Screening and identification of specific markers for bladder transitional cell carcinoma from urine urothelial cells with suppressive subtractive hybridization and cDNA microarray

Abstract

Objective: The objective of this study was to screen and identify differentially expressed genes in invasive bladder transitional cell carcinoma (BTCC). Methods: Voided urine samples were collected from consecutive patients with BTCC and patients under surveillance for bladder cancer recurrence; voided urine samples from patients with nonmalignant diseases served as control. We identified the differentially expressed genes by comparing urine samples of bladder carcinoma to that of the control group with suppressive subtractive hybridization (SSH) and cDNA microarray. The differentially expressed genes were verified by quantitative real-time polymerase chain reaction (QPCR). Results: From the 762 white colonies, a total of 449 positive clones were obtained in which 112 were found to be upregulated in BTCC. Sequencing and homology analysis were performed for these 112 clonies. The detection rates of some known genes (including IGF-1, human telomerase reverse transcriptase [hTERT], bladder cancer specific nuclear matrix protein 4 [BLCA-4] and homeobox A13 [HOXA13]) for BTCC at the Ta, T1 and >T1 stages were 48%, 90% and 100%, respectively, with a specificity of 85%. The test specificity was 80% for the 30 control patients with urinary tract infections. The combination of BLCA-4 and HOXA13 could distinguish between low-and high-grade tumours, with specificity and sensitivity of 80%. Conclusion: We successfully constructed a reliable SSH library of BTCC and found that combination detection insulin-like growth factor 1 (IGF-1), hTERT, BLCA-4 and HOXA13 genes could help to evaluate BTCC at different stages. IntroductionBladder transitional cell carcinoma (BTCC) is the fourth common cancer worldwide.1 The 5-year relative survival rate is about 6% for patients with an advanced stage of bladder cancer.1 Cystoscopy and biopsy of suspected lesions have been regarded as the gold standard in the diagnosis of bladder cancer and in the follow-up of these patients. A variety of studies have focused on developing new non-invasive methods for the diagnosis of bladder cancer and monitoring the progression of the disease. New biomarkers that would increase the detection rate of transitional cell carcinomas (TCCs) before bladder muscle layer invasion would reduce TCC-related morbidity and mortality. Although the detection of telomerase and hyaluronic acid phyaluronidase in the urine is sensitive and specific, their role in bladder cancer still requires validation through multicentre clinical studies.2 Therefore, it is important to find the specific biomarkers for bladder cancer which may improve the early diagnosis and effective postoperative follow-up.Recent studies using high-throughput microarray analysis have shown that cDNA expression is helpful in discriminating bladder tumours from normal urothelium at different stages or grades.3,4 The detection of gene expression in the urine may be promising in the diagnosis of TCC and would provide more information for clinical practice. With the rapid development of molecular biot...