摘要
鼻咽癌(NPC)是我国南方地区常见的一种恶性肿瘤,其发病机制还不清楚。DNA甲基化修饰是基因表达调控的重要表观遗传学机制之一,DNA甲基化修饰导致抑瘤基因等的沉默在癌变过程中发挥重要的作用。因此,筛选NPC的甲基化失活基因将有助于揭示NPC的发病机制,具有重要的理论和应用价值。本研究室前期采用蛋白质组学技术比较激光捕获显微切割纯化的NPC组织与正常鼻咽黏膜组织蛋白质表达谱的差异,发现NPC组织中14-3-3σ、Annexin A1和SCCA1蛋白质表达降低,但其表达下调的机制不清楚。
为探讨NPC 14-3-3σ、Annexin A1和SCCA1基因表达下调是否由启动子甲基化引起,本研究以4株具有不同分化程度和转移潜能的NPC细胞系(CNE1、CNE2、5-8F、6-10B)、1株永生化的鼻咽上皮细胞系NP69,以及75例NPC组织和25例正常鼻咽黏膜组织作为实验对象,采用甲基化特异性PCR(MSP)、RT-PCR、Western blotting和免疫组织化学分别检测14-3-3σ、Annexin A1和SCCA1基因启动子甲基化情况、mRNA和蛋白质的表达水平;利用不同浓度(0、0.1、1、5、10μmol/L)的去甲基化药物5-杂氮-2′-脱氧胞苷(5-aza-2dC)处理NPC细胞株,采用MTT比色检测细胞生长,流式细胞仪检测细胞周期分布和细胞凋亡。
主要结果如下:(1)与正常鼻咽黏膜组织比较,NPC组织14-3-3σ(84%VS 28%)、Annexin A1(92%VS 12%)和SCCA1(87%VS 22%)基因启动子出现高频甲基化;(2)在14-3-3σ、Annexin A1和SCCA1基因启动子完全甲基化NPC组织中,其mRNA和蛋白质的表达缺失;在14-3-3σ、Annexin A1和SCCA1基因启动子不完全甲基化NPC组织中,其mRNA和蛋白质的表达显著低于未甲基化的NPC组织;14-3-3σ、Annexin A1和SCCA1基因甲基化与其表达水平高度负相关;(3)14-3-3σ、Annexin A1、SCCA1基因启动子甲基化与NPC淋巴结转移正相关;(4)NP69细胞系14-3-3σ、Annexin A1和SCCA1基因启动子未甲基化,4株NPC细胞系14-3-3σ、Annexin A1和SCCA1基因启动子均存在不同程度的甲基化,而且其甲基化程度与细胞系的分化程度和转移潜能有关,分化程度越低甲基化程度越高,转移潜能越大甲基化程度越高;(5)NPC细胞系14-3-3σ、Annexin A1和SCCA1mRNA表达水平低于NP69细胞;(6)5-aza-2dC呈浓度依赖性地抑制4株NPC细胞系14-3-3σ、Annexin A1和SCCA1基因甲基化,上调其mRNA和蛋白质的表达;(7)5-aza-2dC呈浓度依赖性地抑制4株NPC细胞系的生长,促进细胞凋亡,导致细胞周期阻滞,而且高分化CNE1和无转移6-10B细胞对药物的敏感性高于低分化CNE2和高转移5-8F细胞。
本研究首次报道14-3-3σ、AnnexinA1和SCCA1基因在NPC中出现高频甲基化,启动子甲基化是导致NPC 14-3-3σ、AnnexinA1和SCCA1基因表达缺失或降低的原因之一,14-3-3σ、AnnexinA1和SCCA1基因是新的NPC甲基化沉默基因;本研究发现14-3-3σ、AnnexinA1和SCCA1启动子甲基化与NPC淋巴结转移正相关,提示14-3-3σ、AnnexinA1和SCCA1启动子甲基化可能成为预测NPC转移的指标;本研究结果显示去甲基化药物5-aza-2dC能抑制NPC细胞生长、促进NPC细胞凋亡,提示去甲基化药物可能具有治疗NPC的作用。
Nasopharyngeal carcinoma(NPC)is one of the most common malignant tumors in southern China.However,the mechanism underlying the pathogenesis of NPC remains unclear.DNA methylation is one of the epigenetics mechanisms of gene expression regulation.And promoter methylation of tumor suppression genes plays a critical role in carcinogenesis.Therefore,screening for methylation silenced genes may help to reveal the pathogenesis of NPC,which has important theoretical and clinical value.Our previous study has found that the expression of 14-3-3σ,Annexin A1 and SCCA1 proteins were downregulated in NPC by laser capture microdissection combined with proteomics,but the mechanisms of their downregulation need to be elucidated.
To explore whether the downregulation of 14-3-3σ,Annexin A1 and SCCA1 in NPC is caused by promoter methylation,four NPC cell lines (CNE1,CNE2,5-8F,6-10B),one immortalized human nasopharyngeal epithelial cell line NP69,75 cases of NPC biopsy tissues and 25 cases of normal nasopharyngeal mucosal biopsy tissues were enrolled for the current study.Methylation specific PCR(MSP)was used to examine methylation levels of 14-3-3σ,Annexin A1 and SCCA1 gene promoters; Semi-quantitative RT-PCR,immunohistochemistry and western blotting were performed to assess the expression of 14-3-3σ,Annexin A1 and SCCA1 at mRNA and protein level,respectively.And demethylation agent 5-aza-2dC of 0,0.1,1,5 and 10μmol/L was used to treat NPC cell lines,and then MTT assay was used to detect the cell growth,and flow cytometry was adopted to detec the cell cycle distribution and apoptosis.
The main results of the current study as following:(1)High frequent promoter methylation was found for 14-3-3σ(84%VS 28%),Annexin A1 (92%VS 12%)and SCCA1(87%VS 22%)genes in NPC tissues when compared with normal nasopharyngeal mucosal tissues;(2)In NPC tissues,complete methylation of 14-3-3σ,Annexin A1 and SCCA1 gene promoters was accompanied with expression loss of their mRNA and proteins,and partial methylation of 14-3-3σ,Annexin A1 and SCCA1 gene promoters was accompanied with significant downregulation of their mRNA and proteins.Methylation of 14-3-3σ,Annexin A1 and SCCA1 gene promoters was negatively correlated with their protein expression levels;(3)Methylation of 14-3-3σ,Annexin A1 and SCCA1 promoter were positively correlated with NPC lymphonode metastasis;(4) Methylation with different degree,s of 14-3-3σ,Annexin A1 and SCCA1 gene promoters was detected in all of the four NPC cell lines but not in NP69 cell line,and the methylation levels were related with the differentiated degree and metastatic potential of NPC cell lines;(5)The expression levels of 14-3-3σ,Annexin A1 and SCCA1 were significantly lower in the four NPC cell lines than those in NP69;(6)5-aza-2dC could inhibit methylation of 14-3-3σ,Annexin A1 and SCCA1 gene promoters in a dose-dependent manner,and upregulate the expression of their mRNA and proteins in the four NPC cell lines;(7)5-aza-2dC could inhibit cell proliferation,promote cell apoptosis,and blocks cell cycle progress in a dose-dependent manner in all of the four NPC cell lines.
The results indicate:(1)Promoters of 14-3-3σ,Annexin A1 and SCCA1 genes are high frequently methylated in NPC;(2)The methylation might be reasons for decreased or lossed expressions of 14-3-3σ,Annexin A1 and SCCA1 genes;(3)14-3-3σ,Annexin A1 and SCCA1 are novel methylation-silenced genes in NPC;(4)The methylation of 14-3-3σ,AnnexinA1 and SCCA1 gene promoters was associated with lymphonode metastasis in NPC,indicating that methylaton of 14-3-3σ,Annexin A1 and SCCA1 gene promoters might be a biomarker for NPC metastasis;(5)5-aza-2dC could decrease the methylation levels of 14-3-3σ,AnnexinA1 and SCCA1 gene promoters, upregulate their expressions,inhibit cell proliferation,and promote cell apoptosis in NPC.
引文
[1] Jones P.A, Laird P.W. Cancer epigenetics comes of age. Nat Genet, 1999, 21(2): 163-167
[2] Jones PA. Epigenetics in carcinogenesis and cancer prevention, Ann N Y Acad Sci, 2003, 983: 213-219
[3] Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med, 2003,349(21):2042-2054
[4] Costello JF, Fruhwald MC, Smiraglia DJ, et al. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nat Genet, 2000, 24(2): 132-138
[5] Costello JF, Plass C. Methylation matters. J Med Genet, 2001, 38(5):285-303
[6] Jones PA, Takai D. The role of DNA methylation in mammalian epigenetics. Science, 2001,293(5532): 1068-1070
[7] Antequera F, Bird A. Number of CpG islands and genes in human and mouse. Proc Natl Acad Sci U S A, 1993, 90(24): 11995-11999
[8] Karpf AR, Jones DA. Reactivating the expression of methylation silenced genes in human cancer.Oncogene, 2002, 21(35):5496-5503
[9] Baylin SB, Herman JG, Graff JR, et al. Alternations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res, 1988, 72: 141-196
[10] Nephew KP, Huang TH. Epigenetic gene silencing in cancer initiation and progression. Cancer Letter, 2003,190(2): 125-133
[11]Merlo A, Herman JG, Mao L, et al. 5' CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers. Nat Med, 1995, 1(7):686-692
[12] Hsu HS, Wen CK, Tang YA,et al. Promoter hypermethylation is the predominant mechanism in hMLH1 and hMSH2 deregulation and is a poor prognostic factor in nonsmoking lung cancer. Clin Cancer Res, 2005,11(15):5410-5416
[13]Schildhaus HU, Krockel I, Lippert H, et al. Promoter hypermethylation of p16INK4a, E-cadherin, 06-MGMT, DAPK and FHIT in adenocarcinomas of the esophagus, esophagogastric junction and proximal stomach. Int J Oncol, 2005, 26(6): 1493-1500
[14]Laird PW, Jackson-Grusby L, Fazeli A, et al. Suppression of intestinal neoplasia by DNA hypomethylation. Cell, 1995, 81(2): 197-205
[15]Jeronimo C, Henrique R, Hoque MO, et al. Quantitative RARbeta2 hyperme- thylation: a promising prostate cancer marker. Clin Cancer Res, 2004, 10(12 Pt 1):4010-4014
[16]Belinsky SA, Nikula KJ, Palmisano WA, et al. Aberrant methylation of p16(INK4a) is an early event in lung cancer and a potential biomarker for early diagnosis. Proc Natl Acad Sci U S A, 1998,95(20): 11891-11896
[17]Balch C, Montgomery JS, Paik HI, et al. New anti-cancer strategies: epigenetic therapies and biomarkers. Front Biosci, 2005,10:1897-1931
[18]Kwong J, Lo KW, To KF, et al. Promoter hypermethylation of multiple gene in nasopharyngeal carcinoma. Clin Cancer Res, 2002, 8(1):131-137
[19]Lo KW, Kwong J, Hui AB, et al. High frequency of promoter hypermethylation of RASSF1A in nasopharyngeal carcinoma. Cancer Res, 2001, 61(10):3877-3881
[20]Zhou L, Jiang W, Ren C, et al. Frequent hypermethylation of RASSF1A and TSLC1, and high viral load of Epstein-Barr virus DNA in nasopharyngeal carcinoma and matched tumor-adjacent tissues. Neoplasia, 2005, 7(9):809-815
[21] Cheng AL, Huang WG, Chen ZC, et al.Identification of novel nasopharyngeal carcinoma biomarkers by laser capture microdissection and proteomic analysis. Clin Cancer Res. 2008,14(2):435-45
[22]Mhawech P. 14-3-3 proteins-an update. Cell Res, 2005,15(4):228-236
[23]Hermeking H. The 14-3-3 cancer connection. Nat Rev Cancer, 2003, 3(12):931-943
[24] Simooka H, Oyama T, Sano T, Horiguchi J, Nakajima T. Immunohistochemical analysis of 14-3-3 sigma and related proteins in hyperplastic and neoplastic breast lesions, with particular reference to early carcinogenesis. Pathol Int, 2004, 54(8):595-602
[25]Tanaka K, Hatada T, Kobayashi M, Mohri Y, Tonouchi H, Miki C,Nobori T, Kusunoki M. The clinical implication of 14-3-3 sigma expression in primary gastrointestinal malignancy. Int J Oncol 2004,25(6): 1591-1597
[26] Yang H, Zhao R, Lee MH. 14-3-3 sigma, a p53 regulator, suppresses tumor growth of nasopharyngeal carcinoma. Mol Cancer Ther 2006, 5(2):253-260
[27] Buckingham JC,John CD,Solito E,et al.Annexin 1, Glucocorticoids, and the Neuroendocrine-Immune Interface.Ann N Y Acad Sci.2006, 1088: 396-409
[28]Parente L, Solito E. Annexin 1: more than an anti-phospholipase protein. Inflamm Res. 2004,53 (2): 125-132
[29]张敏,曹云山,朱任之.膜联蛋白Al与肿瘤。国外医学·生理、病理科学与临床分册.2005,25(2):139-142
[30]Paweletz CP,Ornstein DK,Roth MJ,et al.Loss of annexin 1 correlates with early onset of tumorigenesis in esophageal and prostate carcinoma.Cancer Res.2000,60(22):6293-6297
[31]Shen D,Nooraie F,Elshimali Y,et al.Decreased expression of annexin Al is correlated with breast cancer development and progression as determined by a tissue microarray analysis.Human Pathology.2006,37(12):1583-1591
[32]Garcia Pedrero JM,Fernandez MP,Morgan RO,et alo Annexin Al down-regulation in head and neck cancer is associated with epithelial differentiation status.Am J Pathol.2004;164(1):73-79
[33]Masumoto K,Sakata Y,Arima K,et al.Inhibitory mechanism of a cross-class serpin,the squamous cell carcinoma antigen 1.J Biol Chem.2003,278(46):45296-45304
[34]Cataltepe S,Gornstein ER,Schick C,et al.Co-expression of the squamous cell carcinoma antigens 1 and 2 in normal adult human tissues and squamous cell carcinomas.J Histochem Cytochem.2000,48(1):113-122
[35]Suminami Y,Nagashima S,Murakami A,et al.Suppression of a squamous cell carcinoma(SCC)-related serpin,SCC antigen,inhibits tumor growth with increased intratumor infiltration of natural killer cells.Cancer Res.2001,61(5):1776-1780
[36]Katagiri C,Nakanishi J,Kadoya K,et al.Serpin squamous cell carcinoma antigen inhibits UV-induced apoptosis via suppression of c-JUN NH2-terminal kinase.J Cell Biol.2006,172(7):983-990
[37]Hashimoto K,Kiyoshima T,Matsuo K,et al.Effect of SCCA1 and SCCA2 on the suppression of TNF-alpha-induced cell death by impeding the release of mitochondrial cytochrome c in an oral squamous cell carcinoma cell line.Tumour Biol.2005,26(4):165-172
[38]Nakashima T,Yasumatsu R,Kuratomi Y,et al.Role of squamous cell carcinoma antigen 1 expression in the invasive potential of head and neck squamous cell carcinoma.Head Neck.2006,28(1):24-30
[39]Hsu KF,Huang SC,Shiau AL,et al.Increased expression level of squamous cell carcinoma antigen 2 and 1 ratio is associated with poor prognosis in early-stage uterine cervical cancer.Int J Gynecol Cancer.2007,17(1):174-181
[40]Akahira J,Sugihashi Y,Suzuki T,et al.Decreased expression of 14-3-3 sigma is associated with advanced disease in human epithelial ovarian cancer:its correlation with aberrant DNA methylation.Clin Cancer Res.2004,10(8):2687-2693
[41]Ferguson AT,Evron E,Umbricht CB,et al.High frequency of hypermethylation at the 14-3-3 sigma locus leads to gene silencing in breast cancer.Proc Natl Acad Sci U S A.2000,97(11):6049-6054
[42]Perathoner A,Pirkebner D,Brandacher G,et a1.14-3-3σ Expression Is an Independent Prognostic Parameter for Poor Survival in Colorectal Carcinoma Patients.Clinical Cancer Research,2005,11(9):3274-3279
[43]Gronbaek K,Hother C,Jones PA.Epigenetic changes in cancer.APMIS.2007,115(10):1039-1159
[44]Goh L,Murphy SK,Muhkerjee S,et al.Genomic sweeping for hypermethylated genes.Bioinformatics,2007,23(3):281-288
[45]Santini V,Kantarjian HM,Issa JP.Changes in DNA methylation in neoplasia:pathophysiology and therapeutic implications.Ann Intern Med,2001,134(7):573-586
[46]曹卡加,范乔阳,刘奕龙,等.广州市2000～2002年恶性肿瘤的发病率与死亡率分析.癌症,2008;27(3):225-230
[47]Kamer S,Esassolak M,Yalman D,et al.Mature results of neoadjuvant chemotherapy followed by radiotherapy in nasopharyngeal cancer:is it really old fashioned? Med Oncol.2008,25(1):93-99
[48]Chua D,Wei WI,Sham JS,et al.Capecitabine monotherapy for recurrent and metastatic nasopharyngeal cancer.Jpn J Clin Oncol.2008,38(4):244-249
[49]Mostafa E,Nasar MN,Rabie NA,et al.Induction chemotherapy with paclitaxel and cisplatin,followed by concomitant cisplatin and radiotherapy for the treatment of locally advanced nasopharyngeal carcinoma.J Egypt Natl Canc Inst.2006,18(4):348-356
[50]谭双香,肖志强。DNA甲基化与NPC.国际肿瘤学杂志.2007,34(12):904-907
[51]Lo KW,Tsang YS,Kwong J,et al.Promoter hypermethylation of the EDNRB gene in nasopharyngeal carcinoma,Int J Cancer.2002,98(5):651-655
[52]张松,孔维佳,王彦君,等.5-杂氮-2'-脱氧胞苷对人NPC裸鼠移植瘤的抑制作用.癌症.2005,24(10):1201-1205
[53]Kong WJ,Zhang S,Guo CK,et al.Effect of methylation-associated silencing of the death-associated protein kinase gene on nasopharyngeal carcinoma. Anticancer Drugs. 2006,17(3):251-259
[54]Yi ZC, Wang H, Zhang GY, et al. Downregulation of connexin 43 in nasopharyngeal carcinoma cells is related to promoter methylation.Oral Oncol. 2007, 43(9):898-904
[55]Tao Q, Chan AT.Nasopharyngeal carcinoma: molecular pathogenesis and therapeutic developments.Expert Rev Mol Med. 2007,9(12): 1-24
[56] Mackintosh C. Dynamic interactions between 14-3-3 proteins and phosphoproteins regulate diverse cellular processes. Biochem J.2004,381(Pt2):329-342
[57]Leffers H, Madsen P, Rasmussen HH, et al. Molecular cloning and expression ofthe transformation sensitive epithelial marker stratifin. A member of a protein family that has been involved in the protein kinase C signaling pathway. J Mol Biol.l993,23(4):982-998
[58]Hermeking H, Lengauer C, Polyak K, et al. 14-3-3σ is a p53-regulated inhibitor of G2/M progression. Mol Cell. 1997,1(1):3-11
[59]Alldridge LC, Bryant CE. Annexin 1 regulates cell proliferation by disruption of cell morphology and inhibition of cyclin D1 expression through sustained activation of the ERK1/2 MAPK signal. Exp Cell Res. 2003,290 (1): 93-107
[60]Solito E, Kamal A, Russo2Marie F, et al. A novel calcium dependent proapoptotic effect of annexin 1 on human neutrophils .FASEB J. 2003, 17(11):1544-1556
[61]Debret R, EIBtaouri H, Duca L, et al. Annexin A1 processing is associated with caspase-dependent apoptosis in BZR cells[ J ]. FEB S L ett. 2003, 546(223): 195-202
[62]Nagaraja GM, Othman M, Fox BP, et al.Gene expression signatures and biomarkers of noninvasive and invasive breast cancer cells: comprehensive profiles by representational difference analysis, microarrays and proteomics. Oncogene.2006,25(16): 2328-2338
[63]Hayslip J, Montero A.Tumor suppressor gene methylation in follicular lymphoma: a comprehensive review.Mol Cancer.2006,5:44
[64]Rodrigo JP, Garcia-Pedrero JM, Fernandez MP, et al. Annexin A1 expression in nasopharyngeal carcinoma correlates with squamous differentiation. Am J Rhinol. 2005, 19(5):483-487
[65] Qi Y, Chiu JF, Wang L, et al.Comparative proteomic analysis of esophageal squamous cell carcinoma.Proteomics.2005,5(11):2960-2971
[66]Roijer E,de Bruijn HW,Dahlen U,et al.Squamous cell carcinoma antigen isoforms in serum from cervical cancer patients.Tumour Biol.2006,27(3):142-152
[1]Ahmad I,Rao DN.Chemistry and biology of DNA methyltransferaseso Crit Rev Biochem Mol Biol.1996,31(5-6):361-380.
[2]Liu K,Wang YF,Cantemir C,et al.Endogenous assays of DNA methyltransferases:Evidence for differential activities of DNMT1,DNMT2,and DNMT3 in mammalian cells in vivo.Mol Cell Biol.2003,23(8):2709-2719.
[3]Oka M,Meacham AM,Hamazaki T,et al.De novo DNA methyltransferases Dnmt3a and Dnmt3b primarily mediate the cytotoxic effect of 5-aza-2'-deoxycytidine.Oncogene.2005,24(19):3091-3099.
[4]Goh L,Murphy SK,Muhkerjee S,et al.Genomic sweeping for hypermethylated genes.Bioinformatics.2007,23(3):281-288.
[5]Robertson KD,Jones PA.DNA methylation:past,present and future directions.Carcinogenesis.2000,21(3):461-467.
[6]Santini V,Kantarjian HM,Issa JP.Changes in DNA methylation in neoplasia:pathophysiology and therapeutic implications.Ann Intem Med.2001,134(7):573-586.
[7]Lande-Diner L,Zhang J,et al.Role of DNA methylation in stable gene repression.J Biol Chem.2007,282(16):12194-21200.
[8]Momparler RL,Bovenzi V.DNA methylation and cancer.J Cell Phsiol.2000,183(2):145-154.
[9]Dehissenko MF,Chen JX,Tang MS,et al.Cytosine methylation determines hot spots of DNA damage in the human P53 gene.Proc Natl Acad Sci USA.1997,94(8):3893-3898.
[10]Weeler JM,Beck NE,Kim HC,et al.Mechanisms of inactivation of mismatch repair genes in human colorectal cancer cell lines:the predominant role of hMLH1.Proc Natl Acad Sci USA.1999,96(18):10296-10301.
[11]Chen RZ,Pettersson V,Beard C,et al.DNA hypomethylation leads to elevated mutation rates.Nature.1998,395(6697):89-93.
[12]Park SY,Kim BH,Kim JH,et al.Methylation profiles of CpG island loci in major types of human cancers. J Korean Med Sci. 2007, 22(2):311-317,
[13]Issa JP. DNA methylation as a therapeutic target in cancer. Clin Cancer Res. 2007, 13(6):1634-1637.
[14]Ernberg I, Falk K, Minarovits J, et al. The role of methylation in the phenotype-dependent modulation of Epstein-Barr nuclear antigen 2 and latent membrane protein genes in cells latently infected with Epstein-Barr virus. J Gen Virol. 1989, 70 ( Pt 11):2989-3002.
[15]Allday MJ, Kundu D, Finerty S, et al. CpG methylation of viral DNA in EBV-associated tumours. Int J Cancer. 1990, 45(6):1125-1130.
[16]Hu LF, Minarovits J, Cao SL, et al. Variable expression of latent membrane protein in nasopharyngeal carcinoma can be related to methylation status of the Epstein-Barr virus BNLF-1 5'-flanking region. J Virol. 1991, 65(3):1558-1567.
[17]Hsueh CT, Dolnick BJ. Regulation of folate-binding protein gene expression by DNA methylation in methotrexate-resistant KB cells. Biochem Pharmacol. 1994, 47(6):1019-1027.
[18]Lo KW, Cheung ST, Leung SF, et al. Hypermethylation of the p16 gene in nasopharyngeal carcinoma. Cancer Res. 1996, 56(12):2721-2725.
[19] Kong WJ, Zhang S, Guo CK, et al. Effect of methylation-associated silencing of the death-associated protein kinase gene on nasopharyngeal carcinoma. Anticancer Drugs. 2006, 17(3):251-259.
[20] Chang HW, Chan A, Kwong DL, et al. Evaluation of hypermethylated tumor suppressor genes as tumor markers in mouth and throat rinsing fluid, nasopharyngeal swab and peripheral blood of nasopharygeal carcinoma patient. Int J Cancer. 2003, 105(6):851-855.
[21] Wong TS, Chang HW, Tang KC,et al. High frequency of promoter hypermethylation of the death-associated protein-kinase gene in nasopharyngeal carcinoma and its detection in the peripheral blood of patients. Clin Cancer Res. 2002, 8(2):433-437.
[22] Kwong J, Lo KW, To KF, et al. Promoter hypermethylation of multiple genes in nasopharyngeal carcinoma. Clin Cancer Res. 2002, 8(1): 131-137.
[23] Zhou L, Jiang W, Ren C, et al. Frequent hypermethylation of RASSF1A and TSLC1, and high viral load of Epstein-Barr Virus DNA in nasopharyngeal carcinoma and matched tumor-adjacent tissues. Neoplasia. 2005, 7(9):809-815.
[24] Pan ZG, Kashuba VI, Liu XQ, et al. High frequency somatic mutations in RASSF1A in nasopharyngeal carcinoma.Cancer Biol Ther.2005,4(10):1116-1122.
[25]Qiu GH,Tan LK,Loh KS,et al.The candidate tumor suppressor gene BLU,located at the commonly deleted region 3p21.3,is an E2F-regulated,stress-responsive gene and inactivated by both epigenetic and genetic mechanisms in nasopharyngeal carcinoma.Oncogene.2004,23(27):4793-4806.
[26]Chow LS,Lo KW,Kwong J,et al.RASSF1A is a target tumor suppressor from 3p21.3 in nasopharyngeal carcinoma.Int J Cancer.2004,109(6):839-847.
[27]Wong TS,Tang KC,Kwong DL,et al.Differential gene methylation in undifferentiated nasopharyngeal carcinoma.Int J Oncol.2003,22(4):869-874.
[28]Lo KW,Kwong J,Hui AB,et al.High frequency of promoter hypermethylation of RASSF1A in nasopharyngeal carcinoma.Cancer Res.2001,61(10):3877-3881.
[29]Zhang Z,Sun D,Van do N,et al.Inactivation of RASSF2A by promoter methylation correlates with lymph node metastasis in nasopharyngeal carcinoma.Int J Cancer.2007,120(1):32-38.
[30]吴宏,王跃建,周明,等.NPC患者p16/MTS1抑癌基因失活研究.耳鼻咽喉-头颈外科.1996,6(6)350-352。
[31]郑珺,李文生,黄若葵,等.NPC肿瘤c2myc基因表达及p16基因失活的研究.中华耳鼻咽喉科杂志.2000,35(6):464-468.
[32]项一宁,张维元.NPC组织中16基因的缺失、高甲基化和蛋白的表达及其临床意义.中华病理学杂志.2005,34(6):358-361。
[33]肖白,朱章菱,刘敬忠,等.上皮源性恶性肿瘤患者p16抑癌基因甲基化研究.肿瘤研究与临床.2000,12(3):147-149.
[34]Cheung HW,Ching YP,Nicholls JM,et al.Epigenetic inactivation of CHFR in nasopharyngeal carcinoma through promoter methylationo Mol Carcinog.2005,43(4):237-245.
[35]Tsao SW,Liu Y,Wang X,et al.The association of E-cadherin expression and the methylation status of the E-cadherin gene in nasopharyngeal carcinoma cells.Eur J Cancer.2003,39(4):524-531.
[36]李智,林素暇,梁英杰.上皮型钙黏素基因启动子甲基化和B2链接素基因突变对NPC细胞侵袭转移的影响.中华肿瘤杂志.2003,25(3):238-242.
[37]Tsai CN,Tsai CL,Tse KP,et al.The Epstein-Barr vires oncogene product,latent membrane protein 1,induces the downregulation of E-cadherin gene expression via activation of DNA methyltransferases.Proc Natl Acad Sci U S A.2002, 99(15):10084-10089.
[38]Sun D,Zhang Z,Van DN,et al.Aberrant methylation of CDH13 gene in nasopharyngeal carcinoma could serve as a potential diagnostic biomarker.Oral Oncol.2006,43(1):82-87.
[39]Ying J,Li H,Seng TJ,et al.Functional epigenetics identifies a protocadherin PCDH10 as a candidate tumor suppressor for nasopharyngeal,esophageal and multiple other carcinomas with frequent methylation.Oncogeneo 2006,25(7):1070-1080.
[40]Lung HL,Cheng Y,Kumaran MK,et al.Fine mapping of the 11q22-23 tumor suppressive region and involvement of TSLC1 in nasopharyngeal carcinoma,Int J Cancer.2004,112(4):628-635.
[41]Hui AB,Lo KW,Kwong J,et al.Epigenetic inactivation of TSLC1 gene in nasopharyngeal carcinoma.Mol Carcinog.2003,38(4):170-1788.
[42]Kwong J,Lo KW,Chow LS,et al.Epigenetic silencing of cellular retinol-binding proteins in nasopharyngeal carcinoma.Neoplasia.2005,7(1):67-74.
[43]Kwong J,Lo KW,Chow LS,et al.Silencing of the retinoid response gene TIG1by promoter hypermethylation in nasopharyngeal carcinoma,hat J Cancer.2005,113(3):386-392.
[44]Liu XQ,Chen HK,Zhang XS,et al.Alterations of BLU,a candidate tumor suppressor gene on chromosome 3p21.3,in human nasopharyngeal carcinoma.Int J Cancer.2003,106(1):60-65.
[45]Agathanggelou A,Dallol A,Zochbauer-Muller S,et al.Epigenetic inactivation of the candidate 3p21.3 suppressor gene BLU in human cancers.Oncogene.2003,22(10):1580-1588.
[46]刘晓琼,潘志刚,李满枝,等。3p21.3区域NPC相关基因——BLU的抑癌功能。癌症.2003,22(2):128-135。
[47]Ying J,Srivastava G,Hsieh WS,et al.The stress-responsive gene GADD45G is a functional tumor suppressor,with its response to environmental stresses frequently disrupted epigenetically in multiple tumors.Clin Cancer Res.2005,11(18):6442-6449.
[48]Lin YC,You L,Xu Z,et al.Wnt signaling activation and WIF-1 silencing in nasopharyngeal cancer cell lines.Biochem Biophys Res Commun.2006,341(2):635-640.
[49]Ng A,Tang JP,Goh CH,et al.Regulation of the H19 imprinting gene expression in human nasopharyngeal carcinoma by methylationo Int J Cancer.2003,104(2):179-187.
[50]Soo R,Putti T,Tao Q,et al.Overexpression of cyclooxygenase-2 in nasopharyngeal carcinoma and association with epidermal growth factor receptor expression.Arch Otolaryngol Head Neck Surg.2005,131(2):147-152.
[51]Wong TS,Kwong DL,Sham JS,et al.Promoter hyperrnethylation of high-in-normal 1 gene in primary nasopharyngeal carcinoma.Clin Cancer Res.2003,9(8):3042-3046.
[52]Lo KW,Tsang YS,Kwong J,et al.Promoter hypermethylation of the EDNRB gene in nasopharyngeal carcinoma.Int J Cancer.2002,98(5):651-655.
[53]Yi HM,Li H,Peng D,et al.Genetic and epigenetic alterations of LTF at 3p21.3in nasopharyngeal carcinoma.Oncol Res.2006;16(6):261-272.
[54]Peng D,Ren CP,Yi HM,et al.Genetic and epigenetic alterations of DLC-1,a candidate tumor suppressor gene,in nasopharyngeal carcinoma.Acta Biochim Biophys Sin.2006,38(5):349-355.
[55]Seng TJ,Low JS,Li H,et al.The major 8p22 tumor suppressor DLC1 is frequently silenced by methylation in both endemic and sporadic nasopharyngeal,esophageal,and cervical carcinomas,and inhibits tumor cell colony formation.Oncogene..2007,26(6):934-944.
[56]Chang HW,Chan A,Kwong DL,et al.Detection of hypermethylated RIZ1 gene in primary tumor,mouth,and throat rinsing fluid,nasopharyngeal swab,and peripheral blood of nasopharyngeal carcinoma patient.Clin Cancer Res.2003,9(3):1033-8103.
[57]邱元正,田勇泉,肖健云,等.细胞角蛋白基因13在NPC中的作用研究。中华肿瘤杂志.1999,21(6):444-446.
[58]易红梅,任彩萍,彭丹,等。SEMA3B基因在NPC组织中的表达、杂合性丢失和甲基化分析.生命科学研究.2006,10(2):183-188。
