The Gene Expression and DNA Methylation of Glutathione S-Transferase Mu1 in Bladder Cancer Carcinogenesis

• Main Author: 陳泱亦
• Other Authors: 劉怡文
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/49919286515160244355

碩士 === 國立嘉義大學 === 微生物免疫與生物藥學系研究所 === 102 === ABSTRACT 　　Bladder cancer is the 13th most common cancer in Taiwan. After clinical therapy, bladder cancer recurs frequently that leads the efforts on finding early-stage diagnostic technology and therapeutic agents for it. Specific changes in protein expression can provide a diagnostic marker. In our previous study, in order to understand the potential protein expression change during urothelial carcinogenesis, carcinogen BBN was used to induce mouse bladder carcinogenesis and bladder mucosa proteins were analyzed by 2D electrophoresis. Some significantly down-regulated proteins were found including glutathione S-transferase Mu 1 (GSTM1), L-lactate dehydrogenase B chain, fatty acid binding protein 4 (FABP4), alcohol dehydrogenase 1B, annexin A5, strtifin, gelsolin, keratin 8 and 18. After Oncomine analysis, all above genes show down-regulated in bladder cancer patients. In order to identify whether the downregulation was mediated by DNA methylation, we used 5-aza-2’-deoxycytidine (5-aza-dC), a DNA demethylation drug, to treat bladder cancer cell lines and analyzed mRNA expression of above genes. We found that 5-aza-dC significantly increased GSTM1 mRNA expression in RT4 and 5637 cells. Gelsolin was slightly increased in TSGH8301, T24, J82 and 1376 cells. FABP4 was increased in J82 and 5637. Keratin 8, 18 were slightly increased in T24. DNMT3b was increased in RT4, 5637 and 1376. We collected genomic DNA from BBN-treated C57BL/6J mice bladders and 2 different human bladder cancer cell lines to analyze DNA methylation status of GSTM1. Massarray assay was used in mouse study. Mouse GSTM1 CpG island methylation assay was divided into two regions. The methylation ratios were slightly increased in upstream region (from 0.071 to 0.181) and downstream region (from 0.114 to 0.134) by BBN treatment for 14 weeks. Direct PCR product sequencing was used in human GSTM1 analysis in RT4 and T24 cell lines. It presented that most cytosines on GSTM1 CpG island were methylated in RT4 but not in T24 cells. In summary, this study provides some clues for bladder tumor carcinogenesis and the down-regulation of GSTM1 gene. The GSTM1 expression or DNA methylation may provide for diagnosis and prevention of bladder tumor in the future.