Screening And Identification Of Specific Markers For Bladder Carcinoma Stratification From Urine Urothelial Cells With SSH And CDNA Microarray

Objective:To Screen and identify differentially expressed genes in BTCC and normal urine exfoliated urothelial cells. Part of genes could be validated simultaneously with many clinical samples. Methods:Choose 10 definite patients with BTCC at the People's Hospital of Gansu Province from 2005 to 2007 who were recruited for the study and didn't received chemotherapy, radiotherapy or immunotherapy in Preoperative. Meanwhile, urine from 10 healthy volunteers served as controls. Total mRNA was isolated from BTCC and normal exfoliated urothelial cells in urine, respectively. Using SSH technique to construct a forward/reverse subtracted cDNA library and to make cDNA chip of recombinant plasmid. The SSH library was further screened using DIG-labeled PCR amplification products as probe. The selected clone were further sequenced with unsupervised hierarchical clustering based on the standard correlation of logarithmic transformed data and homology analysised in the GenBank, to classify those genes based on defined categories of molecular function and biological processes by using the Expressed Gene Anatomy Database (EGAD). PTEN, RUNX3, hMLH1, hMSH2 and hMSH3 genes could be performed simultaneously with many clinical samples by using RT-PCR. Western blot and methylation-specific PCR (MSP) technique to find a group of BTCC related markers in urine, to evaluate the correlation between the markers and the biological behavior of BTCC. Results:Differentially expressed genes of urine exfoliated urothelial cells were screened by combined with SSH and cDNA microarray technology. Plasmid inserted rate was up to 58.9%. The growth of clone and efficiency of plasmid inserted indicated that each clone contained the possibility of specific fragments. It suggested that the SSH library had a reliable quality. The forward/reverse subtracted cDNA library was further screened using DIG-labeled PCR amplification products as probe. After initial screening, the selected genes were further sequenced, and homology analysis was performed as an additional validation to the microarray. The result showed that 112 clones represent 67 known genes, which were appeared to be a series of complicated molecular events in the development of BTCC. The detection method of cDNA microarray was developed in this study. The expression of PTEN, RUNX3 mRNA and protein in the normal bladder tissues and BTCC tissue were 93.8%/15.6%, 93.8%/34.4%, respectively (P<0.05). hMLH1,hMSH2 and hMSH3H mRNA in the normal bladder tissues and BTCC tissue were21.9%/46.9%,65.6%/37.5%,0%/18.8%, respectively (P<0.05). The frequence of Promoter methylation of PTEN and RUNX3 genes was 65.6% and 46.9% in BTCC tissues, but it was not found in normal bladder tissues. The frequence of Promoter methylation of hMSHl and hMSH2 gene in BTCC tissues was 37.5%,53.1%, respectively, and existed part of methylation, but hMSH3 methylation was not found in all bladder tissues. The frequence of hMSHl, hMSH2 methylation were declined with the increasing tumor pathological grades (P<0.05), However, it was not correlated with its clinical stages (P>0.05). Conclusions:Using low-throughput cDNA microarray in urine exfoliated urothelial cells analysis have shown that mRNA expression profiles are a powerful method for discriminating between bladder tumors and normal urothelium. The informativeness of these expression profiles and the high frequency of tumor cell exfoliation into urine have raised the prospect of using multiple gene expression signatures obtained from urine total RNA to better diagnose bladder cancer and provide more information on disease characteristics at the time of diagnosis.