NANOGP8基因在肺癌细胞系中甲基化水平与表达研究

doi:10.13523/j.cb.20140204

中国生物工程杂志

2014, Vol. 34

Issue (2): 21-25 DOI: 10.13523/j.cb.20140204

研究报告

NANOGP8基因在肺癌细胞系中甲基化水平与表达研究

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

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

Methylation Regulation of NANOGP8 Expression in Lung Cancer Cell Line

WANG Ai-e¹, WANG Ying¹, LI Zhao-xia¹, SONG Yun-xi¹, WANG Dong-xia¹, GAN Le-wen¹, MA Jian-xin¹, CHANG Yan¹, ZHANG Sui-yang¹, JIANG Xiao-xia²

1. Department of Respiratory, General Hospital of the Secondary Artillery, PLA, Beijing 100088, China;
2. Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China

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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。

关键词： NANOGP8; 肺癌细胞系A549; 甲基化; 调控; 表达

Abstract: 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 cells were performed. Genomic DNA was extracted from these cells 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 E.coli DH5α and eight clones for each region were sequenced and blasted. There are no significant changes 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 cells. 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.

Key words: NANOGP8 A549 cell line Methylation Regulation Expression

收稿日期: 2013-11-20 出版日期: 2014-02-25

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通讯作者: 王英 E-mail: 13641083406@163.com

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引用本文:

王爱娥, 王英, 李朝霞, 宋云熙, 王东霞, 甘乐文, 马建新, 常艳, 张睢扬, 江小霞. NANOGP8基因在肺癌细胞系中甲基化水平与表达研究[J]. 中国生物工程杂志, 2014, 34(2): 21-25.

WANG Ai-e, WANG Ying, LI Zhao-xia, SONG Yun-xi, WANG Dong-xia, GAN Le-wen, MA Jian-xin, CHANG Yan, ZHANG Sui-yang, JIANG Xiao-xia. Methylation Regulation of NANOGP8 Expression in Lung Cancer Cell Line. China Biotechnology, 2014, 34(2): 21-25.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140204 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I2/21

[1] Soon Y Y, Stockler M R, Askie L M, et al. Duration of chemotherapy for advanced non small cell lung cancer: a systematic review and meta analysis of randomized trials. J Clin Oncol, 2009, 27(20): 3277-3283.
[2] 宋东颖,王毅,孙岚,等. 肿瘤干细胞理论及肿瘤干细胞分离和鉴定研究进展. 中国药理学与毒理学杂志, 2012, 26(5): 674-679. Song D Y,Wang Y,Sun L,et al. Progress in tumor-stem cell theory and tumor stem cells isolation and identification. Chin J Pharmacol Toxicol,2012, 26(5): 674-679.
[3] 冯缤,冯起校. 肿瘤学说对肺癌研究的价值. 职业与健康,2012, 28(5): 613-614. Feng B,Feng Q X. Value of cancer stem cell theory in lung cancer related studies. Occu Pand Health,2012, 28(5): 613-614.
[4] Park I H, Zhao R, West J A, et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature, 2008, 451(7175): 141-146.
[5] Miyoshi N, Ishii H, Nagai K, et al. Defined factors induce programming of gastrointestinal cancer cells. Proc Natl Acad Sci USA, 2010, 107: 40-45.
[6] Jeter C R, Badeaux M, Choy G, et al. Functional evidence that the self-renewal gene NANOG regulates human tumor development. Stem W Cells, 2009, 27: 993-1005.
[7] Luo W, Li S, Peng B, et al. Embryonic stem cells markers SOX2, OCT4 and Nanog expression and their correlations with epithelial-mesenchymal transition in nasopharyngeal carcinoma. PLoS One, 2013, 8(2): 1-8.
[8] Lee M T, Bonneau A R, Takacs C M, et al. Nanog, Pou5f1 and SoxB1 activate zygotic gene expression during the maternal-to-zygotic transition. Nature, 2013, 503(7476): 360-364.
[9] Mitsui K, Tokuzawa Y, Itoh H, et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell, 2003, 113: 631-642.
[10] Chambers I, Colby D, Robertson M, et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell, 2003, 113: 643-655.
[11] Costa Y, Ding J, Theunissen T W, et al. NANOG-dependent function of TET1 and TET2 in establishment of pluripotency. Nature, 2013, 495(7441): 370-374.
[12] Booth H A, Holland P W. Eleven daughters of Nanog. Genomics, 2004, 84: 229-238.
[13] Zhang J, Wang X, Li M, et al. NANOGP8 is a retrogene expressed in cancers. FEBS J, 2006, 273(8): 1723-1730.
[14] Palla A R, Piazzolla D, Abad M, et al. Reprogramming activity of NANOGP8, a NANOG family member widely expressed in cancer. Oncogene, 2013, 32(8): 1723-1730.
[15] Ishiguro T, Sato A, Ohata H, et al. Differential expression of nanog1 and nanogp8 in colon cancer cells. Biochem Biophys Res Commun, 2012, 418(2): 199-204.
[16] Jeter C R, Liu B, Liu X, et al. NANOG promotes cancer stem cell characteristics and prostate cancer resistance to androgen deprivation. Oncogene, 2011, 30(9): 3833-3845.
[17] Zhang J, Espinoza L A, Kinders R J, et al. NANOG modulates stemness in human colorectal cancer. Oncogene, 2013, 32(37): 4397-405.
[18] Liu Y, Wu X, Li X, et al. Blocking mtDNA replication upregulates the expression of stemness-related genes in prostate cancer cell lines. Ultrastruct Pathol, 2013, 37(4): 258-266.
[19] Herman J G, Graff J R, Myohanen S, et al. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A, 1996, 93 (18): 9821-9826.
[20] 张经余,王爱娥,李美香,等. 人NANOGP8蛋白的原核表达及多克隆抗体的制备.中国生物工程杂志, 2009, 29(3): 30-35. Zhang J Y, Wang A E, Li M X, et al. Prokaryotic expression and preparation of polyclonal antibody of human NANOGP8. China Biotechnology, 2009, 29(3): 30-35.

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