肾透明细胞癌中谷胱甘肽S-转移酶M3基因的表达及其启动子区CpG岛的甲基化水平  

Expression of glutathione S-transferase M3 in clear cell renal cell carcinoma and its promoter CpG methylation

作　　者：武旗[1] 侯建国[1] 杜稳斌[1] 常文军[2] 翟羽佳[2] 林丽萍[2] 谭晓洁[2] 曹广文[2] 

机构地区：[1]第二军医大学长海医院泌尿外科,上海200433 [2]第二军医大学卫生勤务学系流行病学教研室,上海200433

出　　处：《第二军医大学学报》2008年第11期1273-1278,共6页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(30873041);Supported by National Natural Science Foundation of China(30873041)

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。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 nonmalignant 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 promo

关 键 词：肾脏肿瘤 肿瘤转移 谷胱甘肽转移酶 DNA甲基化 

分 类 号：R737.11[医药卫生—肿瘤]