Combination Of Laser Capture Microdissection And Gene Microarray Analysis To Identify Gene Expression Profiles And Biological Pathways In Low Malignant Bladder Transitional Cell Carcinoma

Objective:Bladder transitional cell carcinomas (BTCC) is the most common urologic tumor in China. Recent study have found that, from biological behaviour to pathological characteristics, BTCC actually has two phenotypes: low malignant and aggressive, the so-called two type bladder transitional cell carcinomas or two-tie grading system. This new classification provide the new molecular base for the study of bladder cancer. Further study may elucidate the molecular mechanism of bladder carcinogenesis and provide potential clinical diagnosistic and therapeutic targets, and have important significance. This study is for identifying the molecular mechanism, differentially expressed genes and further elucidating the carcinogenesis mechanism of BTCC on the basis of two-tie grading system, this is also one of urgent difficulties in bladder cancer research area.At present, there are many methods for identifying differentially expressed genes, such as ESTs seqauencing, SAGE, subtractive cloning, mRNA differential display, RAD and SSH etc.but the specialty is low and progressive is very slow. While, gene expression microarray can assess the expression of a large number of genes simultaneously, minor amount of mRNA quicly and directly and thereford, provide a highly powerful tool. So far most previous molecular and cytogenetic analyses of bladder cancer were focused on aggressive BTCC. Little is known about the eventsthat lead to the development of low malignant BTCC. To clarify the mechanisms that underlie its development and/or progression, we compared the expression profiles of laser-microdissected low malignant bladder-cancer tissues with corresponding noncancerous mucosae by means of an oligo microarray containing 22000 genes. Methods:1. Identifying the differentially expressed genes between laser-microdissected low malignant bladder-cancer tissues and corresponding noncancerous mucosae by the Combination of laser capture microdissection, oligo microarray analysis and mRNA linear amplification.2. Evaluating the results of gene microarray by bioinformatic analsis and semi-quantitative RT-PCR for the four chosen candidate differentially expressed genes.3 Studying the EZH2 and BMI-1 protein expression and their correlation with clinicpathological factors in bladder transitional cell carcinoma by immunohistochemistry.3. Gene function clustering and biological analysis by using Pathway explorer and GenMAPP analysis tool.Results:1. Differential gene expression profiles: This study identified 215 genes that were commonly up-regulated and 53 genes were commonly down-regulated in the tumors.2. bioinformatic analsis and semi-quantitative RT-PCR support the lightness of gene microarray results.3. Immunohistochemistry analysis on the expression of EZH2 and BMI-1 shows that there are high positive expression of EZH2 and BMI-1 compared with corresponding normal bladder mucosa.(P<0.05). Furthermore, the high expression of EZH2 have correlated with increased pathological grade;The high expression of BMI-1 have correlated with increased tumor recurrence.4. Functional clustering of these differentially expressed genes by Pathway Explorer and GenMAPP in the context of biological pathways revealed that several pathways,especially WNT signal pathway, MAPK signal pathway, ubiquitin mediated proteolysis, Focal adhesion, cell proliferation, tight junction, Calcium signal pathway, apoptosis, cell-cycle control, etc., are actively involved in the carcinogenesis and/or progression of low malignant bladder transitional cell cancer.Conclusion:1. combining laser capture microdissection and microarray is an effective approach to identify differential gene expression profiles.2. bioinformatic analsis and semi-quantitative RT-PCR support the tightness of gene microarray results.3 Immunohistochemistry analysis on the expression of EZH2 and BMI-1 shows thatthere are high positive expression of EZH2 and BMI-1 compared with correspondingnormal bladder mucosa.(P<0.05). Furthermore, the high expression of EZH2 havecorrelated with increased pathological grade;The high expression of BMI-1 havecorrelated with increased tumor recurrence.4. Functional clustering of these differentially expressed genes by Pathway Explorerand GenMAPP in the context of biological pathways can promote there-consideration for oncology on the eye of systematic biology.4. Further study on these differentially expressed genes and underlying biologicalpathways may elucidate the molecular mechanism of bladder carcinogenesis andprovide potential clinical diagnosistic and therapeutic targets.This work was supported by the Applied Basic Research Major Programs of Tianjin Municipal Science and Technology commission (No: 043114211) and 2003 Year PHD Student Innovation Fund of Tianjin Medical University (No: 2005-05)...