【作者】 梁军；

【导师】 康山； 李琰；

【作者基本信息】 河北医科大学 ， 妇产科学， 2008， 硕士

【摘要】 目的:E-钙粘蛋白(E-cadherin,CDH1)是钙粘蛋白家族中最重要也是研究最深入的一种钙粘蛋白,不仅与肿瘤侵袭、转移有关,也与肿瘤的发生有关。其基因启动子区及非编码区存在的多态性位点可通过调控基因转录活性或蛋白表达水平而影响肿瘤的发生和发展。本研究旨在探讨CDH1基因启动子区-160C/A、-347G/GA和3’非编码区(untranslated region,UTR)+54C/T单核苷酸多态性(single nucleotide polymorphism,SNP)与上皮性卵巢癌(epithelial ovarian carcinoma)发病风险的关系。方法:采用病例-对照研究方法,采集207例上皮性卵巢癌患者和256例健康对照个体的静脉抗凝血5ml,同时记录其病史、个人史、肿瘤家族史。以蛋白酶K消化-饱和氯化钠盐析法提取外周血白细胞DNA,采用聚合酶链反应-限制性片段长度多态性(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)方法检测病例-对照组人群CDH1基因启动子区-160C/A、-347G/GA和3′UTR +54C/T三个多态位点的基因型频率分布;采用免疫组织化学SP法检测54例携带CDH1 3′UTR +54C/T不同基因型上皮性卵巢癌组织E-cadherin表达情况。数据统计分析采用SPSS11.5版软件包(SPSS Company, Chicago,Illinois,USA)进行,以P<0.05认为有统计学意义。比较基因型频率观察值与预期值并用卡方检验进行Hardy-Weinberg平衡分析。CDH1基因型及等位基因型在病例组和对照组中的分布比较以行×列表的卡方检验进行。单体型频率及连锁不平衡分析采用EH软件和2LD软件(1.2 version,Rockefeller University,New York)。以非条件Logistic回归法计算经年龄、性别校正的相对风险度的比值比(odds ratio,OR)及其95%可信区间(confidence interval,CI)。CDH1 3′UTR +54C/T位点不同基因型和不同病理类型E-cadherin表达情况的比较应用秩和检验进行。不同临床分期E-cadherin表达情况的比较采用行×列表的卡方检验进行。结果:对照组CDH1 3个SNP的基因型频率分布均符合Hardy-Weinberg平衡。1 CDH1 -160C/A多态位点的C/C、C/A和A/A基因型频率在上皮性卵巢癌患者与对照妇女组间无显著性差异(χ2=0.379,P=0.827);C和A等位基因频率在患者组与健康对照组间亦无显著性差异(χ2=0.229, P=0.632)。与C/C基因型相比,携带A等位基因的基因型(C/A+A/A)不改变上皮性卵巢癌的发病风险,OR值为1.076(95%CI=0.729~1.588)。2 CDH1 -347G/GA多态的G/G、G/GA和GA/GA基因型频率分布在患者与对照组间无显著性差异(χ2=1.548,P=0.461);G和GA等位基因频率在两组间亦无显著性差异(χ2=1.407,P=0.236)。与G/G基因型相比,携带GA等位基因的基因型(G/GA+GA/GA)与上皮性卵巢癌的发病风险无关, OR值为1.207(95%CI=0.835~1.745)。3 CDH1 3′UTR +54C/T多态位点的C/C、C/T和T/T基因型频率分布在患者与健康对照之间有显著性差异(χ2=10.854,P=0.004);C和T等位基因频率在患者组与对照妇女间有显著性差异(χ2=7.220,P=0.007)。与C/C基因型相比,携带T等位基因的基因型(C/T+T/T)能显著增加上皮性卵巢癌的发病风险, OR值为1.846(95%CI=1.267~2.690)。4应用EH和2ld软件对CDH1 -160C/A和-347G/GA SNP位点进行联合分析显示,两多态性位点间存在连锁不平衡现象(D′=0.999582,SD=0.0040)。四种单体型分布在上皮性卵巢癌患者与对照组间存在显著性差异(χ2=33.092,P=0.000)。与-160C/-347G单体型相比,携带-160A/-347GA单体型可显著增加上皮性卵巢癌的发病风险(OR=48.607 , 95%CI=2.912~806.221) ,相反携带-160C/-347GA单体型能降低该病发病风险(OR=0.661, 95%CI=0.455~0.961)。5 CDH1 3′UTR +54C/T C/C基因型E-cadherin表达明显低于携带T等位基因的基因型(C/T+T/T)(χ2=2.182,P=0.029)。6不同临床分期E-cadherin表达之间无显著性差异(χ2=4.828,P=0.185)。7 54例上皮性卵巢癌患者四种病理类型之间E-cadherin表达亦无显著性差异(χ2=2.125,P=0.547)。结论:1 CDH1启动子区-160C/A和-347G/G多态位点可能单独影响上皮性卵巢癌的发病风险无关。2 -160C/A和-347G/GA多态位点间存在紧密连锁关系,且-160A/-347GA和-160C/-347GA单体型可能与上皮性卵巢癌的发病风险相关。3 CDH1 3′UTR +54C/T多态与上皮性卵巢癌的发病风险相关,即C/C基因型可能成为上皮性卵巢癌发病的潜在危险因素。4携带C/C基因型个体的E-cadherin的表达明显高于其他基因型(C/T+T/T)。5上皮性卵巢癌患者不同临床分期以及不同病理类型间E-cadherin表达无显著性差异。更多还原

【Abstract】 Objective: E-cadherin (CDH1), as one of the cadherin members, relates with invasion and metastasis of various cancers also with development of tumor. Polymorphisms in the promoter and untranslated region of CDH1 gene have been associated with tumor development and progression, via modifying its transcriptional activity and protein expression level. This study was designed to investigate the association of single nucleotide polymorphisms on the CDH1 gene with the risk of epithelial ovarian carcinoma.Methods: This hospital-based case-control study included 207 cancer patients and 256 healthy controls, recording their case history, personal history and family medical history. Genomic DNA was extracted by using proteinase K digestion followed by a salting out procedure. CDH1 polymorphisms were analyzed by PCR-restriction fragment length polymorphism analysis (RFLP). Immunohistochemistry was used to measure the level of CDH1 in different genotypes of 3′UTR +54C/T.Statistical analysis was performed using SPSS11.5 software package (SPSS Company, Chicago, Illinois, USA). P<0.05 was considered significant for all statistical analyses. Hardy-Weinberg analysis was performed by comparing the observed and expected genotype frequencies in study groups using Chi-square test. Comparison of the CDH1 genotype, allelotype and haplotype distribution in cancer patients and healthy controls was performed by means of two-sided contingency tables using Chi-square test. The CDH1 haplotype frequencies and linkage disequilibrium coefficient were estimated by using EH linkage software and 2LD software. The odds ratio (OR) and 95% confidence Interval (CI) were calculated using an unconditional logistic regression model and adjusted by age and sex accordingly. Comparison of E-cadherin expression of different 3′UTR +54C/T genotype and different pathological category performed by means of rank test as well Comparison of E-cadherin expression of different 3′UTR+54C/T genotype was performed by means of two-sided contingency tables using Chi-square test.Results: The distribution of the CDH-1 -160 C/A, -347 G/GA, 3′-UTR C/T genotypes in the control group did not significantly deviate from that expected for a Hardy-Weinberg equilibrium. 1 There were no significant difference between patients and control women in genotype distribution of the CDH-1 -160 C/A (χ2=0.379, P=0.827). And there were also no significant difference in allelotype distribution of the CDH-1 -160 C/A between patients and control women (χ2=0.229, P=0.632). Compared with the C/C genotype, the carriers of‘A’ allele were not significantly modified the risk of developing endometriosis. The odds ratios were 1.076 (95%CI=0.729~1.588). 2 There were no significant difference between patients and control women in genotype distribution of the CDH-1 -347 G/GA (χ2=1.548, P=0.461). And there were also no significant difference in allelotype distribution of the CDH-1 -347G/GA between patients and control women (χ2=1.407, P=0.236). Compared with the G/G genotype, the carriers of‘GA’allele were not significantly modified the risk of developing endometriosis. The odds ratios were 1.207 (95%CI=0.835~1.745). 3 There were significant difference between patients and control women in genotype distribution of the CDH-1 3′UTR+54C/T (χ2=10.854, P=0.004). And there were also significant difference in allelotype distribution of the CDH-1 3′UTR+54C/T between patients and control women(χ2=7.220, P=0.007). Compared with the T/T+C/T genotype, the carriers of‘C’allele were not significantly modified the risk of developing endometriosis. The odds ratios were 1.846 (95%CI=1.267~2.690). 4 The results of the 2LD program analysis showed that CDH-1 -160C/A and -347G/GA polymorphism were link disequilibrium (D′=0.999582, SD=0.0040). There were significant difference distribution of the CDH1 haplotype difference between patients and control women (χ2=33.092, P=0.000). Compared with -160C/-347G haplotype, -160A/-347GA haplotype significantly increased susceptibility to epithelial ovarian carcinoma, with adjusted odds ratio of OR=48.607, 95%CI=2.912~806.221. But -160C/-347GA haplotype decreased susceptibility to epithelial ovarian carcinoma, with adjusted odds ratio of 0.661, 95%CI=0.455~0.961. -160A/-347G would not change onset risk of epithelial ovarian carcinoma, with adjusted odds ratio of 0.743, 95%CI=0.542~1.017. 5 In the tissue of patients with C/C genotype of CDH1 3′UTR+54C/T, the expression of CDH1 is significantly higher than that with the other genotypes (C/T+T/T) (χ2=2.182, P=0.029). 6 There were no significant difference between E-cadherin expression of different 3′UTR+54C/T genotype and different clinical stage (χ2=4.828, P=0.185). 7 There were no significant difference between E-cadherin expression of different 3′UTR+54C/T genotype and different pathological category(χ2=2.125, P=0.547).Conclusions:1 The CDH1 -160C/A and -347G/G polymorphism may not be associated with Susceptibility to epithelial ovarian carcinoma.2 The CDH1 -160C/A and -347G/GA SNP was imperfectly in linkage disequilibrium. The -160C allele tends to be linked to the -347G allele. The -160A/-347GA haplotype significantly increased the risk of developing epithelial ovarian carcinoma, compared with -160C/-347G haplotype.3 The CDH1 3′UTR +54C/T polymorphism may be associated with Susceptibility to epithelial ovarian carcinoma, and the C/C genotype of CDH1 3′UTR +54C/T might significantly influence onset risk of epithelial ovarian carcinoma. 4 There were significant difference between E-cadherin expression of the C/C genotype of CDH1 3′UTR +54C/T and the other genotypes (C/T+T/T).5 There were no significant difference between E-cadherin expression of different 3′UTR +54C/T genotype and different clinical stage and there were also no significant difference between E-cadherin expression of different 3′UTR+54C/T genotype and different pathological category.更多还原