采用蛋白质组学技术筛选鼻咽癌细胞的甲基化沉默基因  

Identification of Hypermethylated Genes in Nasopharyngeal Carcinoma Cell Line by Proteomics

作　　者：张文静[1] 易斌[1] 易红[1] 张鹏飞[1] 李茂玉[1] 李萃[1] 阮林[1] 陈主初[1] 李建玲[1] 肖志强[1] 

机构地区：[1]中南大学湘雅医院卫生部肿瘤蛋白质组学重点实验室,长沙410008

出　　处：《生物化学与生物物理进展》2008年第4期410-417,共8页Progress In Biochemistry and Biophysics

基　　金：国家重点基础研究发展计划(973)(2001CB5120);芙蓉学者特聘教授科研奖励基金(湘教通[2007]362号);教育部跨世纪优秀人才培养计划基金(教计函[2002]48);湖南省科技厅重大科技专项(06SK2004)资助项目~~

摘　　要：为筛选鼻咽癌的甲基化沉默基因,采用二维凝胶电泳(2-DE)技术分离甲基转移酶抑制剂5-杂氮-2′-脱氧胞苷(5-aza-2-dC)处理与未处理鼻咽癌细胞5-8F的蛋白质,PDquest图像分析软件识别差异蛋白质点,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)鉴定差异蛋白质.然后采用Westernblotting和RT-PCR检测差异蛋白质nm23-H1在药物处理与未处理5-8F细胞中的表达水平,采用甲基化特异性PCR(MS-PCR)检测nm23-H1基因在药物处理与未处理5-8F细胞中的甲基化水平.建立了5-aza-2-dC处理与未处理5-8F细胞蛋白质的2-DE图谱,识别了49个差异表达的蛋白质点,鉴定了33个差异表达的蛋白质,其中包括nm23-H1在内的15个蛋白质在5-aza-2-dC处理后的5-8F细胞中表达上调,而18个蛋白质表达下调.Westernblotting和RT-PCR结果显示,nm23-H1在5-aza-2-dC处理5-8F细胞后表达上调,MS-PCR结果显示,在5-aza-2-dC处理5-8F细胞后nm23-H1基因甲基化水平下降,结果证实,nm23-H1基因是5-8F细胞中的甲基化沉默基因.15个5-aza-2-dC处理后表达上调的基因可能是5-8F细胞中的甲基化沉默基因,为筛选鼻咽癌甲基化失活基因提供了科学依据.To screen for methylation silenced genes in nasopharyngeal carcinoma cell line 5-8F, two-dimensional gel electrophoresis （2-DE） was performed to separate the proteins of treated and untreated 5-8F cells with demethylating agent 5-aza-2-dC, PDQuest software was used to analyze 2-DE images, and MALDI-TOF-MS was used to identify the differentially expressed proteins between the treated and untreated 5-8F cells. Then RT-PCR and Western blotting were performed to examine the expression levels of nm23-H1 mRNA and protein, one of the differential expression proteins, in the treated and untreated 5-8F cells, respectively. Methylation-specific PCR （MS-PCR） was performed to detect the methylated level of nm23-H1 gene in the treated and untreated 5-8F cells. 2-DE patterns of the treated and untreated 5-8F cells with 5-aza-2-dC were established, and a total of forty-nine differential protein spots were found in treated and untreated 5-8F cells. Thirty-three non-redundant differential proteins were identified by MS, 15 proteins of which were up-regulated after 5-aza-2-dC treatment. The results of Western blotting, RT-PCR and MS-PCR showed that nm23-H1 is a methylation silenced gene in 5-8F cell line. Encoding genes of 15 up-regulated proteins after 5-aza-2-dC treatment may be methylation silenced genes in NPC cell line 5-8F. The data will be helpful to screen for methylation silenced genes in nasopharyngeal carcinoma.

关 键 词：鼻咽癌 甲基化沉默基因 蛋白质组学 甲基化特异PCR NM23-H1 

分 类 号：R318.0[医药卫生—生物医学工程]