Study of DNA Methylation Affecting Glutathione S-Transferase Mu 1 Gene Expression in Bladder Cancer

• Main Authors: Pei-Wen Zhao, 趙珮雯
• Other Authors: Yi-Wen Liu
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/10903142615435050641

碩士 === 國立嘉義大學 === 微生物免疫與生物藥學系研究所 === 103 === According to the statistics of Taiwan Cancer Registration, bladder cancer is one of the common urinary system cancers. The most efficient method for diagnosing cancer of the bladder is cystoscopy along with biopsy of suspicious lesions. However, the process of examination is invasive and uncomfortable. Many studies of bladder cancer detection tend to focus on DNA modification in the voiding bladder epithelial cells. Detection of specific DNA methylation can be a diagnostic and prognostic information of bladder cancer. For example, DAPK, RARβ, E-cadherin and p16 genes have been shown to have a high degree of methylation in the early stage of bladder cancer. In our previous study, we found that the glutathione-S-transferase Mu1 (GSTM1) protein expression is significantly down-regulated in carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-treated mice bladder. In order to study whether the down-regulated GSTM1 protein was due to gene methylated on its 5' CpG islands or not, we designed experiments in vitro and in vivo. In vitro study, we observed that the mRNA and protein expression of GSTM1 in 7 human bladder cancer cell lines, which are identified in tumor grade I~III. It showed that GSTM1 gene expression was up-regulated under a treatment of demethylation drug 5-aza-2’-deoxycytidine or zebularine in J82 cells. The data suggested that GSTM1 may be down-regulated by DNA methylation in this cell line. Next, we used bisulfite conversion and PCR followed by T&;A cloning and sequencing to analyze the CpG islands methylation site of GSTM1 gene promoter in 6 human bladder cancer cell lines. The results revealed that high DNA methylation level was present in human GSTM1 5' CpG islands in all cell lines except T24. Besides, the DNA methylation level of J82 GSTM1 gene was also studied after demethylation agent treatment. The result revealed that DNA methylation level decreases slightly in demethylation agent-treated J82 cells (about 13.5%). Although the GSTM1 gene expression of J82 cell lines up-regulated under the treatment of 5-aza-2’-deoxycytidine and trichostatin A, the GSTM1 DNA methylation level decreased a little. Otherwise, GSTM1 gene expression in mice bladder cancer cell lines MB49 was affected slightly by DNA methylation. But GSTM1 gene expression increased under the treatment of 5-aza-2’-deoxycytidine and trichostatin A combination more than 5-aza-2’-deoxycytidine alone. In animal model study, we observed the GSTM1 protein expression are down-regulated obviously in BBN-treated mice. However, the DNA methylation level of mice GSTM1 gene CpG island was always low. The average of GSTM1 methylation level was a little increased in BBN-treated mice (1.32%) compared to control mice (0.5%). Based on these findings, it suggested that GSTM1 protein down-regulation was mediated by DNA methylation in a little part in BBN-treated mice. In human bladder cancer cells, 5 of 6 cell lines have high methylation level in GSTM1 gene CpG island. Therefore, GSTM1 gene methylation level maybe a diagnostic target in human bladder cancer, but not in BBN-induced mice bladder cancer.