急性白血病细胞EDNRB基因启动子区甲基化状态研究
|
摘要
背景与目的
脊椎动物唯一的DNA自然修饰方式是甲基化修饰,DNA甲基化修饰是表观遗传学(Epigenetics)的重要组成部分,在基因结构的稳定、基因表达调控等方面起重要作用,DNA甲基化状态的改变与肿瘤的发生发展密切相关。白血病细胞中某些基因,主要是抑癌基因(如ER, p15,p16等)其启动子区CpG岛发生异常甲基化,导致这些抑癌基因表达下调或封闭,影响细胞的正常功能,最终导致疾病的发生。
EDNRB基因即内皮素受体B(Endouthelin Receptor B)基因,定位于13q22,长约24kb,是癌肿中一个经常出现基因缺失的区域,含7个外显子和6个内含子,编码一个442个氨基酸的蛋白,是G蛋白偶联的跨膜受体蛋白,该基因所编码的内皮素是一种血管活性肽,具有三种不同形式ET1,ET2,和ET3。此外,EDNRB参与内皮素激活NO合成酶的过程。有研究发现,该基因与正常生长发育相关。EDNRB基因作为一种肿瘤抑制基因,在多种肿瘤的发生发展中起重要作用,在许多肿瘤组织中该基因表达下调,已被确认该基因在多种肿瘤中存在等位基因杂合性丢失(LOH)和REP(复制错误)的现象。
研究发现：肝细胞癌、膀胱癌、鼻咽癌、前列腺癌、乳腺癌等癌肿组织中存在EDNRB基因启动子区CpG岛的异常高甲基化,而相应良性病变组织及正常组织中均无该基因相应区域的高甲基化,用去甲基化药物处理后可使该基因表达恢复。台湾的Hsiao等报道了在初治白血病及多发性骨髓瘤细胞中p16基因和EDNRB基因的高甲基化状态,本实验将进一步检测EDNRB基因在初治急性白血病、完全缓解的急性白血病及复发难治性白血病中的甲基化状态,并分析该基因与临床特征之间的关系,进而探讨EDNRB基因启动子区CPG岛甲基化模式的改变与白血病发生发展的关系。
实验材料与方法
1.标本来源：所有病例组标本共39例,均取自2009年2月至9月间郑州大学第一附属医院血液科门诊及住院的急性白血病(AL)患者,均经细胞形态学、组织化学、免疫学、分子生物学和(或)细胞遗传学确诊的病例。男21例,女18例,年龄16-70岁。
2.实验分组：2.1初诊未治疗急性白血病组17例(AML 10例,ALL7例)；2.2完全缓解组13例；2.3复发难治性急性白血病组9例；2.4正常对照组8例。其中初诊未治疗组,完全缓解组及复发难治组均留取患者骨髓血2ml。正常对照组留取正常人为造血干细胞移植患者配型的健康供者骨髓血2ml,均用EDTA管-20℃保存备用。
3.方法：采用甲基化特异性聚合酶链反应(MS-PCR)对正常对照组骨髓血,以及初诊未治组、完全缓解组、复发难治组骨髓血单个核细胞EDNRB基因启动子区CpG岛甲基化状态进行检测。
4.数据处理：运用SPSS17.0软件对实验数据进行统计学处理,采用x2检验,以P=0.05为检验标准。
结果
1.EDNRB基因在正常对照组无一例出现甲基化。
2.EDNRB基因在初诊未治疗AL组、完全缓解AL组及复发难治性AL组中的甲基化比例分别为82%(14/17)、31%(4/13)、89%(8/9)。三组相比差异有统计学意义,p=0.003。进一步做两两比较,则初治组与完全缓解组；复发难治组与完全缓解组相比,差异均有统计学意义,p<0.05。初治组与复发难治组相比,差异无统计学意义,p>0.05。
3.EDNRB基因甲基化状态与患者的性别、年龄、白血病分型之间无显著相关性,差异无统计学意义,p>0.05。
结论
1.EDNRB基因异常高甲基化模式参与了AL的发生发展。
2.EDNRB基因异常甲基化可能与白血病初治、复发难治以及完全缓解的状态有关,而与患者的性别、年龄、白血病分型之间无显著相关性。
3.MSP是一种检测EDNRB基因甲基化状态敏感的实验方法。
BACKGROUND and PURPOSE
The only natural way of DNA modification of vertebrate is DNA methylation, DNA Methylation is an important part of the Epigenetics, and play an important role in the stability of genetic structure, gene expression and regμlation, etc,and are closely linked with tumor occurrence and development. Some genes found in Leukemia cells, mainly are tumor suppressor genes (such as ER, p15,p16, etc.),if the promoter region of CpG islands occurs abnormal methylation,will resμlt in gene expression reduced or closed down,affect the normal function of cells,and at last leading to the occurrence of the disease.
The gene of Endouthelin Receptor B is located in 13q22, Approximately 24kb, it often has gene deletion of many cancer in this area, containing seven exons and six introns, Encoding a 442 amino acid protein, is a G protein-coupled transmembrane receptor protein.Endothelin is a vasoactive peptide, has three different forms of ET1, ET2,and ET3.In addition, EDNRB involved in the process of activating NO synthase. Studies have found that the genes associated with normal growth and development. EDNRB gene as a tumor suppressor gene, play an important role in a variety of tumor occurrence and development, the gene's expression reduced in many tumors. It has been confirmed that it has allelic loss of heterozygosity (LOH) and replication error (REP) in variety of tumors.
The study found:The tumor tissue in hepatocellμlar carcinoma, bladder cancer, nasopharyngeal cancer, prostate cancer, breast cancer, etc, exists aberrant hypermethylation in the promoter CpG island of EDNRB gene, while the corresponding benign tissue and normal tissue had no hypermethylation of the gene, after demethylation of drug treatment can restore the expression of the gene.Taiwan's Hsiao has reported the hypermethylation status of the p16 and the EDNRB gene in the untreated leukemia and mμltiple myeloma cells. In this study, we will further tests the methylation status of the EDNRB gene in newly diagnosed acute leukemia, complete remission of acute leukemia and relapsed leukemia, And analysis the relationship between the gene and clinical features, and then further explore the relationship between the methylation pattern's change of the EDNRB gene promoter CPG islands and the occurrence and development of leukemia.
MATERIALS AND METHODS
Object
all the specimens of cases were 39, all derived from Hematology outpatient and inpatient who have diagonosed acute leukemia (AL) in the First Affiliated Hospital of Zhengzhou University on February to September 2009,all the confirmed cases were characterized by cell morphology, histochemistry, immunology, molecμlar biology and (or) cell genetics.21 males and 18 females aged 16-70 years old.
Experimental group
1.The untreated group of acute leukemia have 17 patients (AML 10 cases, ALL 7 cases);2.Complete remission group have 13 cases; 3.the relapsed or refractory group of acute leukemia have 9 cases; 4. The normal control group has 8 cases.2ml bone marrow blood specimen frome each patient of the untreatment group, complete remission group and relapsed or refractory group,In the normal control group,2ml bone marrow specimens from the healthy donors who present hematopoietic stem cell transplantation for patients, EDTA tubes were used to save and backup in -20℃.
Methods
Using methylation-specific polymerase chain reaction (MS-PCR) to detected methylation status of the promoter CpG islands of the EDNRB gene in mononuclear cells of the normal bone marrow blood, As well as the blood of patients with acute leukemia bone marrow.
Data processing
Using SPSS17.0 software to analysis the experimental data statistically, Using x2 test for the statistics, P=0.05 for the test.
RESMLTS
1.There is no methylation of EDNRB gene in any cases of the normal control group.
2.The methylation rate of EDNRB was 82%(14/17),31%(4/13),89%(8/ 9).respectively in untreated AL group, complete remission AL group and in relapsed AL group.There was significant difference amoun the three groups, p=0.003.Further compared between the two, we found that the untreated group compared with the complete remission group, the relapsed or refractory group compared with the complete remission group, there were significant differences, p<0.05.There was no significant difference between the untreated group and the relapsed or refractory group, p>0.05.
3.The methylation of EDNRB gene was not related to gender, age, and the type of leukemia, There was no significant difference, p>0.05.
CONCLUSIONS
1.The aberrant hypermethylation pattern of EDNRB gene involved in the pathogenesis of AL.
2.The aberrant methylation of EDNRB gene may be related to the states of untreated AL, complete remission AL and relapsed AL, well there was not correlated with gender, age, and the type of leukemia.
3.Methylation-specific polymerase chain reaction (MS-P) is a sensitive experimental method to detecte the methylation status of EDNRB gene.
脊椎动物唯一的DNA自然修饰方式是甲基化修饰,DNA甲基化修饰是表观遗传学(Epigenetics)的重要组成部分,在基因结构的稳定、基因表达调控等方面起重要作用,DNA甲基化状态的改变与肿瘤的发生发展密切相关。白血病细胞中某些基因,主要是抑癌基因(如ER, p15,p16等)其启动子区CpG岛发生异常甲基化,导致这些抑癌基因表达下调或封闭,影响细胞的正常功能,最终导致疾病的发生。
EDNRB基因即内皮素受体B(Endouthelin Receptor B)基因,定位于13q22,长约24kb,是癌肿中一个经常出现基因缺失的区域,含7个外显子和6个内含子,编码一个442个氨基酸的蛋白,是G蛋白偶联的跨膜受体蛋白,该基因所编码的内皮素是一种血管活性肽,具有三种不同形式ET1,ET2,和ET3。此外,EDNRB参与内皮素激活NO合成酶的过程。有研究发现,该基因与正常生长发育相关。EDNRB基因作为一种肿瘤抑制基因,在多种肿瘤的发生发展中起重要作用,在许多肿瘤组织中该基因表达下调,已被确认该基因在多种肿瘤中存在等位基因杂合性丢失(LOH)和REP(复制错误)的现象。
研究发现：肝细胞癌、膀胱癌、鼻咽癌、前列腺癌、乳腺癌等癌肿组织中存在EDNRB基因启动子区CpG岛的异常高甲基化,而相应良性病变组织及正常组织中均无该基因相应区域的高甲基化,用去甲基化药物处理后可使该基因表达恢复。台湾的Hsiao等报道了在初治白血病及多发性骨髓瘤细胞中p16基因和EDNRB基因的高甲基化状态,本实验将进一步检测EDNRB基因在初治急性白血病、完全缓解的急性白血病及复发难治性白血病中的甲基化状态,并分析该基因与临床特征之间的关系,进而探讨EDNRB基因启动子区CPG岛甲基化模式的改变与白血病发生发展的关系。
实验材料与方法
1.标本来源：所有病例组标本共39例,均取自2009年2月至9月间郑州大学第一附属医院血液科门诊及住院的急性白血病(AL)患者,均经细胞形态学、组织化学、免疫学、分子生物学和(或)细胞遗传学确诊的病例。男21例,女18例,年龄16-70岁。
2.实验分组：2.1初诊未治疗急性白血病组17例(AML 10例,ALL7例)；2.2完全缓解组13例；2.3复发难治性急性白血病组9例；2.4正常对照组8例。其中初诊未治疗组,完全缓解组及复发难治组均留取患者骨髓血2ml。正常对照组留取正常人为造血干细胞移植患者配型的健康供者骨髓血2ml,均用EDTA管-20℃保存备用。
3.方法：采用甲基化特异性聚合酶链反应(MS-PCR)对正常对照组骨髓血,以及初诊未治组、完全缓解组、复发难治组骨髓血单个核细胞EDNRB基因启动子区CpG岛甲基化状态进行检测。
4.数据处理：运用SPSS17.0软件对实验数据进行统计学处理,采用x2检验,以P=0.05为检验标准。
结果
1.EDNRB基因在正常对照组无一例出现甲基化。
2.EDNRB基因在初诊未治疗AL组、完全缓解AL组及复发难治性AL组中的甲基化比例分别为82%(14/17)、31%(4/13)、89%(8/9)。三组相比差异有统计学意义,p=0.003。进一步做两两比较,则初治组与完全缓解组；复发难治组与完全缓解组相比,差异均有统计学意义,p<0.05。初治组与复发难治组相比,差异无统计学意义,p>0.05。
3.EDNRB基因甲基化状态与患者的性别、年龄、白血病分型之间无显著相关性,差异无统计学意义,p>0.05。
结论
1.EDNRB基因异常高甲基化模式参与了AL的发生发展。
2.EDNRB基因异常甲基化可能与白血病初治、复发难治以及完全缓解的状态有关,而与患者的性别、年龄、白血病分型之间无显著相关性。
3.MSP是一种检测EDNRB基因甲基化状态敏感的实验方法。
BACKGROUND and PURPOSE
The only natural way of DNA modification of vertebrate is DNA methylation, DNA Methylation is an important part of the Epigenetics, and play an important role in the stability of genetic structure, gene expression and regμlation, etc,and are closely linked with tumor occurrence and development. Some genes found in Leukemia cells, mainly are tumor suppressor genes (such as ER, p15,p16, etc.),if the promoter region of CpG islands occurs abnormal methylation,will resμlt in gene expression reduced or closed down,affect the normal function of cells,and at last leading to the occurrence of the disease.
The gene of Endouthelin Receptor B is located in 13q22, Approximately 24kb, it often has gene deletion of many cancer in this area, containing seven exons and six introns, Encoding a 442 amino acid protein, is a G protein-coupled transmembrane receptor protein.Endothelin is a vasoactive peptide, has three different forms of ET1, ET2,and ET3.In addition, EDNRB involved in the process of activating NO synthase. Studies have found that the genes associated with normal growth and development. EDNRB gene as a tumor suppressor gene, play an important role in a variety of tumor occurrence and development, the gene's expression reduced in many tumors. It has been confirmed that it has allelic loss of heterozygosity (LOH) and replication error (REP) in variety of tumors.
The study found:The tumor tissue in hepatocellμlar carcinoma, bladder cancer, nasopharyngeal cancer, prostate cancer, breast cancer, etc, exists aberrant hypermethylation in the promoter CpG island of EDNRB gene, while the corresponding benign tissue and normal tissue had no hypermethylation of the gene, after demethylation of drug treatment can restore the expression of the gene.Taiwan's Hsiao has reported the hypermethylation status of the p16 and the EDNRB gene in the untreated leukemia and mμltiple myeloma cells. In this study, we will further tests the methylation status of the EDNRB gene in newly diagnosed acute leukemia, complete remission of acute leukemia and relapsed leukemia, And analysis the relationship between the gene and clinical features, and then further explore the relationship between the methylation pattern's change of the EDNRB gene promoter CPG islands and the occurrence and development of leukemia.
MATERIALS AND METHODS
Object
all the specimens of cases were 39, all derived from Hematology outpatient and inpatient who have diagonosed acute leukemia (AL) in the First Affiliated Hospital of Zhengzhou University on February to September 2009,all the confirmed cases were characterized by cell morphology, histochemistry, immunology, molecμlar biology and (or) cell genetics.21 males and 18 females aged 16-70 years old.
Experimental group
1.The untreated group of acute leukemia have 17 patients (AML 10 cases, ALL 7 cases);2.Complete remission group have 13 cases; 3.the relapsed or refractory group of acute leukemia have 9 cases; 4. The normal control group has 8 cases.2ml bone marrow blood specimen frome each patient of the untreatment group, complete remission group and relapsed or refractory group,In the normal control group,2ml bone marrow specimens from the healthy donors who present hematopoietic stem cell transplantation for patients, EDTA tubes were used to save and backup in -20℃.
Methods
Using methylation-specific polymerase chain reaction (MS-PCR) to detected methylation status of the promoter CpG islands of the EDNRB gene in mononuclear cells of the normal bone marrow blood, As well as the blood of patients with acute leukemia bone marrow.
Data processing
Using SPSS17.0 software to analysis the experimental data statistically, Using x2 test for the statistics, P=0.05 for the test.
RESMLTS
1.There is no methylation of EDNRB gene in any cases of the normal control group.
2.The methylation rate of EDNRB was 82%(14/17),31%(4/13),89%(8/ 9).respectively in untreated AL group, complete remission AL group and in relapsed AL group.There was significant difference amoun the three groups, p=0.003.Further compared between the two, we found that the untreated group compared with the complete remission group, the relapsed or refractory group compared with the complete remission group, there were significant differences, p<0.05.There was no significant difference between the untreated group and the relapsed or refractory group, p>0.05.
3.The methylation of EDNRB gene was not related to gender, age, and the type of leukemia, There was no significant difference, p>0.05.
CONCLUSIONS
1.The aberrant hypermethylation pattern of EDNRB gene involved in the pathogenesis of AL.
2.The aberrant methylation of EDNRB gene may be related to the states of untreated AL, complete remission AL and relapsed AL, well there was not correlated with gender, age, and the type of leukemia.
3.Methylation-specific polymerase chain reaction (MS-P) is a sensitive experimental method to detecte the methylation status of EDNRB gene.
引文
[1]Ballestar E, Wolffe AP, Methyl-CpG-binding proteins. Targeting specific gene repression. Eur J Biochem,2001,268:1-6.
[2]Dahl C, Gμldberg P. DNA methylation analysis techniques [J].Biogerontology,2003,4(4): 233-250.
[3]Cottrell SE. Molecμlar diagnostic applications of DNA methylation technology [J].Clin Biochem,2004,37(7):595-604.
[4]Jones PA, Baylin SB.The fundamental role of epigenetic events in cancer [J].Nat Rev Genet,2002,3(6):415-28.
[5]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.
[6]洪凡真,王波,李晓明.卵巢上皮性肿瘤EDNRB基因甲基化状态的研究.中华妇产科杂志,2006,41(9)：630-632.
[7]Lo KW, Tsang YS, Kwong J, et al. Promoter hypermethylation of the EDNRB gene in nasopharyngeal carcinoma..Int J Cancer,2002,98:651-655.
[8]Pao MM, Tsutsumi M, Liang G, et al. The endothelin receptor B (EDNRB) promoter displays heterogeneous site specific methylation patterns in normal and tumor cells. Hum Mol Genet,2001,10:903-910.
[9]Jongsma AP, Piek JM, Zweemer RP, et al. Molecular evidence for putative tumor suppressor genes on chromosome 13q specific to BRCA1 related ovarian and fallopian tube cancer. Mol Pathol,2002,55(5):305-309.
[10]Arai H, Nakao K, Takaya K, et al. The human endothelin-B receotor gene, structural organization and chromosomal assignment. J Biolchem,1993,268(5):3463-3470.
[11]Shin MK, Levorse JM, Ingram RS, et al. The temporal requirement for endothelin receptor-B signaling during neural crest development. Nature,1999,402(6761):496-501.
[12]Ford HL, et al.Homeobox genes:A link between development, cell cycle, and cancer. Cell Biol Int,1998,22(6):397-400.
[13]Hyytinen ER, Frierson HF, Boyd JC, et al. Three distinct regions of allelic lose at 13q14, 13q21,22, and 13q33 in prostate cancer[J].Genes Chromosomes Cancer,1999,25(2): 108-114.
[14]Hsiao PC,Liu MC, Chen LM, et al. Peomoter methylation of P16 and EDNRB gene in leukemia patients in taiwan. Chinese Journal of Physiology,2008,51(1):27-31.
[15]Kwok-Wai Lo, Yuen-Shan Tsang, Joseph Kwong, Promoter hypermethylation of the EDNRB gene in nasopharyngeal carcinoma. Cancer,2002,98,651-655.
[16]Hiroshi Arai,Kazuwa Nakao,Kazuhiko Takaya,et al.The Human Endothelin-B Receptor Gene. The Journal of Biological Chemistry.1993,268(5):463-3470.
[17]Kuo K C,McCune R A, Gehrke C W, et al. Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA [J].Nucleic Acids Res,1980,8:4763-4776.
[18]Fraga MF, Uriol E, Borja DL, et al. High-performance capillary electrophoretic method for the quantification of 5-methyl 2-deoxycytidine in genomic DNA:application to plant, animal and human cancer tissues [J].Electrophoresis,2002,23:1677-1681.
[19]Oefner, PJ, Bonn GK, Huber CG, et al. Comparative study of capillary zone electrophoresis and high-performance liquid chromatography in the analysis of oligonucleotides and DNA. [J].Chromatogr,1992,625(2):331-3401.
[20]邓大君,邓国仁,吕有勇等.变性高效液相色谱法检测CpG岛胞嘧啶甲基化[J].中华医学杂志,2001,80(2),158-1611.
[21]Xiong Z, Laird PW COBRA:A sensitive and quantitative DNA methylation assay [J]. Nucleic Acids Res,1997,25:2532-2534.
[22]Maekawa M, Sugano K, Kashiwabara H, et al. DNA methylation analysis using bisμlfite treatment and PCR-single-strand conformation polymorphism in colorectal cancer showing microsatellite instability [J].Biochem Biophys Res Commun,1999,262:671-676.
[23]武立鹏,朱卫国.DNA甲基化的生物学应用及检测方法进展[J].中国检验医学杂志,2004,27(7)：468-474.
[24]Frommer M, McDonald LE, Millar DS, et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands [J].Proc Natl Acad Sci USA,1992,89:1827-1831.
[25]朱燕.DNA的甲基化的分析与状态检测[J].现代预防医学,2005,32(9)：1070-1073.
[26]黄琼晓,金帆,黄荷凤.DNA甲基化的研究方法学[J].国外医学遗传学分册,2004,27(6)：354-358.
[27]叶雪石,刘霆,崔旭等.骨髓增生异常综合症p15INK4B基因甲基化剂药物去甲基化作用.四川大学学报(医学版),2007,38(1)：57-59.
[1]Ballestar E, Wolffe AP, Methyl-CpG-binding proteins. Targeting specific gene repression. Eur J Biochem,2001,268:1-6.
[2]Dahl C, Gμldberg P. DNA methylation analysis techniques [J].Biogerontology,2003,4(4): 233-250.
[3]黄庆,郭颖,府伟灵.人类表观基因组计划[J].生命的化学,2004,24(2)：101-102.
[4]武立鹏,朱卫国.DNA甲基化的生物学应用及检测方法进展[J].中国检验医学杂志,2004,27(7)：468-474.
[5]Antequera F, Bird A. Number of CpG islands and genes in human and mouse. Proc Natl Acad Sci USA,1993,90(24):11995-11999.
[6]Reik W, Murrell A. Genomic imprinting. Silence across the border. Nature,2000,405: 408-409.
[7]Jones PA, Baylin SB.The fundamental role of epigenetic events in cancer [J].Nat Rev Genet,2002,3(6):415-28.
[8]Fang JY, Lu YY, et al. Effects of histoneacethlation and DNA methylation on P21 (WAF1) regμlation. World J Gastroenterol,2002,8(3):400-5.
[9]Hollstein M, Shomer B, Greenblatt M, et al. Somatic point mutations in the p53 gene of human tumors and cell lines:updated compilation. Nucleic Acids Res,1996,24(1): 141-146.
[10]Nephew KP, Huang TH. Epigenetic gene silencing in cancer initiation and progression. Cancer Lett,2003,190:125-133.
[11]Chen RZ, Pettersoon V, Beard C, et al. DNA hypomethylation leads to elevated mutationates.Nature,1998,395(6697):89-93.
[12]Schuffenhauer S, Bartsch O, Stumm M, et al. DNA, FISH and complementation studies in ICF syndrome:A hypomethylation of repetitive and single copy loci and eeidence for a trans acting factor.Hum Genet,1995;96(5):562-571.
[13]Almeida A, Kokalj-Vokac N, Lefrancois D, et al. Hypomethylation of classical satellite DNA and chromosome instability lymphoblastoid cell lines. Hum Genet,1993,91(6): 538-546.
[14]Paμlsen M, Ferguson-Smith AC. DNA methylation in genomic imprinting, development, and diseased.[J]Pathol,2001,195(1):97-110.
[15]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.
[16]Nuovo GJ, Plaia TW, Belinsky SA, et al. In situ detection of the hypermethylation-induced inactivation of the p16 gene as an early event in oncogenesis [J].Proc Natl Acad Sci USA, 1999,96:12754-12759.
[17]Soares J,Pinto AE, Cunha CV, et al. Global DNA hypomethylation in breast carcinoma: correlation with prognostic factors and tumor ptogression [J].Cancer,1999,85:112-118.
[18]Uhlmann K, Rohde K, Zeller C, et al. Distinct methylation profiles of glioma subtypes. [J] Int Cancer,2003,106(1):52-59.
[19]Strathdee G,Holyoake TL, Sim A. Inactivation of HOXA genes by hypermethylation in myeloid and lymphoid malignancy is frequent and associated with poor prognosis. Clin Cancer Res.2007,13(17):5048-5055.
[20]Martin P, Garcia-Cosio M, Santon A, Aberrant gene promoter methylation in plasma cell dyscrasias. Experrimental and Molecμlar Pathology,2008,84(3):256-561.
[21]Jongsma AP, Piek JM, Zweemer RP, et al. Molecμlar evidence for putative tumor suppressor genes on chromosome 13q specific to BRCA1 related ovarian and fallopian tube cancer. Mol Pathol,2002,55(5):305-309.
[22]Friedrich MG, Weisenberger DJ, Cheng JC, et al. Detection of methylated apoptosis-associated genes in urine sediments of bladder cancer patients. Clin Cancer Res,2004, 10(22):7457-7465.
[23]Chen SC, Lin CY, Cben YH, et al. Aberrent promter methylation of EDNRB in lung cancer in Taiwan. Oncol Rep,2006,15(1):167-72.
[24]Pei-Ching Hsiaol,Min-Chang Liu2,Li-Mien Chen3,Promoter Methylation of p16 and EDNRB Gene in Leukemia Patients in Taiwan.Chinese Journal of Physiology 2008,51(1): 27-31.
[25]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.
[26]Liu M, Taketani T, Li R, et al.Loss of p73 gene expression in lymphoid leukemia cell lines is associated with hpermethylation. Leμl Res,2001,25(6):441-447.
[27]Widschwendter A, Mμller HM, Hubalek MM,et al.Methylation status and expression of human telomerase reverse transcriptase in ovarian and cervical cancer. Gynecol Oncol, 2004,93(2):407-416.
[28].Macleod AR, Szyf M, Expression of antisense to DNA methyltransferases mRNA induces DNA demethylation and inhibits tumorigenesis. J B Chem,1995,270(14):8037-8043.
[29]Jennifer L, Counts JL, Goodman JI. Alterations in DNA methylation may play a variety of roles in carcinogenesis. Cell,1995,83(1):13-15.
[30]Courtier B,Heard E, Avner P. Xce haplotypes show modified methylation in a region of the active X chromosome lying 3'to Xist. Proc Natl Acad Sci USA,1995,92:3531-3535.
[31]Knudson AG. Hereditary cancer:Two hitsrevisited. J Cancer Res Clin Oncology,1996, 122:135-140.
[32]Jnes PA, Laird PW, et al.Cancer epigenetics comes of age [J].Na. Gene,1999,21(2): 163-167.
[2]Dahl C, Gμldberg P. DNA methylation analysis techniques [J].Biogerontology,2003,4(4): 233-250.
[3]Cottrell SE. Molecμlar diagnostic applications of DNA methylation technology [J].Clin Biochem,2004,37(7):595-604.
[4]Jones PA, Baylin SB.The fundamental role of epigenetic events in cancer [J].Nat Rev Genet,2002,3(6):415-28.
[5]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.
[6]洪凡真,王波,李晓明.卵巢上皮性肿瘤EDNRB基因甲基化状态的研究.中华妇产科杂志,2006,41(9)：630-632.
[7]Lo KW, Tsang YS, Kwong J, et al. Promoter hypermethylation of the EDNRB gene in nasopharyngeal carcinoma..Int J Cancer,2002,98:651-655.
[8]Pao MM, Tsutsumi M, Liang G, et al. The endothelin receptor B (EDNRB) promoter displays heterogeneous site specific methylation patterns in normal and tumor cells. Hum Mol Genet,2001,10:903-910.
[9]Jongsma AP, Piek JM, Zweemer RP, et al. Molecular evidence for putative tumor suppressor genes on chromosome 13q specific to BRCA1 related ovarian and fallopian tube cancer. Mol Pathol,2002,55(5):305-309.
[10]Arai H, Nakao K, Takaya K, et al. The human endothelin-B receotor gene, structural organization and chromosomal assignment. J Biolchem,1993,268(5):3463-3470.
[11]Shin MK, Levorse JM, Ingram RS, et al. The temporal requirement for endothelin receptor-B signaling during neural crest development. Nature,1999,402(6761):496-501.
[12]Ford HL, et al.Homeobox genes:A link between development, cell cycle, and cancer. Cell Biol Int,1998,22(6):397-400.
[13]Hyytinen ER, Frierson HF, Boyd JC, et al. Three distinct regions of allelic lose at 13q14, 13q21,22, and 13q33 in prostate cancer[J].Genes Chromosomes Cancer,1999,25(2): 108-114.
[14]Hsiao PC,Liu MC, Chen LM, et al. Peomoter methylation of P16 and EDNRB gene in leukemia patients in taiwan. Chinese Journal of Physiology,2008,51(1):27-31.
[15]Kwok-Wai Lo, Yuen-Shan Tsang, Joseph Kwong, Promoter hypermethylation of the EDNRB gene in nasopharyngeal carcinoma. Cancer,2002,98,651-655.
[16]Hiroshi Arai,Kazuwa Nakao,Kazuhiko Takaya,et al.The Human Endothelin-B Receptor Gene. The Journal of Biological Chemistry.1993,268(5):463-3470.
[17]Kuo K C,McCune R A, Gehrke C W, et al. Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA [J].Nucleic Acids Res,1980,8:4763-4776.
[18]Fraga MF, Uriol E, Borja DL, et al. High-performance capillary electrophoretic method for the quantification of 5-methyl 2-deoxycytidine in genomic DNA:application to plant, animal and human cancer tissues [J].Electrophoresis,2002,23:1677-1681.
[19]Oefner, PJ, Bonn GK, Huber CG, et al. Comparative study of capillary zone electrophoresis and high-performance liquid chromatography in the analysis of oligonucleotides and DNA. [J].Chromatogr,1992,625(2):331-3401.
[20]邓大君,邓国仁,吕有勇等.变性高效液相色谱法检测CpG岛胞嘧啶甲基化[J].中华医学杂志,2001,80(2),158-1611.
[21]Xiong Z, Laird PW COBRA:A sensitive and quantitative DNA methylation assay [J]. Nucleic Acids Res,1997,25:2532-2534.
[22]Maekawa M, Sugano K, Kashiwabara H, et al. DNA methylation analysis using bisμlfite treatment and PCR-single-strand conformation polymorphism in colorectal cancer showing microsatellite instability [J].Biochem Biophys Res Commun,1999,262:671-676.
[23]武立鹏,朱卫国.DNA甲基化的生物学应用及检测方法进展[J].中国检验医学杂志,2004,27(7)：468-474.
[24]Frommer M, McDonald LE, Millar DS, et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands [J].Proc Natl Acad Sci USA,1992,89:1827-1831.
[25]朱燕.DNA的甲基化的分析与状态检测[J].现代预防医学,2005,32(9)：1070-1073.
[26]黄琼晓,金帆,黄荷凤.DNA甲基化的研究方法学[J].国外医学遗传学分册,2004,27(6)：354-358.
[27]叶雪石,刘霆,崔旭等.骨髓增生异常综合症p15INK4B基因甲基化剂药物去甲基化作用.四川大学学报(医学版),2007,38(1)：57-59.
[1]Ballestar E, Wolffe AP, Methyl-CpG-binding proteins. Targeting specific gene repression. Eur J Biochem,2001,268:1-6.
[2]Dahl C, Gμldberg P. DNA methylation analysis techniques [J].Biogerontology,2003,4(4): 233-250.
[3]黄庆,郭颖,府伟灵.人类表观基因组计划[J].生命的化学,2004,24(2)：101-102.
[4]武立鹏,朱卫国.DNA甲基化的生物学应用及检测方法进展[J].中国检验医学杂志,2004,27(7)：468-474.
[5]Antequera F, Bird A. Number of CpG islands and genes in human and mouse. Proc Natl Acad Sci USA,1993,90(24):11995-11999.
[6]Reik W, Murrell A. Genomic imprinting. Silence across the border. Nature,2000,405: 408-409.
[7]Jones PA, Baylin SB.The fundamental role of epigenetic events in cancer [J].Nat Rev Genet,2002,3(6):415-28.
[8]Fang JY, Lu YY, et al. Effects of histoneacethlation and DNA methylation on P21 (WAF1) regμlation. World J Gastroenterol,2002,8(3):400-5.
[9]Hollstein M, Shomer B, Greenblatt M, et al. Somatic point mutations in the p53 gene of human tumors and cell lines:updated compilation. Nucleic Acids Res,1996,24(1): 141-146.
[10]Nephew KP, Huang TH. Epigenetic gene silencing in cancer initiation and progression. Cancer Lett,2003,190:125-133.
[11]Chen RZ, Pettersoon V, Beard C, et al. DNA hypomethylation leads to elevated mutationates.Nature,1998,395(6697):89-93.
[12]Schuffenhauer S, Bartsch O, Stumm M, et al. DNA, FISH and complementation studies in ICF syndrome:A hypomethylation of repetitive and single copy loci and eeidence for a trans acting factor.Hum Genet,1995;96(5):562-571.
[13]Almeida A, Kokalj-Vokac N, Lefrancois D, et al. Hypomethylation of classical satellite DNA and chromosome instability lymphoblastoid cell lines. Hum Genet,1993,91(6): 538-546.
[14]Paμlsen M, Ferguson-Smith AC. DNA methylation in genomic imprinting, development, and diseased.[J]Pathol,2001,195(1):97-110.
[15]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.
[16]Nuovo GJ, Plaia TW, Belinsky SA, et al. In situ detection of the hypermethylation-induced inactivation of the p16 gene as an early event in oncogenesis [J].Proc Natl Acad Sci USA, 1999,96:12754-12759.
[17]Soares J,Pinto AE, Cunha CV, et al. Global DNA hypomethylation in breast carcinoma: correlation with prognostic factors and tumor ptogression [J].Cancer,1999,85:112-118.
[18]Uhlmann K, Rohde K, Zeller C, et al. Distinct methylation profiles of glioma subtypes. [J] Int Cancer,2003,106(1):52-59.
[19]Strathdee G,Holyoake TL, Sim A. Inactivation of HOXA genes by hypermethylation in myeloid and lymphoid malignancy is frequent and associated with poor prognosis. Clin Cancer Res.2007,13(17):5048-5055.
[20]Martin P, Garcia-Cosio M, Santon A, Aberrant gene promoter methylation in plasma cell dyscrasias. Experrimental and Molecμlar Pathology,2008,84(3):256-561.
[21]Jongsma AP, Piek JM, Zweemer RP, et al. Molecμlar evidence for putative tumor suppressor genes on chromosome 13q specific to BRCA1 related ovarian and fallopian tube cancer. Mol Pathol,2002,55(5):305-309.
[22]Friedrich MG, Weisenberger DJ, Cheng JC, et al. Detection of methylated apoptosis-associated genes in urine sediments of bladder cancer patients. Clin Cancer Res,2004, 10(22):7457-7465.
[23]Chen SC, Lin CY, Cben YH, et al. Aberrent promter methylation of EDNRB in lung cancer in Taiwan. Oncol Rep,2006,15(1):167-72.
[24]Pei-Ching Hsiaol,Min-Chang Liu2,Li-Mien Chen3,Promoter Methylation of p16 and EDNRB Gene in Leukemia Patients in Taiwan.Chinese Journal of Physiology 2008,51(1): 27-31.
[25]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.
[26]Liu M, Taketani T, Li R, et al.Loss of p73 gene expression in lymphoid leukemia cell lines is associated with hpermethylation. Leμl Res,2001,25(6):441-447.
[27]Widschwendter A, Mμller HM, Hubalek MM,et al.Methylation status and expression of human telomerase reverse transcriptase in ovarian and cervical cancer. Gynecol Oncol, 2004,93(2):407-416.
[28].Macleod AR, Szyf M, Expression of antisense to DNA methyltransferases mRNA induces DNA demethylation and inhibits tumorigenesis. J B Chem,1995,270(14):8037-8043.
[29]Jennifer L, Counts JL, Goodman JI. Alterations in DNA methylation may play a variety of roles in carcinogenesis. Cell,1995,83(1):13-15.
[30]Courtier B,Heard E, Avner P. Xce haplotypes show modified methylation in a region of the active X chromosome lying 3'to Xist. Proc Natl Acad Sci USA,1995,92:3531-3535.
[31]Knudson AG. Hereditary cancer:Two hitsrevisited. J Cancer Res Clin Oncology,1996, 122:135-140.
[32]Jnes PA, Laird PW, et al.Cancer epigenetics comes of age [J].Na. Gene,1999,21(2): 163-167.