Yonsei Med J.  2008 Apr;49(2):301-310. 10.3349/ymj.2008.49.2.301.

Identification of Up-Regulated Genes in Malignant Glioma with Subtraction Hybridization: Preliminary Screening Studies

• Cho YJ

• KMID: 1084505
• DOI: http://doi.org/10.3349/ymj.2008.49.2.301

Abstract

PURPOSE
This investigation is intended to obtain differentially expressed genes related to human malignant glioma using Subtractive hybridization. MATERIALS AND METHODS: Subtractive hybridization is potentially faster methods for identifying differentially expressed genes associated with a particular disease state. We identified 7 over-expressed genes which were not homologous to any of the known genes in the Genbank(TM) database. RESULTS: Using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), the mRNA expression levels of these 7 genes were higher in human glioblastomas tissue than in non-tumor brain tissue. In order to learn more about the expression profile of these genes, RT-PCR was performed using various commercially available human carcinoma cell lines. Some of these new genes were over-expressed in human glioma cell line, but not the expressed in other human cancer cell line. CONCLUSION: Theses cloned new genes may play a role in brain tumorigenesis. Further studies including verification of oncogene, cancer protein, and glioblastoma induction in animal model are needed.

Keyword

Glioblastoma; gene; subtraction hybridization

MeSH Terms

Aged
Brain Neoplasms/*genetics/pathology
Cell Line, Tumor
Female
*Gene Expression Regulation, Neoplastic
Glioma/*genetics/pathology
Humans
Male
Middle Aged
RNA, Messenger/genetics/metabolism
Reverse Transcriptase Polymerase Chain Reaction
Up-Regulation/genetics

Figure

• 

  Fig. 1 RT-PCR analysis for differential expression of 21 clones between glioblastoma and non-tumor brain tissue. We selected seven genes for RT-PCR. N, non-tumor brain tissue; T, glioblastoma.

• 

  Fig. 2 Expression of seven genes' mRNA levels from glioblastoma tissues by RT-PCR. The expressions of clone 7 were higher than those in non-tumor brain tissues. N, non-tumor brain tissues; T, glioblastoma tissues; 18srRNA, control.

• 

  Fig. 3 Expressions of seven genes' mRNA from glioma line by RT-PCR. The expressions of mRNA of seven clones were significantly higher than those in non-tumor brain tissues. In glioma cell lines, two different types of gene expression profile were observed. One type was genes that were overexpressed by all the cell lines (clone 7, 25, and 28), whereas the other was genes that showed variable expression across the cell lines without any distinct pattern (clone 10, 15, 43, and 47). 18srRNA (control) demonstrates equal expression across the panel. NBT, non-tumor brain tissue.

• 

  Fig. 4 Expressions of seven genes' mRNA from various human cancer cell line by RT-PCR. Lane 1, normal lung (WI-38); Lane 2, SV40-immortalzed (WI-38 VA13); Lane 3, Lung carcinoma (NCI-H596); Lane 4, Normal colon (CCD-18CO); Lane 5, Colon carcinoma (KM 1214); Lane 6, Normal prostate (RWPE-1); Lane 7, Prostate carcinoma cell line (DU 145); and control (18srRNA).

• 

  Fig. 5 Serum deprivation and stimulation. The expressions of seven genes were significantly increased 0.5 and 1 hr after 10% FBS treatment. After 2 hr of treatment, the levels of seven genes' mRNA a drastically decreased.

Reference

1. Dai C, Holland EC. Astrocyte differentiation states and gliomas formation. Cancer J. 2003. 9:72–81.

2. Sallinen SL, Sallinen PK, Haapasalo HK, Helin HJ, Helén PT, Schraml P, et al. Identification of differentially expressed genes in human gliomas by DNA microarray and tissue chip techniques. Cancer Res. 2000. 60:6617–6622.

3. Christensen HC, Kosteljanetz M, Johansen C. Incidences of gliomas and meningiomas in Denmark, 1943 to 1997. Neurosurgery. 2003. 52:1327–1333. discussion 1333-4.

4. Velculescu VE, Zhang L, Zhou W, Vogelstein J, Basrai MA, Bassett DE Jr, et al. Characterization of the yeast transcriptome. Cell. 1997. 88:243–251.

5. Chook YM, Blobel G. Karyopherins and nuclear import. Curr Opin Struct Biol. 2001. 11:703–715.

6. Nutt CL, Zerillo CA, Kelly GM, Hockfield S. Brain enriched hyaluronan binding (BEHAB)/brevican increases aggressiveness of CNS-1 gliomas in Lewis rats. Cancer Res. 2001. 61:7056–7059.

7. Palackal NT, Lee SH, Harvey RG, Blair IA, Penning TM. Activation of polycyclic aromatic hydrocarbon trans-dihydrodiol proximate carcinogens by human aldo-keto reductase (AKR1C) enzymes and their functional overexpression in human lung carcinoma (A549) cells. J Biol Chem. 2002. 277:24799–24808.

8. Rehman I, Cross SS, Azzouzi AR, Catto JW, Deloulme JC, Larre S, et al. S100A6 (Calcyclin) is a prostate basal cell marker absent in prostate cancer and its precursors. Br J Cancer. 2004. 91:739–744.

9. Welsh J, Chada K, Dalal SS, Cheng R, Ralph D, McClelland M. Arbitrarily primed PCR fingerprinting of RNA. Nucleic Acids Res. 1992. 20:4965–4970.

10. Smith JM, Bowles J, Wilson M, Koopman P. HMG box transcription factor gene Hbp1 is expressed in germ cells of the developing mouse testis. Dev Dyn. 2004. 230:366–370.

11. van Nocker S, Ludwig P. The WD-repeat protein superfamily in Arabidopsis: conservation and divergence in structure and function. BMC Genomics. 2003. 4:50.

12. Porkka KP, Visakorpi T. Detection of differentially expressed genes in prostate cancer by combining suppression subtractive hybridization and cDNA library array. J Pathol. 2001. 193:73–79.

13. Wandrey M, Trevaskis B, Brewin N, Udvardi MK. Molecular and cell biology of a family of voltagedependent anion channel porins in Lotus japonicus. Plant Physiol. 2004. 134:182–193.

14. Zeindl-Eberhart E, Haraida S, Liebmann S, Jungblut PR, Lamer S, Mayer Mayer, et al. Detection and identification of tumor-associated protein variants in human hepatocellular carcinomas. Hepatology. 2004. 39:540–549.

15. Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam F, Huang B, et al. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci U S A. 1996. 93:6025–6030.

16. Hufton SE, Moerkerk PT, Brandwijk R, de Bruïne AP, Arends JW, Hoogenboom HR. A profile of differentially expressed genes in primary colorectal cancer using suppression subtractive hybridization. FEBS Lett. 1999. 463:77–82.

17. Ji W, Wright MB, Cai L, Flament A, Lindpaintner K. Efficacy of SSH PCR in isolating differentially expressed genes. BMC Genomics. 2002. 3:12.

18. Lee SJ, Suh MC, Kim S, Kwon JK, Kim M, Paek KH, et al. Molecular cloning of a novel pathogen-inducible cDNA encoding a putative acyl-CoA synthetase from Capsicum annuum L. Plant Mol Biol. 2001. 46:661–671.

19. Shridhar V, Sen A, Chien J, Staub J, Avula R, Kovats S, et al. Identification of underexpressed genes in earlyand late-stage primary ovarian tumors by suppression subtraction hybridization. Cancer Res. 2002. 62:262–270.

20. Swearingen ML, Sun D, Bourner M, Weinstein EJ. Detection of differentially expressed HES-6 gene in metastatic colon carcinoma by combination of suppression subtractive hybridization and cDNA library array. Cancer Lett. 2003. 198:229–239.

21. Liang P, Pardee AB. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science. 1992. 257:967–971.

22. Dong Y, Zhang H, Hawthorn L, Ganther HE, Ip C. Delineation of the molecular basis for seleniuminduced growth arrest in human prostate cancer cells by oligonucleotide array. Cancer Res. 2003. 63:52–59.