肝细胞癌中SLIT2、BLU、ppENK和TMS1基因甲基化状态研究
摘要
目的：了解肝细胞癌(hepatocellular carcinoma，HCC)中SLIT2、BLU、ppENK和TMS1基因的甲基化状态及临床意义。方法：采用甲基化特异性聚合酶链反应(methylation-specific PCR，MSP)技术，检测50例HCC癌组织及相应的癌旁组织，4个HCC细胞株，8例肝血管瘤旁和4例供肝组织，了解SLIT2、BLU、ppENK和TMS1基因在上述组织中的甲基化状态，并分析其与临床资料间的相关性。结果：4例供肝组织均未发生甲基化，8例肝血管瘤旁组织有1例发生BLU甲基化，4个HCC细胞株分别有3、3、1和2个发生SLIT2、BLU、ppENK和TMS1甲基化，50例HCC癌组织及其相应的癌旁组织中，分别检测到39例(78％)、26例(52％)发生SLIT2甲基化，33例(66％)、18例(36％)发生BLU甲基化，14例(28％)、8例(16％)发生ppENK甲基化，14例(28％)、7例(14％)发生TMS1甲基化。癌组织与癌旁组织甲基化率比较，SLIT2、BLU差异有显著性(χ~2=7.429，P=0.006；χ~2=9.004，P=0.003)，ppENK、TMS1差异无显著性(χ~2=2.008，P=0.148；χ~2=2.954，P=0.086)。100％的癌和78％的癌旁组织至少有1个基因发生了甲基化。癌组织中只含1种基因甲基化的标本为15份(30％)，含2种基因甲基化的标本为20份(40％)，3种基因甲基化的标本为15份(30％)，4种基因甲基化的标本为0份。癌组织与癌旁组织SLIT2、BLU、ppENK和TMS1基因甲基化发生情况之间无统计学相关性(P＞0.05)。TNM Ⅲ期患者癌组织SLIT2甲基化率高于TNM Ⅰ／Ⅱ期患者(χ~2=4.432，P=0.035)。肝功能Child B级患者癌组织BLU甲基化率高于Child A级患者(χ~2=4.193，P=0.041)。癌组织BLU甲基化阳性患者的肿瘤直径大于BLU甲基化阴性患者(t=3.038，P=0.004)。癌旁组织BLU甲基化阴性患者的血清AFP水平高于BLU甲基化阳性者(t=2.244，P=0.029)。癌组织BLU甲基化阳性患者
Objective: To investigate methylation of SLIT2, BLU, ppENK and TMS1 genes in hepatocellular carcinoma. Methods: Methylation-specific PCR was adopted to investigate methylation of SLIT2, BLU, ppENK and TMS1 genes in 50 cases of HCC and corresponding non-tumor liver tissue, 4 HCC cell lines, 8 normal liver tissue in liver angioma and 4 liver donors. The relationship between methylation of SLIT2, BLU, ppENK and TMS1 genes and clinical data was analyzed. Results: 4 liver donors have no methylation of these genes. 1 of 8 normal liver tissue in liver angioma was detected methylation in BLU. No normal liver tissue in liver angioma was detected methylation in SLIT2, ppENK and TMS1. Methylation of SLIT2, BLU, ppENK and TMS1 was found in 3, 3, 1 and 2 of 4 HCC cell lines, respectively. In 50 cases of HCC and corresponding non-tumor liver tissue, the frequency of SLIT2 methylation was 78%, 52%, respectively; the frequency of BLU methylation was 66%, 38%, respectively; the frequency of ppENK methylation was 28%, 16%, respectively; the frequency of TMS1 methylation was 28%, 14%, respectively; Methylation of SLIT2 and BLU was more frequent in HCC than in non-tumor liver tissue(x~2=7.429, P=0.006; x~2=9.004, P=0.003). There was no significant difference between methylation of ppENK and TMS 1 in HCC and non-tumor liver tissue(x~2=2.098, P=0.148; x~2=2.954, P=0.086). At least one gene was methylated in 100% of HCC and 78% of non-tumor liver tissue. One gene was methylated in 15 of HCC (30%) . Two genes were methylated in 20 of HCC (40%) . Three genes were methylated in 15 of HCC (30%). No cases had methylation of 4 genes.
Objective: To investigate methylation of SLIT2, BLU, ppENK and TMS1 genes in hepatocellular carcinoma. Methods: Methylation-specific PCR was adopted to investigate methylation of SLIT2, BLU, ppENK and TMS1 genes in 50 cases of HCC and corresponding non-tumor liver tissue, 4 HCC cell lines, 8 normal liver tissue in liver angioma and 4 liver donors. The relationship between methylation of SLIT2, BLU, ppENK and TMS1 genes and clinical data was analyzed. Results: 4 liver donors have no methylation of these genes. 1 of 8 normal liver tissue in liver angioma was detected methylation in BLU. No normal liver tissue in liver angioma was detected methylation in SLIT2, ppENK and TMS1. Methylation of SLIT2, BLU, ppENK and TMS1 was found in 3, 3, 1 and 2 of 4 HCC cell lines, respectively. In 50 cases of HCC and corresponding non-tumor liver tissue, the frequency of SLIT2 methylation was 78%, 52%, respectively; the frequency of BLU methylation was 66%, 38%, respectively; the frequency of ppENK methylation was 28%, 16%, respectively; the frequency of TMS1 methylation was 28%, 14%, respectively; Methylation of SLIT2 and BLU was more frequent in HCC than in non-tumor liver tissue(x~2=7.429, P=0.006; x~2=9.004, P=0.003). There was no significant difference between methylation of ppENK and TMS 1 in HCC and non-tumor liver tissue(x~2=2.098, P=0.148; x~2=2.954, P=0.086). At least one gene was methylated in 100% of HCC and 78% of non-tumor liver tissue. One gene was methylated in 15 of HCC (30%) . Two genes were methylated in 20 of HCC (40%) . Three genes were methylated in 15 of HCC (30%). No cases had methylation of 4 genes.
引文
1 赵仲堂，贾存显，肝癌流行病学．见：刘倩，王文奇，主编．肝癌．第1版．北京：人民卫生出版社，2000：221-222．
2 Hannhan D, Weinberg RA. The hallmarks of cancer. Cell, 2000, 100(1): 57-70.
3 Wajed SA, Laird PW, Demeester TR. DNA methylation: an alternative pathyway to cancer. Ann Surg, 2001, 234(1): 10-20.
4 Mizuno S, Chijiwa T, Okamura T, et al. Expression of DNA methyltransferases DNMT1, 3A, and 3B in normal hematopoiesis and in acute and chronic myelogenous leukemia. Blood, 2001, 97(5): 1172-1179.
5 Robertson KD, Jones PA. DNA methylation: past, present and future directions. Carcinogenesis, 2000, 21(3): 461-467.
6 Paulsen M, Ferguson-Smith AC. DNA methylation in genomic imprinting, development, and disease. J Pathol, 2001, 195(1): 97-110.
7 Liu L. Wylie RC, Andrews LG, et al. Aging, cancer and nutrition: the DNA methylation connection. Mech Ageing Dev, 2003, 124(10-12): 989-998.
8 Yu J, Zhang HY, Ma ZZ, et al. Methylation profiling of twenty four genes and the concordant methylation behaviours of nineteen genes that may contribute to hepatocellular carcinogenesis. Cell Res, 2003, 13(5): 319-33.
9 WHO: pathology and genetics. Tumours of the digestive system. In: Hamilton SR, Aaltonen LA, eds. Lyon: IARC, 2000. 173-180.
10 UICC: TNM classification of malignant tumors. In: Sobin LH, Wittekind C, eds. 6th ed. New York: Wiley-Liss, 2002.
11 Rein T, DePamphilis ML, Zorbas H. Identifying 5-methylcytosine and related modifications in DNA genomes. Nucleic Acids Res, 1998, 26(110): 2255-2264.12 Frommer M, McDonald LE, Millar DS, et al. Agenomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci USA, 1992, 89(5): 1827-1831
13 Sadri R, Hornsby PJ. Rapid analysis of DNA methylation using new restriction enzyme sites created by bisulfite modification. Nucleic Acids Res, 1996, 24(24): 5058-5059.
14 Herman JG, Graff JR, Myohanen S, et al. Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA, 1996, 93(18): 9821-9826.
15 Coleman WB. Mechanisms of Human Hepatocarcinogenesis. Curr Mol Med, 2003, 3(6): 573-588.
16 Kanai Y, Ushijima S, Hui AM, et al. The E-cadherin gene is silenced by CpG methylation in human hepatocellular carcinomas. Int J Cancer, 1997, 71(3): 355-359.
17 赵中海，耿小平，朱立新，等．肝细胞癌组织中Ras相关区域家族蛋白1A、肿瘤高甲基化基因1和p73基因的异常甲基化．中华外科杂志，2005，43(23)：1528-1532．
18 钱波，朱立新，耿小平等．肝细胞癌MGMT、DAPK、THBS1和R IZ1基因甲基化研究．中华普通外科杂志，2005，20(5)：291-294
19 Wong IH, Lo YM, Zhang J, et al. Detection of aberrant p16 methylation in the plasma and serum of liver cancer patients. Cancer Res, 1999, 59(1): 71-73.
20 Chu HJ, Heo J, Seo SB, et al. Detection of aberrant p16INK4A methylation in sera of patients with liver cirrhosis and hepatocellular carcinoma. J Korean Med Sci, 2004, 19(1): 83-86.
21 Wong IH, Zhang J, Lai PB, et al. Quantitative analysis of tumor-derived methylated p16INK4a sequences in plasma, serum,and blood cells of hepatocellular carcinoma patients. Clin Cancer Res, 2003, 9(3): 1047-1052.22 Wong IH, Johnson PJ, Lai PB, et al. Tumor-derived epigenetic changes in the plasma and serum of liver cancer patients. Implications for cancer detection and monitoring. Ann N Y Acad Sci, 2000, 906: 102-105.
23 Wu JY, Feng L, Park HT, et al. The neuronal repellent Slit inhibits leukocyte chemotaxis induced by chemotactic factors. Nature, 2001, 410(6831): 948-952.
24 Dallol A, Da Silva NF, Viacava P, et al. SLIT2, a human homologue of the Drosophila Slit2 gene, has tumor suppressor activity and is frequently inactivated in lung and breast cancers. Cancer Res, 2002, 62(20): 5874-5880.
25 Dallol A, Morton D, Maher ER, et al. SLIT2 axon guidance molecule is frequently in activated in colorectal cancer and suppresses growth of colorectal carcinoma cells. Cancer Res, 2003, 63(5): 1054-1058.
26 Dallol A, Krex D, Hesson L, et al. Frequent epigenetic inactivation of the SLIT2 gene in gliomas. Oncogene, 2003, 22(29): 4611-4616.
27 Astuti D, Da Silva NF, Dallol A, et al. SLIT2 promoter methylation analysis in neuroblastoma, Wilms' tumour and renal cell carcinoma. Br J Cancer, 2004, 90(2): 515-521.
28 Dammann R, Stnmnikova M, Schagdarsurengin U, et al. CpG island methylation and expression of tumour-associated genes in lung carcinoma. Eur J Cancer, 2005, 41(8): 1223-1236.
29 任同，王忠诚，张亚卓，等．SLIT2基因启动子甲基化与星形细胞肿瘤关系的研究．中国实验诊断学，2005，9(1)：34-36．
30 Lerman MI, Minna JD. The 630-kb lung cancer homozygous deletion region on human chromosome 3p21.3: identification and evaluation of the resident candidate tumor suppressor genes. The International Lung Cancer Chromosome 3p21.3 Tumor Suppressor Gene Consortium. Cancer Res, 2000, 60(21): 6116-6133.
31 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): 4793-806.
32 Tischoff I, Markwarth A, Witzigmann H, et al. Allele loss and epigenetic inactivation of 3p21.3 in malignant liver tumors. Int J Cancer, 2005, 115(5): 684-689.
33 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.
34 Marsit CJ, Kim DH, Liu M, et al. Hypermethylation of RASSF1A and BLU tumor suppressor genes in non-small cell lung cancer: implications for tobacco smoking during adolescence. Int J Cancer, 2005, 114(2): 219-223.
35 Zagon IS, McLaughlin PJ. Opioid growth factor(OGF) inhibits anchorage- independent growth in human cancer cells. Int J Oncol, 2004, 24(6): 1443-1448.
36 Sato N, Ueki T, Fukushima N, et al. Aberrant methylation of CpG islands in intraductal papillary mucinous neoplasms of the pancreas. Gastroenterology, 2002, 123(1): 365-372.
37 McConnell BB,Vertino PM. TMS1/ASC: the cancer connection. Apoptosis. 2004, 9(1): 5-18.
38 Suzuki M, Toyooka S, Shivapurkar N, et al. Aberrant methylation profile of human malignant mesotheliomas and its relationship to SV40 infection. Oncogene, 2005, 24(7): 1302-1308.
39 Stone AR, Bobo W, Brat DJ, et al. Aberrant methylation and down-regulation of TMS1/ASC in human glioblastoma. Am J Pathol, 2004,165(4): 1151-1161.
40 Alaminos M, Davalos V, Cheung NK, et al. Clustering of gene hypermethylation associated with clinical risk groups in neuroblastoma. J Natl Cancer Inst, 2004, 96(16): 1208-1219.
41 Terasawa K, Sagae S, Toyota M, et al. Epigenetic inactivation of TMS1/ASC in ovarian cancer. Clin Cancer Res, 2004,10(6): 2000-2006.42 Lee S, Lee HJ, Kim JH, et al. Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis. Am J Pathol, 2003, 163(4): 1371-1378.
43 Herman JG. Circulating methylated DNA. Ann N Y Acad Sci, 2004, 1022: 33-39.
1 Coleman WB. Mechanisms of Human Hepatocarcinogenesis. Curr Mol Med, 2003, 3(6): 573-588.
2 Wajed SA, Laird PW, Demeester TR. DNA methylation: an alternative pathyway to cancer. Ann Surg, 2001, 234(1): 10-20.
3 Mizuno S, Chijiwa T, Okamura T, et al. Expression of DNA methyltransferases DNMT1, 3A, and 3B in normal hematopoiesis and in acute and chronic myelogenous leukemia. Blood, 2001, 97(5): 1172-1179.
4 Robertson KD, Jones PA. DNA methylation: past, present and future directions. Carcinogenesis, 2000, 21 (3): 461-467.
5 Paulsen M, Ferguson-Smith AC. DNA methylation in genomic imprinting, development, and disease. J Pathol, 2001, 195(1): 97-110.
6 Liu L, Wylie RC, Andrews LG, et al. Aging, cancer and. nutrition: the DNA methylation connection. Mech Ageing Dev. 2003, 124(10-12): 989-998.
7 Iwata N, Yamamoto H, Sasaki S, et al. Frequent hypermethylation of CpG islands and loss of expression of the 14-3-3 sigma gene in human hepatocellular carcinoma. Oncogene, 2000, 19(46): 5298-5302.
8 Yang B, Guo M, Herman JG, et al. Aberrant promoter methylation profiles of tumor suppressor genes in hepatocellular carcinoma. Am J Pathol, 2003, 163(3): 1101-1107.
9 Lee S, Lee HJ, Kim JH, et al. Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis. Am J Pathol, 2003, 163(4): 1371-1378.
10 Kubo T, Yamamoto J, Shikauchi Y, et al. Apoptotic speck protein-like, a highly homologous protein to apoptotic speck protein in the pyrin domain, is silenced byDNA methylation and induces apoptosis in human hepatocellular carcinoma. Cancer Res, 2004, 64(15): 5172-5177.
11 Yu J, Ni M, Xu J, et al. Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis. BMC Cancer, 2002, 2(1): 29.
12 Sengupta PK, Smith EM, Kim K, et al. DNA hypermethylation near the transcription start site of collagen alpha2(I) gene occurs in both cancer cell lines and primary colorectal cancers. Cancer Res, 2003, 63(8): 1789-1797.
13 Shen L, Ahuja N, Shen Y, et al. DNA methylation and environmental exposures in human hepatocellular carcinoma. J Natl Cancer Inst, 2002, 94(10): 755-761.
14 Wong CM, Lee JM, Ching YP, et al. Genetic and epigenetic alterations of DLC-1 gene in hepatocellular carcinoma. Cancer Res, 2003, 63(22): 7646-7651.
15 Kanai Y, Ushijima S, Hui AM, et al. The E-cadherin gene is silenced by CpG methylation in human hepatocellular carcinomas. Int J Cancer, 1997, 71(3): 355-359.
16 Tchou JC, Lin X, Freije D, et al. GSTP1 CpG island DNA hypermethylation in hepatocellular carcinomas. Int J Oncol, 2000,16(4): 663-676.
17 Fukai K, Yokosuka O, Chiba T, et al. Hepatocyte growth factor activator inhibitor 2/placental bikunin (HAI-2/PB) gene is frequently hypermethylated in human hepatocellular carcinoma. Cancer Res, 2003, 63(24): 8674-8679.
18 Zhang CJ, Li HM, Yau LM, et al. Methylation of mismatch repair gene (MMR) in primary hepatocellular carcinoma. Zhonghua Bing Li Xue Za Zhi, 2004, 33(5): 433-436.
19 Hanafusa T, Yumoto Y, Nouso K, et al. Reduced expression of insulin-like growth factor binding protein-3 and its promoter hypermethylation in human hepatocellular carcinoma. Cancer Lett, 2002, 176(2): 149-158.
20 Zhang YJ, Chen Y, Ahsan H, et al. Inactivation of the DNA repair gene O~6- methylguanine-DNA methyltransferase by promoter hypermethylation and its??relationship to aflatoxin B1-DNA adducts and p53 mutation in hepatocellular carcinoma. Int J Cancer, 2003,103(4): 440-444.
21 Herath NI, Kew MC, Walsh MD, et al. Reciprocal relationship between methylation status and loss of heterozygosity at the p14(ARF) locus in Australian and South African hepatocellular carcinomas. J Gastroenterol Hepatol, 2002, 17(3): 301-307.
22 Wong IH, Lo YM, Yeo W, et al. Frequent p15 promoter methylation in tumor and peripheral blood from hepatocellular carcinoma patients. Clin Cancer Res. 2000, 6(9): 3516-3521.
23 Huang J, Shen W, Li B, et al. Molecular and immunohistochemical study of the inactivation of the p16 gene in primary hepatocellular carcinoma. Chin Med J (Engl), 2000, 113(10): 889-893.
24 Roncalli M, Bianchi P, Bruni B, et al. Methylation framework of cell cycle gene inhibitors in cirrhosis and associated hepatocellular carcinoma. Hepatology, 2002, 36(2): 427-432.
25 Pogribny IP, James SJ. Reduction of p53 gene expression in human primary hepatocellular carcinoma is associated with promoter region methylation without coding region mutation. Cancer Lett, 2002,176(2): 169-174.
26 Edamoto Y, Hara A, Biernat W, et al. Alterations of RB1, p53 and Wnt pathways in hepatocellular carcinomas associated with hepatitis C, hepatitis B and alcoholic liver cirrhosis. Int J Cancer, 2003, 106(3): 334-341.
27 Xiao WH, Liu WW. Hemizygous deletion and hypermethylation of RUNX3 gene in hepatocellular carcinoma. World J Gastroenterol, 2004, 10(3): 376-380.
28 Xiao WH, Liu WW. Analysis of methylation and loss of heterozygosity of RUNX3 gene in hepatocellular carcinoma and its clinical significance. Zhonghua Gan Zang Bing Za Zhi, 2004, 12(4) :227-230.
29 Miyoshi H, Fujie H, Moriya K, et al. Methylation status of suppressor of cytokine signaling-1 gene in hepatocellular carcinoma. J Gastroenterol, 2004, 39(6): 563-??569.
30 Lu GL, Wen JM, Xu JM, et al. Relationship between TIMP-3 expression and promoter methylation of TIMP-3 gene in hepatocellular carcinoma. Zhonghua Bing Li Xue Za Zhi, 2003, 32(3): 230-233.
31 Chaubert P, Gayer R, Zimmermann A, et al. Germ-line mutations of the p16INK4 (MTS1) gene occur in a subset of patients with hepatocellular carcinoma. Hepatology, 1997,25(6): 1376-1381.
32 Liew CT, Li HM, Lo KW, et al. High frequency of p16INK4A gene alterations in hepatocellular carcinoma. Oncogene, 1999, 18(3): 789-795.
33 Matsuda Y, Ichida T, Matsuzawa J, et al. pl6(INK4) is inactivated by extensive CpG methylation in human hepatocellular carcinoma. Gastroenterology, 1999, 116(2): 394-400.
34 Liu LH, Xiao WH, Liu WW. Effect of 5-Aza-2'-deoxycytidine on the P16 tumor suppressor gene in hepatocellular carcinoma cell line HepG2. World J Gastroenterol, 2001,7(1): 131-135.
35 Narimatsu T, Tamori A, Koh N, et al. pl6 promoter hypermethylation in human hepatocellular carcinoma with or without hepatitis virus infection. Intervirology, 2004, 47(1): 26-31.
36 Yoshikawa H, Matsubara K, Qian GS, et al. SOCS-1,a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat Genet, 2001, 28(1): 29-35.
37 Okochi O, Hibi K, Sakai M, et al. Methylation-mediated silencing of SOCS-1 gene in hepatocellular carcinoma derived from cirrhosis. Clin Cancer Res, 2003, 9(14): 5295-5298.
38 Yoshida T, Ogata H, Kamio M, et al. SOCS1 is a suppressor of liver fibrosis and hepatitis-induced carcinogenesis. J Exp Med, 2004,199(12): 1701-1707.
39 Zhang YJ, Ahsan H, Chen Y, et al. High frequency of promoter hypermethylation of??RASSF1A and p16 and its relationship to aflatoxin B1-DNA adduct levels in human hepatocellular carcinoma. Mol Carcinog, 2002, 35(2): 85-92.
40 Schagdarsurengin U, Wilkens L, Steinemann D, et al. Frequent epigenetic inactivation of the RASSFIA gene in hepatocellular carcinoma. Oncogene, 2003, 22(12): 1866-1871.
41 Zhong S, Yeo W, Tang MW, et al. Intensive hypermethylation of the CpG island of Ras association domain family 1A in hepatitis B virus-associated hepatocellular carcinomas. Clin Cancer Res, 2003, 9(9): 3376-3382.
42 Zhong S, Tang MW, Yeo W, et al. Silencing of GSTP1 gene by CpG island DNA hypermethylation in HBV-associated hepatocellular carcinomas. Clin Cancer Res, 2002, 8(4): 1087-1092.
43 Rein T, DePamphilis ML, Zorbas H. Identifying 5-methylcytosine and related modif- ications in DNA genomes. Nucleic Acids Res, 1998, 26(10): 2255-2264.
44 Frommer M, McDonald LE, Millar DS, et al. Agenomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.Proc Natl Acad Sci USA, 1992, 89(5): 1827-1831.
45 Sadri R, Hornsby PJ. Rapid analysis of DNA methylation using new restriction enzyme sites created by bisulfite modification.Nucleic Acids Res, 1996, 24(24):5058-5059.
46 Herman JG, Graff JR, Myohanen S, et al. Methylation-specific PCR:A novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA, 1996, 93(18): 9821-9826.
47 Wong IH, Lo YM, Zhang J, et al. Detection of aberrant p16 methylation in the plasma and serum of liver cancer patients. Cancer Res, 1999, 59(1): 71-73.
48 Chu HJ, Heo J, Seo SB, et al. Detection of aberrant p16INK4A methylation in sera of patients with liver cirrhosis and hepatocellular carcinoma. J Korean Med Sci, 2004,19(1): 83-86.49 Wong IH, Zhang J, Lai PB, et al. Quantitative analysis of tumor-derived methylated p16INK4a sequences in plasma, serum and blood cells of hepatocellular carcinoma patients. Clin Cancer Res, 2003, 9(3): 1047-1052.
50 Wong IH, Johnson PJ, Lai PB, et al. Tumor-derived epigenetic changes in the plasma and serum of liver cancer patients. Implications for cancer detection and monitoring. Ann N YAcad Sci, 2000, 906: 102-105.
51 Cheng JC, Yoo CB, Weisenberger DJ, et al. Preferential response of cancer cells to zebularine. Cancer Cell, 2004, 6(2): 151-158.
52 Yoo CB, Cheng JC, Jones PA. Zebularine: a new drug for epigenetic therapy. Biochem Soc Trans, 2004, 32(Pt6): 910-912.
53 Lin XH, Asgari K, Putzi MJ, et al. Reversal of GSTPl CpG island hypermethylation and reactivation of GSTPl expression in human prostate cancer cells by treatment with procainamide. Cancer Res, 2001, 61(24): 8611-8616.
54 Scheinbart LS, Johnson MA, Gross MA, et al. Procainamide inhibits DNA methyltr- ansferase in human T cell line. J Rheumatol, 1991,18(4): 530-534.
2 Hannhan D, Weinberg RA. The hallmarks of cancer. Cell, 2000, 100(1): 57-70.
3 Wajed SA, Laird PW, Demeester TR. DNA methylation: an alternative pathyway to cancer. Ann Surg, 2001, 234(1): 10-20.
4 Mizuno S, Chijiwa T, Okamura T, et al. Expression of DNA methyltransferases DNMT1, 3A, and 3B in normal hematopoiesis and in acute and chronic myelogenous leukemia. Blood, 2001, 97(5): 1172-1179.
5 Robertson KD, Jones PA. DNA methylation: past, present and future directions. Carcinogenesis, 2000, 21(3): 461-467.
6 Paulsen M, Ferguson-Smith AC. DNA methylation in genomic imprinting, development, and disease. J Pathol, 2001, 195(1): 97-110.
7 Liu L. Wylie RC, Andrews LG, et al. Aging, cancer and nutrition: the DNA methylation connection. Mech Ageing Dev, 2003, 124(10-12): 989-998.
8 Yu J, Zhang HY, Ma ZZ, et al. Methylation profiling of twenty four genes and the concordant methylation behaviours of nineteen genes that may contribute to hepatocellular carcinogenesis. Cell Res, 2003, 13(5): 319-33.
9 WHO: pathology and genetics. Tumours of the digestive system. In: Hamilton SR, Aaltonen LA, eds. Lyon: IARC, 2000. 173-180.
10 UICC: TNM classification of malignant tumors. In: Sobin LH, Wittekind C, eds. 6th ed. New York: Wiley-Liss, 2002.
11 Rein T, DePamphilis ML, Zorbas H. Identifying 5-methylcytosine and related modifications in DNA genomes. Nucleic Acids Res, 1998, 26(110): 2255-2264.12 Frommer M, McDonald LE, Millar DS, et al. Agenomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci USA, 1992, 89(5): 1827-1831
13 Sadri R, Hornsby PJ. Rapid analysis of DNA methylation using new restriction enzyme sites created by bisulfite modification. Nucleic Acids Res, 1996, 24(24): 5058-5059.
14 Herman JG, Graff JR, Myohanen S, et al. Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA, 1996, 93(18): 9821-9826.
15 Coleman WB. Mechanisms of Human Hepatocarcinogenesis. Curr Mol Med, 2003, 3(6): 573-588.
16 Kanai Y, Ushijima S, Hui AM, et al. The E-cadherin gene is silenced by CpG methylation in human hepatocellular carcinomas. Int J Cancer, 1997, 71(3): 355-359.
17 赵中海，耿小平，朱立新，等．肝细胞癌组织中Ras相关区域家族蛋白1A、肿瘤高甲基化基因1和p73基因的异常甲基化．中华外科杂志，2005，43(23)：1528-1532．
18 钱波，朱立新，耿小平等．肝细胞癌MGMT、DAPK、THBS1和R IZ1基因甲基化研究．中华普通外科杂志，2005，20(5)：291-294
19 Wong IH, Lo YM, Zhang J, et al. Detection of aberrant p16 methylation in the plasma and serum of liver cancer patients. Cancer Res, 1999, 59(1): 71-73.
20 Chu HJ, Heo J, Seo SB, et al. Detection of aberrant p16INK4A methylation in sera of patients with liver cirrhosis and hepatocellular carcinoma. J Korean Med Sci, 2004, 19(1): 83-86.
21 Wong IH, Zhang J, Lai PB, et al. Quantitative analysis of tumor-derived methylated p16INK4a sequences in plasma, serum,and blood cells of hepatocellular carcinoma patients. Clin Cancer Res, 2003, 9(3): 1047-1052.22 Wong IH, Johnson PJ, Lai PB, et al. Tumor-derived epigenetic changes in the plasma and serum of liver cancer patients. Implications for cancer detection and monitoring. Ann N Y Acad Sci, 2000, 906: 102-105.
23 Wu JY, Feng L, Park HT, et al. The neuronal repellent Slit inhibits leukocyte chemotaxis induced by chemotactic factors. Nature, 2001, 410(6831): 948-952.
24 Dallol A, Da Silva NF, Viacava P, et al. SLIT2, a human homologue of the Drosophila Slit2 gene, has tumor suppressor activity and is frequently inactivated in lung and breast cancers. Cancer Res, 2002, 62(20): 5874-5880.
25 Dallol A, Morton D, Maher ER, et al. SLIT2 axon guidance molecule is frequently in activated in colorectal cancer and suppresses growth of colorectal carcinoma cells. Cancer Res, 2003, 63(5): 1054-1058.
26 Dallol A, Krex D, Hesson L, et al. Frequent epigenetic inactivation of the SLIT2 gene in gliomas. Oncogene, 2003, 22(29): 4611-4616.
27 Astuti D, Da Silva NF, Dallol A, et al. SLIT2 promoter methylation analysis in neuroblastoma, Wilms' tumour and renal cell carcinoma. Br J Cancer, 2004, 90(2): 515-521.
28 Dammann R, Stnmnikova M, Schagdarsurengin U, et al. CpG island methylation and expression of tumour-associated genes in lung carcinoma. Eur J Cancer, 2005, 41(8): 1223-1236.
29 任同，王忠诚，张亚卓，等．SLIT2基因启动子甲基化与星形细胞肿瘤关系的研究．中国实验诊断学，2005，9(1)：34-36．
30 Lerman MI, Minna JD. The 630-kb lung cancer homozygous deletion region on human chromosome 3p21.3: identification and evaluation of the resident candidate tumor suppressor genes. The International Lung Cancer Chromosome 3p21.3 Tumor Suppressor Gene Consortium. Cancer Res, 2000, 60(21): 6116-6133.
31 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): 4793-806.
32 Tischoff I, Markwarth A, Witzigmann H, et al. Allele loss and epigenetic inactivation of 3p21.3 in malignant liver tumors. Int J Cancer, 2005, 115(5): 684-689.
33 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.
34 Marsit CJ, Kim DH, Liu M, et al. Hypermethylation of RASSF1A and BLU tumor suppressor genes in non-small cell lung cancer: implications for tobacco smoking during adolescence. Int J Cancer, 2005, 114(2): 219-223.
35 Zagon IS, McLaughlin PJ. Opioid growth factor(OGF) inhibits anchorage- independent growth in human cancer cells. Int J Oncol, 2004, 24(6): 1443-1448.
36 Sato N, Ueki T, Fukushima N, et al. Aberrant methylation of CpG islands in intraductal papillary mucinous neoplasms of the pancreas. Gastroenterology, 2002, 123(1): 365-372.
37 McConnell BB,Vertino PM. TMS1/ASC: the cancer connection. Apoptosis. 2004, 9(1): 5-18.
38 Suzuki M, Toyooka S, Shivapurkar N, et al. Aberrant methylation profile of human malignant mesotheliomas and its relationship to SV40 infection. Oncogene, 2005, 24(7): 1302-1308.
39 Stone AR, Bobo W, Brat DJ, et al. Aberrant methylation and down-regulation of TMS1/ASC in human glioblastoma. Am J Pathol, 2004,165(4): 1151-1161.
40 Alaminos M, Davalos V, Cheung NK, et al. Clustering of gene hypermethylation associated with clinical risk groups in neuroblastoma. J Natl Cancer Inst, 2004, 96(16): 1208-1219.
41 Terasawa K, Sagae S, Toyota M, et al. Epigenetic inactivation of TMS1/ASC in ovarian cancer. Clin Cancer Res, 2004,10(6): 2000-2006.42 Lee S, Lee HJ, Kim JH, et al. Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis. Am J Pathol, 2003, 163(4): 1371-1378.
43 Herman JG. Circulating methylated DNA. Ann N Y Acad Sci, 2004, 1022: 33-39.
1 Coleman WB. Mechanisms of Human Hepatocarcinogenesis. Curr Mol Med, 2003, 3(6): 573-588.
2 Wajed SA, Laird PW, Demeester TR. DNA methylation: an alternative pathyway to cancer. Ann Surg, 2001, 234(1): 10-20.
3 Mizuno S, Chijiwa T, Okamura T, et al. Expression of DNA methyltransferases DNMT1, 3A, and 3B in normal hematopoiesis and in acute and chronic myelogenous leukemia. Blood, 2001, 97(5): 1172-1179.
4 Robertson KD, Jones PA. DNA methylation: past, present and future directions. Carcinogenesis, 2000, 21 (3): 461-467.
5 Paulsen M, Ferguson-Smith AC. DNA methylation in genomic imprinting, development, and disease. J Pathol, 2001, 195(1): 97-110.
6 Liu L, Wylie RC, Andrews LG, et al. Aging, cancer and. nutrition: the DNA methylation connection. Mech Ageing Dev. 2003, 124(10-12): 989-998.
7 Iwata N, Yamamoto H, Sasaki S, et al. Frequent hypermethylation of CpG islands and loss of expression of the 14-3-3 sigma gene in human hepatocellular carcinoma. Oncogene, 2000, 19(46): 5298-5302.
8 Yang B, Guo M, Herman JG, et al. Aberrant promoter methylation profiles of tumor suppressor genes in hepatocellular carcinoma. Am J Pathol, 2003, 163(3): 1101-1107.
9 Lee S, Lee HJ, Kim JH, et al. Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis. Am J Pathol, 2003, 163(4): 1371-1378.
10 Kubo T, Yamamoto J, Shikauchi Y, et al. Apoptotic speck protein-like, a highly homologous protein to apoptotic speck protein in the pyrin domain, is silenced byDNA methylation and induces apoptosis in human hepatocellular carcinoma. Cancer Res, 2004, 64(15): 5172-5177.
11 Yu J, Ni M, Xu J, et al. Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis. BMC Cancer, 2002, 2(1): 29.
12 Sengupta PK, Smith EM, Kim K, et al. DNA hypermethylation near the transcription start site of collagen alpha2(I) gene occurs in both cancer cell lines and primary colorectal cancers. Cancer Res, 2003, 63(8): 1789-1797.
13 Shen L, Ahuja N, Shen Y, et al. DNA methylation and environmental exposures in human hepatocellular carcinoma. J Natl Cancer Inst, 2002, 94(10): 755-761.
14 Wong CM, Lee JM, Ching YP, et al. Genetic and epigenetic alterations of DLC-1 gene in hepatocellular carcinoma. Cancer Res, 2003, 63(22): 7646-7651.
15 Kanai Y, Ushijima S, Hui AM, et al. The E-cadherin gene is silenced by CpG methylation in human hepatocellular carcinomas. Int J Cancer, 1997, 71(3): 355-359.
16 Tchou JC, Lin X, Freije D, et al. GSTP1 CpG island DNA hypermethylation in hepatocellular carcinomas. Int J Oncol, 2000,16(4): 663-676.
17 Fukai K, Yokosuka O, Chiba T, et al. Hepatocyte growth factor activator inhibitor 2/placental bikunin (HAI-2/PB) gene is frequently hypermethylated in human hepatocellular carcinoma. Cancer Res, 2003, 63(24): 8674-8679.
18 Zhang CJ, Li HM, Yau LM, et al. Methylation of mismatch repair gene (MMR) in primary hepatocellular carcinoma. Zhonghua Bing Li Xue Za Zhi, 2004, 33(5): 433-436.
19 Hanafusa T, Yumoto Y, Nouso K, et al. Reduced expression of insulin-like growth factor binding protein-3 and its promoter hypermethylation in human hepatocellular carcinoma. Cancer Lett, 2002, 176(2): 149-158.
20 Zhang YJ, Chen Y, Ahsan H, et al. Inactivation of the DNA repair gene O~6- methylguanine-DNA methyltransferase by promoter hypermethylation and its??relationship to aflatoxin B1-DNA adducts and p53 mutation in hepatocellular carcinoma. Int J Cancer, 2003,103(4): 440-444.
21 Herath NI, Kew MC, Walsh MD, et al. Reciprocal relationship between methylation status and loss of heterozygosity at the p14(ARF) locus in Australian and South African hepatocellular carcinomas. J Gastroenterol Hepatol, 2002, 17(3): 301-307.
22 Wong IH, Lo YM, Yeo W, et al. Frequent p15 promoter methylation in tumor and peripheral blood from hepatocellular carcinoma patients. Clin Cancer Res. 2000, 6(9): 3516-3521.
23 Huang J, Shen W, Li B, et al. Molecular and immunohistochemical study of the inactivation of the p16 gene in primary hepatocellular carcinoma. Chin Med J (Engl), 2000, 113(10): 889-893.
24 Roncalli M, Bianchi P, Bruni B, et al. Methylation framework of cell cycle gene inhibitors in cirrhosis and associated hepatocellular carcinoma. Hepatology, 2002, 36(2): 427-432.
25 Pogribny IP, James SJ. Reduction of p53 gene expression in human primary hepatocellular carcinoma is associated with promoter region methylation without coding region mutation. Cancer Lett, 2002,176(2): 169-174.
26 Edamoto Y, Hara A, Biernat W, et al. Alterations of RB1, p53 and Wnt pathways in hepatocellular carcinomas associated with hepatitis C, hepatitis B and alcoholic liver cirrhosis. Int J Cancer, 2003, 106(3): 334-341.
27 Xiao WH, Liu WW. Hemizygous deletion and hypermethylation of RUNX3 gene in hepatocellular carcinoma. World J Gastroenterol, 2004, 10(3): 376-380.
28 Xiao WH, Liu WW. Analysis of methylation and loss of heterozygosity of RUNX3 gene in hepatocellular carcinoma and its clinical significance. Zhonghua Gan Zang Bing Za Zhi, 2004, 12(4) :227-230.
29 Miyoshi H, Fujie H, Moriya K, et al. Methylation status of suppressor of cytokine signaling-1 gene in hepatocellular carcinoma. J Gastroenterol, 2004, 39(6): 563-??569.
30 Lu GL, Wen JM, Xu JM, et al. Relationship between TIMP-3 expression and promoter methylation of TIMP-3 gene in hepatocellular carcinoma. Zhonghua Bing Li Xue Za Zhi, 2003, 32(3): 230-233.
31 Chaubert P, Gayer R, Zimmermann A, et al. Germ-line mutations of the p16INK4 (MTS1) gene occur in a subset of patients with hepatocellular carcinoma. Hepatology, 1997,25(6): 1376-1381.
32 Liew CT, Li HM, Lo KW, et al. High frequency of p16INK4A gene alterations in hepatocellular carcinoma. Oncogene, 1999, 18(3): 789-795.
33 Matsuda Y, Ichida T, Matsuzawa J, et al. pl6(INK4) is inactivated by extensive CpG methylation in human hepatocellular carcinoma. Gastroenterology, 1999, 116(2): 394-400.
34 Liu LH, Xiao WH, Liu WW. Effect of 5-Aza-2'-deoxycytidine on the P16 tumor suppressor gene in hepatocellular carcinoma cell line HepG2. World J Gastroenterol, 2001,7(1): 131-135.
35 Narimatsu T, Tamori A, Koh N, et al. pl6 promoter hypermethylation in human hepatocellular carcinoma with or without hepatitis virus infection. Intervirology, 2004, 47(1): 26-31.
36 Yoshikawa H, Matsubara K, Qian GS, et al. SOCS-1,a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat Genet, 2001, 28(1): 29-35.
37 Okochi O, Hibi K, Sakai M, et al. Methylation-mediated silencing of SOCS-1 gene in hepatocellular carcinoma derived from cirrhosis. Clin Cancer Res, 2003, 9(14): 5295-5298.
38 Yoshida T, Ogata H, Kamio M, et al. SOCS1 is a suppressor of liver fibrosis and hepatitis-induced carcinogenesis. J Exp Med, 2004,199(12): 1701-1707.
39 Zhang YJ, Ahsan H, Chen Y, et al. High frequency of promoter hypermethylation of??RASSF1A and p16 and its relationship to aflatoxin B1-DNA adduct levels in human hepatocellular carcinoma. Mol Carcinog, 2002, 35(2): 85-92.
40 Schagdarsurengin U, Wilkens L, Steinemann D, et al. Frequent epigenetic inactivation of the RASSFIA gene in hepatocellular carcinoma. Oncogene, 2003, 22(12): 1866-1871.
41 Zhong S, Yeo W, Tang MW, et al. Intensive hypermethylation of the CpG island of Ras association domain family 1A in hepatitis B virus-associated hepatocellular carcinomas. Clin Cancer Res, 2003, 9(9): 3376-3382.
42 Zhong S, Tang MW, Yeo W, et al. Silencing of GSTP1 gene by CpG island DNA hypermethylation in HBV-associated hepatocellular carcinomas. Clin Cancer Res, 2002, 8(4): 1087-1092.
43 Rein T, DePamphilis ML, Zorbas H. Identifying 5-methylcytosine and related modif- ications in DNA genomes. Nucleic Acids Res, 1998, 26(10): 2255-2264.
44 Frommer M, McDonald LE, Millar DS, et al. Agenomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.Proc Natl Acad Sci USA, 1992, 89(5): 1827-1831.
45 Sadri R, Hornsby PJ. Rapid analysis of DNA methylation using new restriction enzyme sites created by bisulfite modification.Nucleic Acids Res, 1996, 24(24):5058-5059.
46 Herman JG, Graff JR, Myohanen S, et al. Methylation-specific PCR:A novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA, 1996, 93(18): 9821-9826.
47 Wong IH, Lo YM, Zhang J, et al. Detection of aberrant p16 methylation in the plasma and serum of liver cancer patients. Cancer Res, 1999, 59(1): 71-73.
48 Chu HJ, Heo J, Seo SB, et al. Detection of aberrant p16INK4A methylation in sera of patients with liver cirrhosis and hepatocellular carcinoma. J Korean Med Sci, 2004,19(1): 83-86.49 Wong IH, Zhang J, Lai PB, et al. Quantitative analysis of tumor-derived methylated p16INK4a sequences in plasma, serum and blood cells of hepatocellular carcinoma patients. Clin Cancer Res, 2003, 9(3): 1047-1052.
50 Wong IH, Johnson PJ, Lai PB, et al. Tumor-derived epigenetic changes in the plasma and serum of liver cancer patients. Implications for cancer detection and monitoring. Ann N YAcad Sci, 2000, 906: 102-105.
51 Cheng JC, Yoo CB, Weisenberger DJ, et al. Preferential response of cancer cells to zebularine. Cancer Cell, 2004, 6(2): 151-158.
52 Yoo CB, Cheng JC, Jones PA. Zebularine: a new drug for epigenetic therapy. Biochem Soc Trans, 2004, 32(Pt6): 910-912.
53 Lin XH, Asgari K, Putzi MJ, et al. Reversal of GSTPl CpG island hypermethylation and reactivation of GSTPl expression in human prostate cancer cells by treatment with procainamide. Cancer Res, 2001, 61(24): 8611-8616.
54 Scheinbart LS, Johnson MA, Gross MA, et al. Procainamide inhibits DNA methyltr- ansferase in human T cell line. J Rheumatol, 1991,18(4): 530-534.