Identification of Differentially Expressed Genes in Human Bladder Cancer using Oligonucleotide Microarray

Bladder cancer is one of the most common malignancies in developed countries while transitional cell carcinomas (Tee) are the origin of more than 90% of diagnosed bladder cancers. Patients diagnosed with bladder cancer basically belong to two clinically distinct groups, namely non-muscle invasive (which are papillary pattern) and muscle invasive (which are solid). These carcinomas pose the greatest clinical problems due to the high recurrence of non-muscle invasive tumors even after transurethral resection of the tumors. At present, there are no clinically useful markers available for identifying bladder cancer patients with a high risk of disease recurrence or progressIon. Multiple molecular events take place when normal epithelial cells are transformed into tumor tissues. These can now be monitored simultaneously, by using oligonucleotide rnicroarrays and the expression patterns of three different grades of Tee namely Tee WHO Grade I, Tee WHO Grade II and Tee WHO Grade III (according to WHO classification) can be established. In this study, individual cell suspensions were prepared from bladder tumor biopsies. Pools of cells were also prepared from noncancerous tissues. Total RNA was isolated and reverse transcribed into eDNA. In vitro transcription into cRNA was carried out with the incorporation of Cy3 and Cy5 dyes. Labeled cRNA probes were co-hybridized to the microarray slide containing 1,853 cancer related genes. Following hybridization procedure, scanning of the array slides was carried out to identify the gene expression levels in each of the sample investigated. To determine the co-expression patterns displayed between different stages of the bladder cancer, hierarchical clustering analysis that group tumors according to similarity in their expression profile was used. Hierarchical clustering demonstrated an unambiguous separation of TCC WHO Grade I from Grades II and III of these urothelial tumors. In addition, based on the gene function, nine clusters of genes were identified. These genes are associated with cell adhesion molecules, protein synthesis, oncogenes, apoptosis markers, growth factors, immunology, cell cycle regulators, transcription factors and angiogenesis. Fold-change analysis of gene expression revealed 106, 49 and 51 genes that are over-expressed and 13, 186 and 132 that are suppressed in TCC Grades I, II and III, respectively. A gene is considered differentially expressed if its relative expression is two-fold or greater. Because of the inherent limitations in the reliability of microarray, genes identified as differentially expressed were validated with a PCR-based method. Real-Time peR was able to confirmed 75% of the microarray data. These results were in concordance to that previously reported. This study indicates that gene expression patterns can be identified in bladder cancer by combining oligonucleotide arrays and Real-Time PCR analysis. These data combined with other data from similar research, could provide insights into the extent of expression differences underlying malignancy of the bladder cancer and reveal genes that may prove useful as diagnostic or prognostic markers.

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