital of Harbin Medical UniversityHarbinChina

Personalised recommendations