高危型HPV感染、DNMT1表达和DAPK基因启动子甲基化状态在宫颈病变中相关性的研究
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摘要
目的：
1.检测宫颈病变细胞中DNMT1蛋白表达状态和抑癌基因DAPK启动子区的甲基化状态，探讨其在宫颈病变发生发展中的作用；
2.探讨高危型HPV的感染、DNMT1表达和DAPK启动子区甲基化状态间的相互关系，可进一步了解宫颈病变的发生机制，对该病早期筛查及病情发展的监测提供帮助。
方法：
1.研究对象随机选择2011年1月至2011年12月我院妇科门诊患者中均行TCT和阴道镜检查的150例患者，根据阴道镜宫颈活检的病理诊断结果，分为子宫颈上皮内瘤变Ⅰ级（CINⅠ）30例，子宫颈上皮内瘤变Ⅱ级（CINⅡ）30例，子宫颈上皮内瘤变Ⅲ级（CINⅢ）30例，鳞状细胞癌（SCC）30例作为研究组；另取未见恶性细胞和上皮内病变细胞（NILM）30例为对照组。
2.检测高危型HPV-DNA感染情况取患者的TCT剩余标本，提取DNA，SPR方法检测高危型HPV，运用生物传感芯片阅读仪读取高危型HPV-DNA感染情况。
3.检测DNMT1蛋白表达状态取上述所选患者的TCT剩余标本（保存期不超过3周），经Thinprep2000系统重新制片1张，置于95%酒精中室温固定24小时，空气中晾干，置冰箱4℃储存备用拟行DNMT1蛋白的免疫细胞化学检测。显微镜下观察细胞图片，细胞质及细胞间质出现棕褐色颗粒者为阳性。
4．检测抑癌基因DAPK启动子区的甲基化状态取入组患者的TCT剩余标本，采用MSP技术，检测患者宫颈脱落细胞DAPK基因启动子区的甲基化状况。摄像观察8% PAGE上电泳结果：获得甲基化产物，无论是否出现非甲基化产物，均判定为甲基化阳性；只出现非甲基化产物判定为甲基化阴性；甲基化产物与非甲基化产物均未获得判定该标本实验失败。
5.统计学分析采用SPSS18.0软件包进行统计分析，检验水准α= 0.05。
结果：
1. 150例宫颈各病变阶段宫颈脱落细胞中高危型HPV-DNA的检测情况：高危型HPV感染阳性率为80.67%（121∕150），随着病变程度的进展，各组间高危型HPV感染率存在统计学差异（χ2=30.658，P＜0.05），且呈现出增高的趋势（趋势χ2=30.658，P＜0.05）。
2.宫颈病变各阶段DNMT1蛋白检测结果：NILM组6.67%；CINⅠ组56.67%；CINⅡ组63.33%；CINⅢ组66.67%；SCC组76.67%。DNMT1蛋白在各宫颈病变阶段组中的阳性表达率差异有显著性（P﹤0.05），且随着病变程度的进展，DNMT1蛋白表达率呈现出增高的趋势（P﹤0.05）。
3.本实验中随着病变程度的进展，DAPK基因启动子区CpG岛的甲基化率分别为3.33%、10.00%、13.33%、46.67%、60.00%，各组间有显著性差异（P<0.05），且呈现出增高的趋势（P<0.05）。
4.在宫颈病变的过程中，高危型HPV感染、DNMT1蛋白表达与DAPK基因甲基化两两间相关系数分别为0.340、0.214、0.444，互呈正相关（P均< 0.05）。
结论：
随着宫颈病变级别的升高，高危型HPV持续性感染、DNMT1蛋白异常高表达、DAPK抑癌基因的异常高甲基化，各指标间可能存在相互间的促进关系，可以为宫颈病变的发现和病变程度的监测提供新思路、新方法。
Objective：
1. To detect the expression of DNMT1 protein and the methylation status of suppressorgene DAPK promoter in cervical lesions, discuss their role in the development of cervical lesions
2. To Study the relationship among infection of high-risk type HPV, expression of DNMT1and methylation status of DAPK promoter in cervical lesions, and to further understand thepathogenesis of the disease, to help early screening and monitoring of disease progression.
Methods:
1.Source of specimen We randomly collected the 150 patients were treated with TCT andcolposcopic examinations from 2011 January to 2011 December. According to the results ofcervical biopsy pathologic diagnosis under colposcopy, divided into cervical intraepithelialneoplasia grade I ( CIN I ) in 30 cases, cervical intraepithelial neoplasia grade II ( CINII )30cases, cervical intraepithelial neoplasia ( CIN III ) in 30 cases, squamous cell carcinoma( SCC ) in 30 cases as study group; the other with negative for intraepithelial lesion ormalignancy ( NILM ) as the control group of 30 cases.
2. Detection of high-risk type HPV-DNA infection For patients with TCT residualpreservation solution, DNA was extracted, the SPR method for detection of HPV, HPV-DNAinfection was detected by the biosensor chip.
3. Detection of the expression of DNMT1 protein state The selected the TCT residualpreservation solution of 150 patients (preservation period of not more than 3weeks), via theThinprep2000 system to produce 1cell slides. Then put the sliders under 95% alcohol fixation for24 hours at room temperature, then dry them in the air, store them in the refrigerator at 4 degreesbackup to DNMT1 protein was with immunocytochemistry. Cells picture were observed under amicroscope. The case with brown particles in cytoplasm and stromal cells was positive.
4. Detection of tumor suppressor gene DAPK promoter methylation status Patients’TCTresidual preservation solution, with MSP technology，were detected the methylation status on thecervical exfoliated cells DAPK gene promoter region. Determination of results PCR productswere walking in 8% PAGE. Get methylated product, whether they appear non-methylatedproduct, both determinated to be methylated positive; only a non-methylated product were foundfor unmethylated; has not been methylated product and non-methylated product is the sign of thefailure of the experiment.
5. Statistical analysis Using SPSS18.0 software package for the Statistical analysis,α= 0.05 of inspection standard.
Result:
1.150cases of cervical lesions in cervical exfoliated cells in each phase of high-risk typeHPV-DNA detection: Total high-risk type HPV positive rate was 80.67% (121/ 150), trend ofχ2= 30.658, P < 0.05,with the extent of the lesion progression, HPV infection rate is alsoshowing a trend of increasing.
2. Expression of DNMT1protein in each group: In 6.67% of NILM group; CIN group56.67%;CIN II group 63.33% group66.67%; CIN; SCC76.67%DNMT1 protein expression. DNMT1protein in various types of cervical lesions in the positive expression rates have significantdifference (P < 0.05), and with the extent of the lesion progression, DNMT1 protein expressionrate showed increasing trend (P < 0.05).
3. The experiment of promoter region of DAPK gene in CpG island methylation positive ratewas 26.67% (40/ 150), with the extent progression of the lesion, DAPK gene methylation rateswere 3.33%, 10.00%, 13.33%, 46.67%, 60.00%, there are significant differences among thegroups ( P < 0.05), and showed a trend of increasing( P < 0.05).
4. In cervical lesions during the development of sick, fection of high-risk type HPV,DNMT1protein expression and DAPK gene methylation every two were positively correlated ( P< 0.05), The correlation coefficient is respectively: 0.340、0.214、0.444.
Conclusion：
With the rise of the level of cervical lesions, high-risk type HPV persistent infection,abnormal high expression of DNMT1protein and DAPK tumor suppressor gene abnormalhypermethylation, the promotion of relations may exist between between each Indicators. Newideas and new methods were proposed for the discovery and monitoring of the severity ofcervical lesions.
1.检测宫颈病变细胞中DNMT1蛋白表达状态和抑癌基因DAPK启动子区的甲基化状态，探讨其在宫颈病变发生发展中的作用；
2.探讨高危型HPV的感染、DNMT1表达和DAPK启动子区甲基化状态间的相互关系，可进一步了解宫颈病变的发生机制，对该病早期筛查及病情发展的监测提供帮助。
方法：
1.研究对象随机选择2011年1月至2011年12月我院妇科门诊患者中均行TCT和阴道镜检查的150例患者，根据阴道镜宫颈活检的病理诊断结果，分为子宫颈上皮内瘤变Ⅰ级（CINⅠ）30例，子宫颈上皮内瘤变Ⅱ级（CINⅡ）30例，子宫颈上皮内瘤变Ⅲ级（CINⅢ）30例，鳞状细胞癌（SCC）30例作为研究组；另取未见恶性细胞和上皮内病变细胞（NILM）30例为对照组。
2.检测高危型HPV-DNA感染情况取患者的TCT剩余标本，提取DNA，SPR方法检测高危型HPV，运用生物传感芯片阅读仪读取高危型HPV-DNA感染情况。
3.检测DNMT1蛋白表达状态取上述所选患者的TCT剩余标本（保存期不超过3周），经Thinprep2000系统重新制片1张，置于95%酒精中室温固定24小时，空气中晾干，置冰箱4℃储存备用拟行DNMT1蛋白的免疫细胞化学检测。显微镜下观察细胞图片，细胞质及细胞间质出现棕褐色颗粒者为阳性。
4．检测抑癌基因DAPK启动子区的甲基化状态取入组患者的TCT剩余标本，采用MSP技术，检测患者宫颈脱落细胞DAPK基因启动子区的甲基化状况。摄像观察8% PAGE上电泳结果：获得甲基化产物，无论是否出现非甲基化产物，均判定为甲基化阳性；只出现非甲基化产物判定为甲基化阴性；甲基化产物与非甲基化产物均未获得判定该标本实验失败。
5.统计学分析采用SPSS18.0软件包进行统计分析，检验水准α= 0.05。
结果：
1. 150例宫颈各病变阶段宫颈脱落细胞中高危型HPV-DNA的检测情况：高危型HPV感染阳性率为80.67%（121∕150），随着病变程度的进展，各组间高危型HPV感染率存在统计学差异（χ2=30.658，P＜0.05），且呈现出增高的趋势（趋势χ2=30.658，P＜0.05）。
2.宫颈病变各阶段DNMT1蛋白检测结果：NILM组6.67%；CINⅠ组56.67%；CINⅡ组63.33%；CINⅢ组66.67%；SCC组76.67%。DNMT1蛋白在各宫颈病变阶段组中的阳性表达率差异有显著性（P﹤0.05），且随着病变程度的进展，DNMT1蛋白表达率呈现出增高的趋势（P﹤0.05）。
3.本实验中随着病变程度的进展，DAPK基因启动子区CpG岛的甲基化率分别为3.33%、10.00%、13.33%、46.67%、60.00%，各组间有显著性差异（P<0.05），且呈现出增高的趋势（P<0.05）。
4.在宫颈病变的过程中，高危型HPV感染、DNMT1蛋白表达与DAPK基因甲基化两两间相关系数分别为0.340、0.214、0.444，互呈正相关（P均< 0.05）。
结论：
随着宫颈病变级别的升高，高危型HPV持续性感染、DNMT1蛋白异常高表达、DAPK抑癌基因的异常高甲基化，各指标间可能存在相互间的促进关系，可以为宫颈病变的发现和病变程度的监测提供新思路、新方法。
Objective：
1. To detect the expression of DNMT1 protein and the methylation status of suppressorgene DAPK promoter in cervical lesions, discuss their role in the development of cervical lesions
2. To Study the relationship among infection of high-risk type HPV, expression of DNMT1and methylation status of DAPK promoter in cervical lesions, and to further understand thepathogenesis of the disease, to help early screening and monitoring of disease progression.
Methods:
1.Source of specimen We randomly collected the 150 patients were treated with TCT andcolposcopic examinations from 2011 January to 2011 December. According to the results ofcervical biopsy pathologic diagnosis under colposcopy, divided into cervical intraepithelialneoplasia grade I ( CIN I ) in 30 cases, cervical intraepithelial neoplasia grade II ( CINII )30cases, cervical intraepithelial neoplasia ( CIN III ) in 30 cases, squamous cell carcinoma( SCC ) in 30 cases as study group; the other with negative for intraepithelial lesion ormalignancy ( NILM ) as the control group of 30 cases.
2. Detection of high-risk type HPV-DNA infection For patients with TCT residualpreservation solution, DNA was extracted, the SPR method for detection of HPV, HPV-DNAinfection was detected by the biosensor chip.
3. Detection of the expression of DNMT1 protein state The selected the TCT residualpreservation solution of 150 patients (preservation period of not more than 3weeks), via theThinprep2000 system to produce 1cell slides. Then put the sliders under 95% alcohol fixation for24 hours at room temperature, then dry them in the air, store them in the refrigerator at 4 degreesbackup to DNMT1 protein was with immunocytochemistry. Cells picture were observed under amicroscope. The case with brown particles in cytoplasm and stromal cells was positive.
4. Detection of tumor suppressor gene DAPK promoter methylation status Patients’TCTresidual preservation solution, with MSP technology，were detected the methylation status on thecervical exfoliated cells DAPK gene promoter region. Determination of results PCR productswere walking in 8% PAGE. Get methylated product, whether they appear non-methylatedproduct, both determinated to be methylated positive; only a non-methylated product were foundfor unmethylated; has not been methylated product and non-methylated product is the sign of thefailure of the experiment.
5. Statistical analysis Using SPSS18.0 software package for the Statistical analysis,α= 0.05 of inspection standard.
Result:
1.150cases of cervical lesions in cervical exfoliated cells in each phase of high-risk typeHPV-DNA detection: Total high-risk type HPV positive rate was 80.67% (121/ 150), trend ofχ2= 30.658, P < 0.05,with the extent of the lesion progression, HPV infection rate is alsoshowing a trend of increasing.
2. Expression of DNMT1protein in each group: In 6.67% of NILM group; CIN group56.67%;CIN II group 63.33% group66.67%; CIN; SCC76.67%DNMT1 protein expression. DNMT1protein in various types of cervical lesions in the positive expression rates have significantdifference (P < 0.05), and with the extent of the lesion progression, DNMT1 protein expressionrate showed increasing trend (P < 0.05).
3. The experiment of promoter region of DAPK gene in CpG island methylation positive ratewas 26.67% (40/ 150), with the extent progression of the lesion, DAPK gene methylation rateswere 3.33%, 10.00%, 13.33%, 46.67%, 60.00%, there are significant differences among thegroups ( P < 0.05), and showed a trend of increasing( P < 0.05).
4. In cervical lesions during the development of sick, fection of high-risk type HPV,DNMT1protein expression and DAPK gene methylation every two were positively correlated ( P< 0.05), The correlation coefficient is respectively: 0.340、0.214、0.444.
Conclusion：
With the rise of the level of cervical lesions, high-risk type HPV persistent infection,abnormal high expression of DNMT1protein and DAPK tumor suppressor gene abnormalhypermethylation, the promotion of relations may exist between between each Indicators. Newideas and new methods were proposed for the discovery and monitoring of the severity ofcervical lesions.
引文
[1] Goncalves A, Fabbro M, Lhomme C, et al. A phaseⅡtrial to evaluate gefitinib as second or third-linetreatment in patients with recurring locoregionally advanced or metastatic cervical cancer[J]. GynecolOncol, 2008, 108(1):42-46.
[2]乐杰.第七版妇产科学[M].北京:人民卫生出版社, 2008: 261-.
[3]张亚萍.宫颈上皮内瘤样变的诊治现状[J].辽宁中医药大学学报, 2008, 10(1): 47.
[4] Castle PE，Cox JT，Schiffman M，et al．Factors influencing histologic confirmation of high-grade squamousintraepithelial lesion cytology[J]．Obstet Gynecol，2008，112(3)：637-645.
[5] Cai HB，Dins XH，Chen CC．Prevalence of single and multiple human papillomavirus types in cervicalcancer and precursor lesions in Hubei，China[J]．Oncology，2009，76(3)：157-161.
[6] Guo M，Lin CY，ConS Y，et al．Human papillomavirus genotyping for the eight oncogenic types canimprove specificity of HPV testing in women with mildly abnormal Pap results[J]．Mod Pathul，2008，21(8)：1037-1043
[7] Montero AJ, Díaz-Montero CM, Mao L, et al: Epigenetic inactivation of EGFR by CpG islandhypermethylationin cancer[J]. Cancer Biol Ther. 2006, 5(1):494-501.
[8]杨夕粉.叶酸与5-Aza-CdR对CIN阶段DAPK抑癌基因启动子区甲基化的影响[D].山西医科大学硕士论文. 2011.
[9] Khuloud Bajbouj, Angela Poehlmann, Doerthe Kuester, et al. Identification of Phosphorylated p38 as aNovel DAPK-Interacting Partner during TNF -Induced Apoptosis in Colorectal Tumor Cells [J]. CellInjury, Repair, Aging and Apoptosis. 2009, 2(175): 557-570.
[10]éva G m ri, József Pál, Bernadett Kovács, et al. Concurrent hypermethylation of DNMT1, MGMT andEGFR genes in progression of gliomas [J]. Diagnostic Pathology 2012,1 (7):8-17.
[11] Chen R H, Chen C H, Wang W J, et al. Bidirectional signals transduced by DAPK-ERK interactionpromote the apoptotic effect of DAPK[J]. Embo J. 2005,3(24), 294-304.
[12] Lee TS, Kim JW, Kang GH, et al. DNA hypermethylation of CAGE promotors in squamous cellcarcinoma of uterine cervix[J]. Ann NYA cad Sc,i 2006, 1091: 218- 224.
[13] Jing F, Zhang J, Tao J, et al. Hyperm ethlylation of tumor suppressor genes BRCA1, p16 and 14. 3.3sigma in serum of sporadicbreast cancer patients [J]. Onko log ine, 2007, 30( 1- 2) : 14- 19.
[14] Jones PA, Laird PW. Cancer epigenetics comes of age [J] .Nat Genet, 1999, 21( 2) : 163-167.
[15]艾军华,吕凤林,左国伟等.人乳头瘤病毒11型DNA中CpG基序甲基化状态分析及生物活性的研究[J] .免疫学杂志, 2005, 1( 21) : 29-32.
[16] Etoh T, Kanai Y, Ushijima S, et al. Increased DNA Methyltransferase 1 ( DNMT1) protein expressioncorrelates significantly with poorer tumor differentiation and frequent DNA hypermethylation of multipleCpG islands in gastric cancers [J] .Am J Pathol, 2004, 164(2) : 689-699.
[17] Solomon D, Davey D, Kurman R, et al. The 2001 Bethesda System: terminology for reporting results ofcervical cytology [J]. JAMA, 2002; 287: 2114-2119
[18] Reddy AN, Jiang WW, Kim M, et al. Death-associated protein kinase promoter hyper-methylation innormal human lymphocytes[J]. Cancer Res, 2003, 63(22): 7694-7698.
[19] Widschw endter A, Muller H M, Fiegl H, et al. DNA methylation in serum and tumors of cervical cancerpatients[J]. Clin Cancer Res, 2004, 10(2): 565-571.
[20] Yam aguchi S, Asao T, Nakamura J, et al. High frequency of DAP- kinase gene promoter methylation incolorectal cancer specimens and its identification in serum [J].Cancer Lett, 2003, 194(1): 99-105.
[21] Toyooka S, Toyooka K O, Miyajima K, et al. Epigenetic down-regulation of death-associated proteinkinase in lung cancers [J]. Clin Cancer Res, 2003, 9(8): 3034-3041.
[22]赵先兰,孟志英等.宫颈癌组织中DAPK基因启动子甲基化的研究[J].癌症, 2008,27(9):919-923
[23] Yang Liu, Weimin Gao, Jill M Siegfried, et al. Promoter methylation of RASSF1A and DAPK andmutations of K-ras, p53, and EGFR in lung tumors from smokers and never-smokers [J]. BMC Cancer2007, 3(7):74-82.
[24]顾婷婷,张明忠,郑鹏生. DNA甲基化研究方法的回顾与评价[J].中国妇幼健康研究, 2006, 17(6):555-560.
[25] Mittag F, Kuester D, Vreth M, et al. DAPK promoter methylation is an early eventin colorectalcarcinogenesis [J].Cancer Lett, 2006, 240 (l): 69-75.
[26]刘晓碗，玛依努尔尼牙孜，阿布力孜阿布杜拉等．新疆维吾尔族妇女宫颈病变中DAPK表达的研究[J].中国肿瘤,2010,19（3）: 203-206.
[27] Widau R C, Jin Y, Dixon S A, et al. Protein phosphatase 2A (PP2A) holoenzymes regulatedeath-associated protein kinase (DAPK) in ceramide-induced anoikis [J]. J. Biol. Chem. 2010, 2(85),13827-13838.
[28] Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer[J]. Nat Rev Genet,2002, 3:415–428.
[29]张勇敢,刘保池. DNA甲基转移酶的作用及甲基化在肿瘤发生中的作用[J].国际外科学杂志. 2008,35(8): 537-540.
[30] Eden A, Gaudet F, Waghmare A, Jaenisch R. Chromosomal instability and tumors promoted by DNAhypomethylation[J]. Science. 2003, 300: 455.
[31] Karpf AR, Matsui S (2005) Genetic disruption of cytosine DNA methyltransferase enzymes induceschromosomal instability in human cancer cells[J]. Cancer Res. 2005, 65: 8635–8639.
[32]齐翀,李建芳,瞿颖,等.胃癌及癌旁组织多个肿瘤相关基因超甲基化[J].上海交通大学学报(医学版), 2007, 27(5): 555-558.
[33] Nakagawa T, Kanai Y, Ushijima S, et al. DNA hypermethylation on multiple CpG islands associated withincreased DNA methyltransferase DNMT1 protein expression during multistage urothelialcarcinogenesis[J]. J Urol 2005, 4(173): 1767-71.
[34] .Lin, Y., Hupp, T. R. and Stevens, C. Death-associated protein kinase (DAPK) and signal transduction:additional roles beyond cell death[J]. FEBS J. 2010,277, 48-57.
[35] Bovellan, M., Fritzsche, M., Stevens, C. and Charras, G.. Death-associated protein kinase (DAPK) andsignal transduction: blebbing in programmed cell death[J]. FEBS J. 2010,277, 58-65.
[36] Kimchi, A. A cell death-promoting kinase[J]. Nat. Struct. Biol.2001, 8, 824-826.
[37] Gao WM, Mady HH, Yu GY, et al. Comparison of p53 mutations between adenocarcinoma and squamouscell carcinoma of the lung: unique spectra involving G to A transitions and G to T transversions in bothhistologic types[J]. Lung Cancer 2003, 40:141-150.
[38] Zhang W, Stabile LP, Keohavong P, et al. Mutation and polymorphism in the EGFR-TK domainassociated with lung cancer[J]. J Thorac Oncol 2006, 1:635-647.
[39] Liu Xiaofang, Tang Kun, Yu Shaoping, et al. Correlation between promoter methylation ofp14ARF,TMS1/ASC, and DAPK, and p53 mutationwith prognosis in cholangiocarcinoma[J]. World Journal ofSurgical Oncology, 2012, 1(10): 5-13.
[40] Zbigniew Jab onowski, Edyta Reszka, Jolanta Gromadzińska et al. Hypermethylation of p16 and DAPKpromoter generegions in patients with non-invasive urinary bladdercancer [J]. Arch Med Sci, 2011,7,(3):512-516
[41] Devrim Gozuacik, Adi Kimchi. DAPk Protein Family and Cancer [J]. Spotlight on Cancer, 2006, 2(2):74-79.
[42] Esteller M. Relevance of DNA methylation in the managementof cancer[J] . Lancet Oncol, 2003, 4 ( 6) :351-358.
[1] Esteller M. DNA methylation and cancer therapy: new developments and expectations[J]. Curr OpinOncol,2005, 17(l):55-60.
[2] Das PM, Singal R. DNA methylation and caneer[J]. Clin Oncol, 2004, 22(22):4632-4642.
[3] Davis CD, Uthus EO. DNA methylation，caneer susceptibility，and nutrient interactions. Exp BiolMed(Maywood), 2004, 229(10):988-995
[4] Jones PA,Takai D. The role of DNA methylation in mammalian epigenetics [J]. Science, 2001, 293 ( 5532):1068- 1070.
[5] Issa JP. Aging, DNA methylation and cancer[J]. CritRev Oncol Hematol, 1999, 32(1): 31- 43.
[6] Jones PA, Laird PW. Cancer epigenetics comes of age[J]. Nat Genet, 1999, 21(2) : 163- 167
[7] Singal R, Ginder GD. DNA methylation [J]. Blood,1999, 93(12): 4059- 4070.
[8] Bestor, T.H., The DNA methyltransferases of mamrnals. Hum Mol Genet, 2000. 9(l6): P. 2395-402.
[9] Rhee, I., etal., DNMT1 and DNMT3b cooperate to silence genes in human cancer cells. Nature, 2002.416(6880): p. 552-6.
[10] Ahluwalia, A., etal., DNA methylation in ovarian caneer.Ⅱ. Expression of DNA methyltransferasesin ovarian cancer cell lines and normal ovarian epithelial cells. Gynecol Oneol, 2001. 82(2): P. 299-304.
[11] Robertson, K.D., DNA methylation, methyltransferases, and caneer. Oncogene, 2001. 20(24): P. 3139-55.
[12] Wu, Y., etal., Aberrant expression of deoxyribonucleic acid methyltransferases DNMT1, DNMT3a, andDNMT3b in women with endometriosis. Fertil Steril, 2007. 87(l): P. 24-32.
[13] Sawada, M., etal., Inereased expression of DNA methyltransferase l (DNMT1) Protein in uterine cervixsquamous cell carcinoma and its precursor lesion. Cancer Lett, 2007. 251(2): p. 211-9.
[14]石小燕，DNA甲基转移酶1在子宫颈癌组织和细胞中的表达及意义.解放军医学杂志，2008. 42(12):p. 834-9.
[15]赵先兰，宫颈癌组织中DNA甲基转移酶lmRNA的表达.郑州大学学报(医学版)，2009. 251(2): p.211-9.
[16] Szyf, M., The role of DNA methyltransferase 1 in growth contro1. Front Biosci, 2001. 6: P. D599-609
[17] Harder J，Opitz OG, Brabender J，etal. Quantitative promoter methylation analysis of hepatocellularcarcinoma，cirrhotic and normal liver. Int J Cancer, 2008, 122(12): 2800-2804.
[18] Imai T, Toyota M, Suzuki H, etal. Epigenetic inaetivation of RASSF2 in oral squamous cell carcinoma.Caneer Sci, 2008, 99(5): 958-966.
[19] Lee JH, Byun DS, Lee MG, etal. Frequent epigenetic inactivation of hSRBC in gastric cancer and itsimplication in attenuated P53 response to stresses. Int J Caneer, 2008, 122(7): 1573-1584，
[20] Kitkumthom N, Yanatatsanajit P, KiatPonqsans S, etal. Cyelin A1 promote hypermethylation in humanpapillomavirus-associated cervical cancer. BMCaneer, 2006, 6(l): 55-59.
[21] Dowdy SC, Gostout BS, Shridhar V, etal. Biallelic methylation and silencing of paternally expressed gene3(PEG3) in gyneeologic cancer cell lines. Gyneeo Oneol, 2005, 99(l): 126-134.
[22] Sova P, Feng Q, Geiss G, etal. Discovery of novel methylation biomarkers in cervical carcinoma by globaldemethylation and microarray analysis. Caneer Epidemiol Biomarkers Prev, 2006, 15(l): 114-123.
[23] Yang B, House MG, Guo M, etal. Promoter methylation profiles of tumor suppressor genes in intrahepaticand extrahepatic cholangoearcinoma.Mod Pathol, 2005, 18(3): 412-420.
[24]康新江. DNA甲基化与肿瘤.内江科技，2006，3: 105一108.
[25] Adorian P, Distler J, Lipscher E, etal. Tumor class predietion and discovery by microarray-based DANmethylation analysis. Nucleic Acids Res, 2002, 30(5):e21.
[26] Feng Q, Balasubramanian A, Hawes SE, etal. Deteetion of hypermethylated genes in women with andwithout cervical neoplasia. J Natl Caneer Inst , 2005, 97(4): 273-282.
[27] Maruyama R, Sugio K, Yoshino I, etal. Hypermethylation of FHIT as a prognostic marker in nonsmall celllung carcinoma. Caneer, 2004, 100(7): 1472-1477.
[28] Graziano F, Arduini F, RuzzoA, etal. Prognostic analysis of E-eadherin gene promoter hypermethylationin patients with surgically resected, nod-Positive, diffuse gastric cancer. Clin Caneer Res, 2004, 10(8):2781-2789.
[29] Widsehwendter A, Muller HM, Hubalek MM, etal. Methylation status and expression of humantelomerase reverse transeriptase in ovarian and cervical cancer. Gynceol Oneol 200493(2): 407-416.
[2]乐杰.第七版妇产科学[M].北京:人民卫生出版社, 2008: 261-.
[3]张亚萍.宫颈上皮内瘤样变的诊治现状[J].辽宁中医药大学学报, 2008, 10(1): 47.
[4] Castle PE，Cox JT，Schiffman M，et al．Factors influencing histologic confirmation of high-grade squamousintraepithelial lesion cytology[J]．Obstet Gynecol，2008，112(3)：637-645.
[5] Cai HB，Dins XH，Chen CC．Prevalence of single and multiple human papillomavirus types in cervicalcancer and precursor lesions in Hubei，China[J]．Oncology，2009，76(3)：157-161.
[6] Guo M，Lin CY，ConS Y，et al．Human papillomavirus genotyping for the eight oncogenic types canimprove specificity of HPV testing in women with mildly abnormal Pap results[J]．Mod Pathul，2008，21(8)：1037-1043
[7] Montero AJ, Díaz-Montero CM, Mao L, et al: Epigenetic inactivation of EGFR by CpG islandhypermethylationin cancer[J]. Cancer Biol Ther. 2006, 5(1):494-501.
[8]杨夕粉.叶酸与5-Aza-CdR对CIN阶段DAPK抑癌基因启动子区甲基化的影响[D].山西医科大学硕士论文. 2011.
[9] Khuloud Bajbouj, Angela Poehlmann, Doerthe Kuester, et al. Identification of Phosphorylated p38 as aNovel DAPK-Interacting Partner during TNF -Induced Apoptosis in Colorectal Tumor Cells [J]. CellInjury, Repair, Aging and Apoptosis. 2009, 2(175): 557-570.
[10]éva G m ri, József Pál, Bernadett Kovács, et al. Concurrent hypermethylation of DNMT1, MGMT andEGFR genes in progression of gliomas [J]. Diagnostic Pathology 2012,1 (7):8-17.
[11] Chen R H, Chen C H, Wang W J, et al. Bidirectional signals transduced by DAPK-ERK interactionpromote the apoptotic effect of DAPK[J]. Embo J. 2005,3(24), 294-304.
[12] Lee TS, Kim JW, Kang GH, et al. DNA hypermethylation of CAGE promotors in squamous cellcarcinoma of uterine cervix[J]. Ann NYA cad Sc,i 2006, 1091: 218- 224.
[13] Jing F, Zhang J, Tao J, et al. Hyperm ethlylation of tumor suppressor genes BRCA1, p16 and 14. 3.3sigma in serum of sporadicbreast cancer patients [J]. Onko log ine, 2007, 30( 1- 2) : 14- 19.
[14] Jones PA, Laird PW. Cancer epigenetics comes of age [J] .Nat Genet, 1999, 21( 2) : 163-167.
[15]艾军华,吕凤林,左国伟等.人乳头瘤病毒11型DNA中CpG基序甲基化状态分析及生物活性的研究[J] .免疫学杂志, 2005, 1( 21) : 29-32.
[16] Etoh T, Kanai Y, Ushijima S, et al. Increased DNA Methyltransferase 1 ( DNMT1) protein expressioncorrelates significantly with poorer tumor differentiation and frequent DNA hypermethylation of multipleCpG islands in gastric cancers [J] .Am J Pathol, 2004, 164(2) : 689-699.
[17] Solomon D, Davey D, Kurman R, et al. The 2001 Bethesda System: terminology for reporting results ofcervical cytology [J]. JAMA, 2002; 287: 2114-2119
[18] Reddy AN, Jiang WW, Kim M, et al. Death-associated protein kinase promoter hyper-methylation innormal human lymphocytes[J]. Cancer Res, 2003, 63(22): 7694-7698.
[19] Widschw endter A, Muller H M, Fiegl H, et al. DNA methylation in serum and tumors of cervical cancerpatients[J]. Clin Cancer Res, 2004, 10(2): 565-571.
[20] Yam aguchi S, Asao T, Nakamura J, et al. High frequency of DAP- kinase gene promoter methylation incolorectal cancer specimens and its identification in serum [J].Cancer Lett, 2003, 194(1): 99-105.
[21] Toyooka S, Toyooka K O, Miyajima K, et al. Epigenetic down-regulation of death-associated proteinkinase in lung cancers [J]. Clin Cancer Res, 2003, 9(8): 3034-3041.
[22]赵先兰,孟志英等.宫颈癌组织中DAPK基因启动子甲基化的研究[J].癌症, 2008,27(9):919-923
[23] Yang Liu, Weimin Gao, Jill M Siegfried, et al. Promoter methylation of RASSF1A and DAPK andmutations of K-ras, p53, and EGFR in lung tumors from smokers and never-smokers [J]. BMC Cancer2007, 3(7):74-82.
[24]顾婷婷,张明忠,郑鹏生. DNA甲基化研究方法的回顾与评价[J].中国妇幼健康研究, 2006, 17(6):555-560.
[25] Mittag F, Kuester D, Vreth M, et al. DAPK promoter methylation is an early eventin colorectalcarcinogenesis [J].Cancer Lett, 2006, 240 (l): 69-75.
[26]刘晓碗，玛依努尔尼牙孜，阿布力孜阿布杜拉等．新疆维吾尔族妇女宫颈病变中DAPK表达的研究[J].中国肿瘤,2010,19（3）: 203-206.
[27] Widau R C, Jin Y, Dixon S A, et al. Protein phosphatase 2A (PP2A) holoenzymes regulatedeath-associated protein kinase (DAPK) in ceramide-induced anoikis [J]. J. Biol. Chem. 2010, 2(85),13827-13838.
[28] Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer[J]. Nat Rev Genet,2002, 3:415–428.
[29]张勇敢,刘保池. DNA甲基转移酶的作用及甲基化在肿瘤发生中的作用[J].国际外科学杂志. 2008,35(8): 537-540.
[30] Eden A, Gaudet F, Waghmare A, Jaenisch R. Chromosomal instability and tumors promoted by DNAhypomethylation[J]. Science. 2003, 300: 455.
[31] Karpf AR, Matsui S (2005) Genetic disruption of cytosine DNA methyltransferase enzymes induceschromosomal instability in human cancer cells[J]. Cancer Res. 2005, 65: 8635–8639.
[32]齐翀,李建芳,瞿颖,等.胃癌及癌旁组织多个肿瘤相关基因超甲基化[J].上海交通大学学报(医学版), 2007, 27(5): 555-558.
[33] Nakagawa T, Kanai Y, Ushijima S, et al. DNA hypermethylation on multiple CpG islands associated withincreased DNA methyltransferase DNMT1 protein expression during multistage urothelialcarcinogenesis[J]. J Urol 2005, 4(173): 1767-71.
[34] .Lin, Y., Hupp, T. R. and Stevens, C. Death-associated protein kinase (DAPK) and signal transduction:additional roles beyond cell death[J]. FEBS J. 2010,277, 48-57.
[35] Bovellan, M., Fritzsche, M., Stevens, C. and Charras, G.. Death-associated protein kinase (DAPK) andsignal transduction: blebbing in programmed cell death[J]. FEBS J. 2010,277, 58-65.
[36] Kimchi, A. A cell death-promoting kinase[J]. Nat. Struct. Biol.2001, 8, 824-826.
[37] Gao WM, Mady HH, Yu GY, et al. Comparison of p53 mutations between adenocarcinoma and squamouscell carcinoma of the lung: unique spectra involving G to A transitions and G to T transversions in bothhistologic types[J]. Lung Cancer 2003, 40:141-150.
[38] Zhang W, Stabile LP, Keohavong P, et al. Mutation and polymorphism in the EGFR-TK domainassociated with lung cancer[J]. J Thorac Oncol 2006, 1:635-647.
[39] Liu Xiaofang, Tang Kun, Yu Shaoping, et al. Correlation between promoter methylation ofp14ARF,TMS1/ASC, and DAPK, and p53 mutationwith prognosis in cholangiocarcinoma[J]. World Journal ofSurgical Oncology, 2012, 1(10): 5-13.
[40] Zbigniew Jab onowski, Edyta Reszka, Jolanta Gromadzińska et al. Hypermethylation of p16 and DAPKpromoter generegions in patients with non-invasive urinary bladdercancer [J]. Arch Med Sci, 2011,7,(3):512-516
[41] Devrim Gozuacik, Adi Kimchi. DAPk Protein Family and Cancer [J]. Spotlight on Cancer, 2006, 2(2):74-79.
[42] Esteller M. Relevance of DNA methylation in the managementof cancer[J] . Lancet Oncol, 2003, 4 ( 6) :351-358.
[1] Esteller M. DNA methylation and cancer therapy: new developments and expectations[J]. Curr OpinOncol,2005, 17(l):55-60.
[2] Das PM, Singal R. DNA methylation and caneer[J]. Clin Oncol, 2004, 22(22):4632-4642.
[3] Davis CD, Uthus EO. DNA methylation，caneer susceptibility，and nutrient interactions. Exp BiolMed(Maywood), 2004, 229(10):988-995
[4] Jones PA,Takai D. The role of DNA methylation in mammalian epigenetics [J]. Science, 2001, 293 ( 5532):1068- 1070.
[5] Issa JP. Aging, DNA methylation and cancer[J]. CritRev Oncol Hematol, 1999, 32(1): 31- 43.
[6] Jones PA, Laird PW. Cancer epigenetics comes of age[J]. Nat Genet, 1999, 21(2) : 163- 167
[7] Singal R, Ginder GD. DNA methylation [J]. Blood,1999, 93(12): 4059- 4070.
[8] Bestor, T.H., The DNA methyltransferases of mamrnals. Hum Mol Genet, 2000. 9(l6): P. 2395-402.
[9] Rhee, I., etal., DNMT1 and DNMT3b cooperate to silence genes in human cancer cells. Nature, 2002.416(6880): p. 552-6.
[10] Ahluwalia, A., etal., DNA methylation in ovarian caneer.Ⅱ. Expression of DNA methyltransferasesin ovarian cancer cell lines and normal ovarian epithelial cells. Gynecol Oneol, 2001. 82(2): P. 299-304.
[11] Robertson, K.D., DNA methylation, methyltransferases, and caneer. Oncogene, 2001. 20(24): P. 3139-55.
[12] Wu, Y., etal., Aberrant expression of deoxyribonucleic acid methyltransferases DNMT1, DNMT3a, andDNMT3b in women with endometriosis. Fertil Steril, 2007. 87(l): P. 24-32.
[13] Sawada, M., etal., Inereased expression of DNA methyltransferase l (DNMT1) Protein in uterine cervixsquamous cell carcinoma and its precursor lesion. Cancer Lett, 2007. 251(2): p. 211-9.
[14]石小燕，DNA甲基转移酶1在子宫颈癌组织和细胞中的表达及意义.解放军医学杂志，2008. 42(12):p. 834-9.
[15]赵先兰，宫颈癌组织中DNA甲基转移酶lmRNA的表达.郑州大学学报(医学版)，2009. 251(2): p.211-9.
[16] Szyf, M., The role of DNA methyltransferase 1 in growth contro1. Front Biosci, 2001. 6: P. D599-609
[17] Harder J，Opitz OG, Brabender J，etal. Quantitative promoter methylation analysis of hepatocellularcarcinoma，cirrhotic and normal liver. Int J Cancer, 2008, 122(12): 2800-2804.
[18] Imai T, Toyota M, Suzuki H, etal. Epigenetic inaetivation of RASSF2 in oral squamous cell carcinoma.Caneer Sci, 2008, 99(5): 958-966.
[19] Lee JH, Byun DS, Lee MG, etal. Frequent epigenetic inactivation of hSRBC in gastric cancer and itsimplication in attenuated P53 response to stresses. Int J Caneer, 2008, 122(7): 1573-1584，
[20] Kitkumthom N, Yanatatsanajit P, KiatPonqsans S, etal. Cyelin A1 promote hypermethylation in humanpapillomavirus-associated cervical cancer. BMCaneer, 2006, 6(l): 55-59.
[21] Dowdy SC, Gostout BS, Shridhar V, etal. Biallelic methylation and silencing of paternally expressed gene3(PEG3) in gyneeologic cancer cell lines. Gyneeo Oneol, 2005, 99(l): 126-134.
[22] Sova P, Feng Q, Geiss G, etal. Discovery of novel methylation biomarkers in cervical carcinoma by globaldemethylation and microarray analysis. Caneer Epidemiol Biomarkers Prev, 2006, 15(l): 114-123.
[23] Yang B, House MG, Guo M, etal. Promoter methylation profiles of tumor suppressor genes in intrahepaticand extrahepatic cholangoearcinoma.Mod Pathol, 2005, 18(3): 412-420.
[24]康新江. DNA甲基化与肿瘤.内江科技，2006，3: 105一108.
[25] Adorian P, Distler J, Lipscher E, etal. Tumor class predietion and discovery by microarray-based DANmethylation analysis. Nucleic Acids Res, 2002, 30(5):e21.
[26] Feng Q, Balasubramanian A, Hawes SE, etal. Deteetion of hypermethylated genes in women with andwithout cervical neoplasia. J Natl Caneer Inst , 2005, 97(4): 273-282.
[27] Maruyama R, Sugio K, Yoshino I, etal. Hypermethylation of FHIT as a prognostic marker in nonsmall celllung carcinoma. Caneer, 2004, 100(7): 1472-1477.
[28] Graziano F, Arduini F, RuzzoA, etal. Prognostic analysis of E-eadherin gene promoter hypermethylationin patients with surgically resected, nod-Positive, diffuse gastric cancer. Clin Caneer Res, 2004, 10(8):2781-2789.
[29] Widsehwendter A, Muller HM, Hubalek MM, etal. Methylation status and expression of humantelomerase reverse transeriptase in ovarian and cervical cancer. Gynceol Oneol 200493(2): 407-416.