NANOGP8基因在肺癌细胞系中甲基化水平与表达研究  

Methylation Regulation of NANOGP8 Expression in Lung Cancer Cell Line

作　　者：王爱娥[1] 王英[1] 李朝霞[1] 宋云熙[1] 王东霞[1] 甘乐文[1] 马建新[1] 常艳[1] 张睢扬[1] 江小霞[2] 

机构地区：[1]中国人民解放军第二炮兵总医院,北京100088 [2]军事医学科学院基础医学研究所,北京100850

出　　处：《中国生物工程杂志》2014年第2期21-25,共5页China Biotechnology

摘　　要：目的:研究NANOGP8基因在肺癌细胞系A549中启动子区域甲基化水平及其与基因表达的相关性。方法:利用MethPrimer甲基化岛预测和甲基化引物设计软件,预测NANOGP8启动子区甲基化位点。分别从人成纤维细胞系和肺癌细胞系中提取基因组DNA,经过亚硫酸氢钠处理后,用针对甲基化位点设计的引物进行PCR扩增,获得相应区段DNA后,连接到pGEM-T载体,转化大肠杆菌鉴定阳性克隆后,测序并与GenBank数据库中NANOGP8基因组DNA序列比对,获得其甲基化水平数据。分别提取两个细胞系的总RNA,RT-PCR获得相应cDNA进行PCR扩增,扩增产物经琼脂糖凝胶电泳后,经酶切和测序验证,获取其表达水平数据。结果:成功预测NANOGP8的两个区域有甲基化位点,并检测到人成纤维细胞系和肺癌A549细胞系中NANOGP8启动子甲基化水平分别为59.7%和12.5%,表达检测结果显示在A549细胞系中检测到NANOGP8的基因片段,而在人成纤维细胞系中没有扩增到相应产物。结论:在正常成体细胞中NANOGP8基因由于启动子的高度甲基化而沉默,而在肺癌细胞系中NANOGP8基因启动子去甲基化激活其表达。NANOGP8基因的表达与其启动子区域去甲基化密切相关,同时NANOGP8在肺癌细胞分化过程中发挥重要作用。The nearly identified retrogene NANOGP8 is from a stem cell transcription factor NANOG gene, which expressed in multiple cancers, but generally not in normal tissues and its function is related to cancer development and progression. To investigate the relationship between NANOGP8 expression level and methylation sites at the gene promoters. Bisulphate Sequencing in lung cancer cell line A549 as well as the normal fibroblast ceils were performed. Genomic DNA was extracted from these ceils and treated with bisulphate, then PCR was performed in the NANOG promoter, encompassing a total of 10 CpGs within nucleotides -1449 to -1295 and - 1168 to -952 relative to the transcription start site （TSS） reported by Park. Two regions including 9 CpGs in the potential NANOGP8 promoter, which located within nucleotides -1529 to -1401 and -690 to -587 relative to the TSS of NANOGP8 predicted by Methprimer program were also examined. PCR products were then ligated to pGEM-T vector. The recombinant plasmid was transformed into were sequenced and blasted. There are no significant changes E. coli DH5ot and eight clones for each region of the percentage of clones with methylation in NANOG promoter in A549 cell line compared to in the fibroblast cells （65% vs. 67.5% , P =0.62）. However, the percentage at the two regions of NANOGP8 shows a significant increase from A549 cell line to the fibroblast cells （ 12.5% vs. 59.7%, P 〈 0. 001 ）. NANOGP8 transcriptional level was detected by RT-PCR in both cell lines and found NANOGP8 is highly expressed in A549 cell line but not in the fibroblast ceils. The methylation status of 5＇-flanking regions of human NANOGP8 is associated with the NANOGP8 expression in lung cell line and may relate to lung cancer development and progression.

关 键 词：NANOGP8 肺癌细胞系A549 甲基化 调控 表达 NANOGP8 

分 类 号：Q786[生物学—分子生物学]