Skip to main content
Download PDF
  • Research Article
  • Published:

A microarray gene analysis of peripheral whole blood in normal adult male rats after long-term GH gene therapy

Cellular & Molecular Biology Letters volume 15, pages 177–195 (2010)Cite this article

Abstract

The main aims of this study were to determine the effects of GH gene abuse/misuse in normal animals and to discover genes that could be used as candidate biomarkers for the detection of GH gene therapy abuse/misuse in humans. We determined the global gene expression profile of peripheral whole blood from normal adult male rats after long-term GH gene therapy using CapitalBio 27 K Rat Genome Oligo Arrays. Sixty one genes were found to be differentially expressed in GH gene-treated rats 24 weeks after receiving GH gene therapy, at a two-fold higher or lower level compared to the empty vector group (p < 0.05). These genes were mainly associated with angiogenesis, oncogenesis, apoptosis, immune networks, signaling pathways, general metabolism, type I diabetes mellitus, carbon fixation, cell adhesion molecules, and cytokine-cytokine receptor interaction. The results imply that exogenous GH gene expression in normal subjects is likely to induce cellular changes in the metabolism, signal pathways and immunity. A real-time qRT-PCR analysis of a selection of the genes confirmed the microarray data. Eight differently expressed genes were selected as candidate biomarkers from among these 61 genes. These 8 showed five-fold higher or lower expression levels after the GH gene transduction (p < 0.05). They were then validated in real-time PCR experiments using 15 single-treated blood samples and 10 control blood samples. In summary, we detected the gene expression profiles of rat peripheral whole blood after long-term GH gene therapy and screened eight genes as candidate biomarkers based on the microarray data. This will contribute to an increased mechanistic understanding of the effects of chronic GH gene therapy abuse/misuse in normal subjects.

Abbreviations

GH:

growth hormone

GHD:

growth hormone deficiency

rAAV:

recombinant adeno-associated virus

rAAV2/1:

recombinant adeno-associated viral vectors pseudotyped with viral capsids from serotype 1

References

  1. Götherström, G., Elbornsson, M., Stibrant-Sunnerhagen, K., Bengtsson, B.A., Johannsson, G. and Svensson J. Ten years of growth hormone (GH) replacement normalizes muscle strength in GH deficient adults. J. Clin. Endocrinol. Metab. 94 (2008) 809–816. DOI: 10.1210/jc.2008-1538.

    Article  PubMed  Google Scholar 

  2. Ayuk, J. and Sheppard, M.C. Growth hormone and its disorders. Postgrad. Med. J. 82 (2006) 24–30. DOI: dx.doi.org/10.1136/pgmj.2005.036087.

    Article  CAS  PubMed  Google Scholar 

  3. Rivera, V.M., Ye, X., Courage, N.L., Sachar, J., Cerasoli, F., Wilson, J.M. and Gilman, M. Long-term regulated expression of growth hormone in mice after intramuscular gene transfer. Proc. Natl. Acad. Sci. USA. 96 (1999) 8657–8662. DOI: dx.doi.org/10.1073/pnas.96.15.8657.

    Article  CAS  PubMed  Google Scholar 

  4. Büning, H., Perabo, L., Coutelle, O., Quadt-Humme, S. and Hallek, M. Recent developments in adeno-associated virus vector technology. J. Gene Med. 10 (2008) 717–733. DOI: dx.doi.org/10.1002/jgm.1205.

    Article  PubMed  Google Scholar 

  5. Filipp, F. Is science killing sport? Gene therapy and its possible abuse in doping. EMBO Rep. 8 (2007) 433–435. DOI: dx.doi.org/10.1038/sj.embor.7400968.

    Article  CAS  PubMed  Google Scholar 

  6. Harridge, S.D. and Velloso, C.P. Gene doping. Essays Biochem. 44 (2007) 125–138. DOI: dx.doi.org/10.1042/BSE0440125.

    Article  Google Scholar 

  7. Melmed, S. Medical progress: Acromegaly. N. Engl. J. Med. 355 (2006) 2558–2573. DOI: dx.doi.org/10.1056/NEJMra062453.

    Article  CAS  PubMed  Google Scholar 

  8. McCrory, P. Super athletes or gene cheats? Br. J. Sports Med. 37 (2003) 192–193. DOI: dx.doi.org/10.1136/bjsm.37.3.192.

    Article  CAS  Google Scholar 

  9. Wells, D.J. Gene doping: the hype and the reality. Br. J. Pharmacol. 154 (2008) 623–631. DOI: dx.doi.org/10.1038/bjp.2008.144.

    Article  CAS  PubMed  Google Scholar 

  10. Schena, M., Shalon, D., Davis, R.W. and Brown, P.O. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270 (1995) 467–470. DOI: 10.1126/science.270.5235.467.

    Article  CAS  PubMed  Google Scholar 

  11. Chen, B.P., Li, Y.S., Zhao, Y., Chen, K.D., Li, S., Lao, J., Yuan, S., Shyy, J.Y. and Chien, S. DNA microarray analysis of gene expression in endothelial cells in response to 24-h shear stress. Physiol. Genomics 7 (2001) 55–63.

    Article  CAS  PubMed  Google Scholar 

  12. Yan, H., Guo, Y.H., Zhang, P., Zu, L.Y., Dong, X.Y., Chen, L., Tian, J.W., Fan, X.L., Wang, N.P., Wu, X.B. and Gao, W. Superior neovascularization and muscle regeneration in ischemic skeletal muscles following VEGF gene transfer by rAAV1 pseudotyped vectors. Biochem. Biophys. Res. Commun. 336 (2005) 278–287. DOI: dx.doi.org/10.1016/j.bbrc.2005.08.066.

    Article  Google Scholar 

  13. Snyder, R., Xiao, X. and Samulski, R.J. Production of recombinant adenoassociated viral vectors. in: Current Protocols in Human Genetics (Smith, D. Ed.), Wiley, New York, 1996, 12.1.1–12.2.23.

    Google Scholar 

  14. Livak, K.J. and Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2ΔΔCT method. Methods bd25 (2001) 402–408. DOI: dx.doi.org/10.1006/meth.2001.1262.

    Article  Google Scholar 

  15. Herrington, J. and Carter-Su, C. Signaling pathways activated by the growth hormone receptor. Trends Endocrinol. Metab. 12 (2001) 252–257. DOI: dx.doi.org/10.1016/S1043-2760(01)00423-4.

    Article  CAS  PubMed  Google Scholar 

  16. Lichanska, A.M. and Waters, M.J. How growth hormone controls growth, obesity and sexual dimorphism. Trends Genet. 24 (2007) 41–47. DOI: dx.doi.org/10.1016/j.tig.2007.10.006.

    Article  PubMed  Google Scholar 

  17. Cormier, R.T., Hong, K.H., Halberg, R.B., Hawkins, T.L., Richardson, P., Mulherkar, R., Dove, W.F. and Lander, E.S. Secretory phospholipase Pla2g2a confers resistance to intestinal tumorigenesis. Nat. Genet. 17 (1997) 88–91. DOI: dx.doi.org/10.1038/ng0997-88.

    Article  CAS  PubMed  Google Scholar 

  18. Kugiyama, K., Ota, Y., Sugiyama, S., Kawano, H., Doi, H., Soejima, H., Miyamoto, S., Ogawa, H., Takazoe, K. and Yasue, H. Prognostic value of plasma levels of secretory type II phospholipase A2 in patients with unstable angina pectoris. Am. J. Cardiol. 86 (2000) 718–722. DOI:10.1016/S0002-9149(00)01069-9.

    Article  CAS  PubMed  Google Scholar 

  19. Fijneman, R.J., Peham, J.R., van de Wiel, M.A., Meijer, G.A., Matise, I., Velcich, A. and Cormier, R.T. Expression of Pla2g2a prevents carcinogenesis in Muc2-deficient mice. Cancer Sci. 99 (2008) 2113–2119. DOI: dx.doi.org/10.1111/j.1349-7006.2008.00924.x.

    Article  CAS  PubMed  Google Scholar 

  20. Hong, K.H., Bonventre, J.C., O’Leary, E., Bonventre, J.V. and Lander, E.S. Deletion of cytosolic phospholipase A (2) suppresses Apc(Min)-induced tumorigenesis. Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 3935–3939. DOI: dx.doi.org/10.1073/pnas.051635898.

    Article  CAS  PubMed  Google Scholar 

  21. Touqui, L. and Alaoui-El-Azher, M. Mammalian secreted phospholipases A2 and their pathophysiological significance in inflammatory diseases. Curr. Mol. Med. 1 (2001) 739–754. DOI: dx.doi.org/10.2174/1566524013363258.

    Article  CAS  PubMed  Google Scholar 

  22. Pike, N.B. Flushing out the role of GPR109A (HM74A) in the clinical efficacy of nicotinic acid. J. Clin. Invest. 115 (2005) 3400–3403. DOI: dx.doi.org/10.1172/JCI27160.

    Article  CAS  PubMed  Google Scholar 

  23. Martin, P.M., Ananth, S., Cresci, G., Roon, P., Smith, S. and Ganapathy, V. Expression and localization of GPR109A (PUMA-G/HM74A) mRNA and protein in mammalian retinal pigment epithelium. Mol. Vis. 15 (2009) 362–372.

    CAS  PubMed  Google Scholar 

  24. Chrzanowska-Wodnicka, M., Smyth, S.S., Schoenwaelder, S.M., Fischer, T.H. and White 2nd, G.C. Rap1b is required for normal platelet function and hemostasis in mice. J. Clin. Invest. 115 (2005) 680–687. DOI: 10.1172/JCI22973.

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Clinical Biochemistry, Chinese PLA General Hospital, 28 Fu-Xing Road, Beijing, 100853, P.R. China

    Ying Qin & Ya-Ping Tian

Authors
  1. Ying Qin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ya-Ping Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Ying Qin or Ya-Ping Tian.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Qin, Y., Tian, YP. A microarray gene analysis of peripheral whole blood in normal adult male rats after long-term GH gene therapy. Cell Mol Biol Lett 15, 177–195 (2010). https://doi.org/10.2478/s11658-010-0001-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11658-010-0001-9

Key words

Cellular & Molecular Biology Letters

ISSN: 1689-1392

Contact us