摘要: |
目的:检测肾透明细胞癌(ccRCC)中谷胱甘肽S-转移酶M3(GSTM3)基因表达水平,观察其启动子区CpG岛的甲基化水平,探讨GSTM3基因甲基化与ccRCC发生和转移的关系。方法:半定量RT-PCR检测ccRCC高转移潜能细胞系(RCC05-TXJ)、低转移潜能细胞系(RCC05-ZYJ)、24例原位ccRCC及其癌旁肾组织和14例ccRCC转移组织的GSTM3基因的表达水平。用去甲基化剂5-Aza-CdR干预RCC05-ZYJ,RT-PCR检测处理前后GSTM3基因的表达变化。用巢式BSP(nested bisulfite sequencing PCR)检测10例原位ccRCC及其癌旁肾组织的GSTM3基因启动子区CpG岛甲基化位点。采用巢式MSP(nested methylation specific PCR)检测10例原位ccRCC及其癌旁肾组织、8例ccRCC转移组织甲基化情况。结果:RCC05-TXJ中GSTM3基因表达强度低于RCC05-ZYJ。5-Aza-CdR处理RCC05-ZYJ后,GSTM3基因表达强度高于处理前。24例原位ccRCC中17例GSTM3基因的表达强度低于其在癌旁肾组织中的表达强度,其余7例原位ccRCC中GSTM3基因的表达强度不低于其在癌旁肾组织中的表达。10例原位ccRCC及其癌旁和8例转移组织中,癌组织GSTM3基因启动子甲基化阳性为4例,癌旁组织2例(P=0.628),转移组织1例(P=0.314),差异无统计学意义。结论:GSTM3基因表达与ccRCC的发生、转移密切相关,其启动子区CpG岛甲基化会降低该基因的表达;初步筛选出ccRCC中GSTM3基因启动子区CpG岛甲基化位点,为后续研究奠定了基础。 |
关键词: 肾脏肿瘤 肿瘤转移 谷胱甘肽转移酶 DNA甲基化 |
DOI:10.3724/SP.J.1008.2008.01273 |
投稿时间:2008-03-31修订日期:2008-07-04 |
基金项目:国家自然科学基金(30873041). |
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Expression of glutathione S-transferase M3 in clear cell renal cell carcinoma and its promoter CpG methylation |
WU Qi1,HOU Jian-guo1*,DU Wen-bin1,CHANG Wen-jun2,ZHAI Yu-jia2,LIN Li-ping2,TAN Xiao-jie2,CAO Guang-wen2* |
(1.Department of Urology,Changhai Hospital,Second Military Medical University,Shanghai 200433,China;2.Department of Epidemiology,Faculty of Health Services,Second Military Medical University,Shanghai 200433) |
Abstract: |
Objective:To examine the expression and promoter CpG island methylation of glutathione S-transferase M3 (GSTM3) gene in clear cell renal cell carcinoma (ccRCC),and to evaluate the relationship of expression,methylation of GSTM3 gene with the metastasis and oncogenesis of ccRCC.Methods:Using semi-quantitative RT-PCR technique,we examined GSTM3 expression in surgical specimens of 24 primary ccRCCs and adjacent non-malignant tissues,14 metastatic ccRCCs and 2 ccRCC cell lines (RCC05-TXJ,RCC05-ZTJ) with different metastatic potentials.RCC05-TXJ cells were cultured in RPMI 1640 medium and treated with DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-Aza-CdR).Semi-quantitative RT-PCR was used to examine the expression of GSTM3 in response to 5-Aza-CdR treatment.Nested bisulfite sequencing PCR and DNA sequencing were used to analyze different methylation locuses in GSTM3 gene promoter in 10 primary ccRCCs and adjacent non-malignant tissues.We also examined the methylation level in 10 primary ccRCCs and the corresponding non-malignant tissues as well as 8 metastatic tissues by nested methylation-specific PCR.Results:Expression of GSTM3 gene in metastatic ccRCC cells (RCC05-TXJ) was lower than that in the non-metastatic ccRCC cells (RCC05-ZYJ).Down-regulation of GSTM3 gene expression was found in 17 of the 24 primary ccRCCs as compared with the non-malignant tissue.Expression of GSTM3 in the metastatic ccRCCs was lower than that in primary ccRCCs.5-Aza-CdR treatment increased GSTM3 expression in RCC05-ZYJ.Methylation in GSTM3 promoter was found in 4 of 10 ccRCC tissues,2 of 10 adjacent tissues,and 1 of 8 metastatic tissues.No significant difference was found between ccRCC and adjacent tissues (P=0.628) or ccRCC and metastatic disease (P=0.314) due to limit number of cases.Conclusion:Our findings support that promoter aberrant methylation is one of the major mechanisms of GSTM3 gene down-regulation in ccRCC.The preliminary identification of GSTM3 promoter methylation sites may provide a basis for further study. |
Key words: kidney neoplasms neoplasm metastasis glutathione transferase DNA methylation |