【作者】 沈文静；

【导师】 戴冬秋；

【作者基本信息】 中国医科大学 ， 肿瘤学， 2007， 博士

【摘要】 上皮性卵巢癌的发生是表遗传学与遗传学机制共同参与的涉及多基因改变、通过多阶段变异累积形成的病理过程。目前，在其发生机制中，DNA启动子区CpG岛异常甲基化是一个研究热点。表遗传学是研究没有DNA序列变化、可遗传的基因表达的改变，表遗传学的分子机制包括DNA甲基化、组蛋白修饰、染色质改型和RNA干涉等，它们在基因转录调控过程中起重要作用。作为表遗传学机制的重要内容，DNA甲基化是基因突变及缺失之外肿瘤抑制基因失活的第三种机制，DNA甲基化异常在多种肿瘤的发生中起重要作用。已有研究表明，肿瘤中异常甲基化的基因包括参与细胞周期调控、细胞分化、凋亡调控及肿瘤转移和血管生成相关基因等。DNA甲基化和组蛋白乙酰化是表遗传学调控基因表达的两种主要方式，DNA低甲基化和组蛋白乙酰化可促进基因表达，DNA高甲基化和组蛋白去乙酰化可抑制基因表达，DNA甲基化和组蛋白乙酰化相互影响。卵巢恶性肿瘤是女性生殖器三大恶性肿瘤之一，上皮性卵巢癌占卵巢恶性肿瘤85％-90％，DNA甲基化在上皮性卵巢癌的发生、发展中起重要作用。若干肿瘤抑制基因启动子区异常甲基化与上皮性卵巢癌的形成密切相关，研究表明，DNA甲基化酶抑制剂及组蛋白脱乙酰基酶抑制剂可能用于恶性肿瘤的临床治疗，包括上皮性卵巢癌在内。对肿瘤抑制基因启动子区异常甲基化的深入研究不仅有助于揭示上皮性卵巢癌的发生机制，而且可能促进上皮性卵巢癌临床诊断和治疗的发展。本研究旨在探讨上皮性卵巢癌与表遗传学的关系，选择与细胞凋亡、细胞周期调控、DNA修复及化疗耐药相关的基因RASSF1A、BRCA1、p16、hMLH1及MGMT，研究的第一部分首先通过检测基因启动子区CpG岛甲基化状态及其蛋白表达，寻找上皮性卵巢癌相关的异常甲基化基因，进一步分析基因蛋白表达及与启动子区甲基化的相关性，及与临床病理学特征的关系。研究的第二部分从与遗传学发生机制相结合的角度，分析上皮性卵巢癌组织中MSI状态，以及上述抑癌基因启动子甲基化及蛋白表达与MSI之间的相关性，探讨MMR基因hMLHl启动子甲基化与MSI的相关性以及在上皮性卵巢癌发生及发展中的作用。研究的第三部分以前两部分的实验结果为基础，选择甲基化酶抑制剂5-Aza-CdR及脱乙酰基酶抑制剂NaB两种药物，在观察卵巢癌细胞系生长抑制的同时，分析五个抑癌基因启动子区CpG岛甲基化状态、mRNA及蛋白表达的改变，探讨药物调控的意义及潜在的临床应用价值。材料与方法一、实验材料中国医科大学附属第一医院妇科、辽宁省肿瘤医院妇科1999-2006年手术治疗63例原发散发性上皮性卵巢癌组织、41例相应的盆腹腔转移灶、10例癌旁卵巢组织及20例正常卵巢组织；上皮性卵巢癌细胞株：CAOV3及OVCaR3(中国医科大学细胞生物教研室)，HO-8910(上海肿瘤细胞研究所)，A2780(天津血液病学研究所)：RNA提取试剂TRIZOL Reagent(GIBCO BRL公司)，甲基化酶抑制剂5-aza-2’-deoxycitydine(5-Aza-CdR)及脱乙酰基酶抑制剂Sodium Butyrate(NaB)(Sigma)，RT-PCR试剂盒(TaKaRa)，Wizard DNA Clean-up(Promega公司)，Annexin-v-FITC凋亡检测试剂盒(北京宝赛生物技术有限公司)，RASSF1A羊抗人多克隆抗体、BRCAl兔抗人多克隆抗体、P16鼠抗人单克隆抗体(Santa cruz)，hMLHl兔抗人单克隆抗体及MGMT鼠抗人单克隆抗体(Neomarkers)。二、实验方法1 MSP法检测基因启动子区的甲基化状态2应用MTT法检测上皮性卵巢癌细胞株的增殖活性3流式细胞术检测细胞周期，Annexin V-FITC染色法检测细胞凋亡率4 RT-PCR法及Western-blot法检测基因mRNA及蛋白表达。5 PCR-聚丙烯酰胺凝胶电泳-硝酸银染色法检测卵巢组织MSI状态。三、统计分析采用SPSS11.5统计软件进行分析，率的比较采用X~2检验和Fisher精确概率法；采用Spearman等级相关系数分析相关性；均数统计分析方法为方差分析，两两比较采用LSD法。结果第一部分上皮性卵巢癌多肿瘤抑制基因启动子区异常甲基化及蛋白表达的研究1上皮性卵巢癌组织原发灶及盆腹腔转移灶中RASSF1A、BRCA1、p16、hMLH1及MGMT基因启动子区甲基化发生率在原发灶及转移灶中分别为49.2％、25.4％、20.6％、15.9％、11.1％及58.5％、26.8％、22.0％、12.2％、9.8％，在RASSF1A、BRCA1及p16中显著高于正常卵巢组织(P＜0.05)。2上皮性卵巢癌组织原发灶及盆腹腔转移灶RASSF1A、BRCA1、p16、hMLH1及MGMT蛋白表达率分别为31.7％、28.6％、23.8％、27.0％、44.4％及29.3％、31.7％、22.0％、24.4％、48.8％，显著低于正常卵巢组织及癌旁卵巢组织(P＜0.05)，蛋白表达与其启动子区甲基化之间存在相关性，表明基因启动子区CpG岛异常甲基化是其蛋白失表达的机制之一。3 RASSF1A基因启动子区甲基化发生与上皮性卵巢癌临床分期和细胞分化程度相关，多发生在临床Ⅲ、Ⅳ期和低分化者(P＜0.01)。4上皮性卵巢癌中存在CpG岛甲基化表型，与CIMP阴性癌相比，在不同临床期别、分化程度及病理类型间差异无统计学意义(P＞0.05)。5上皮性卵巢癌中可能有2种不同甲基化类型肿瘤，一组易发生RASSF1A、hMLH1甲基化；另一组易发生BRCA1、p16，可能还包括MGMT的甲基化。第二部分上皮性卵巢癌微卫星不稳定性与肿瘤抑制基因异常甲基化的研究1上皮性卵巢癌组织存在MSI，MSI-H发生率在原发灶及盆腹腔转移灶中分别为22.2％及19.5％，两者之间无统计学差异(P＞0.05)。2上皮性卵巢癌原发灶中MSI-H发生率在粘液性癌(38.9％)显著高于浆液性癌(8.8％)(P＜0.05)，在非浆液性癌组(37.9％) (粘液性癌、内膜样癌及透明细胞癌)显著高于浆液性癌组(P＜0.01)。3上皮性卵巢癌中hMLH1基因启动子区异常甲基化导致的蛋白失表达与MSI-H密切相关(P＜0.01)。第三部分联合应用5-Aza-CdR及NaB调控卵巢癌细胞TSG去甲基化及表达的实验研究1 5-Aza-CdR及NaB抑制四株上皮性卵巢癌细胞增殖，联合用药抑制作用增强。2 5-Aza-CdR及NaB处理后四株上皮性卵巢癌细胞呈现G1期阻滞，联合用药阻滞作用增强。3 5-Aza-CdR及NaB诱导四株上皮性卵巢癌细胞凋亡率显著增加，联合用药细胞凋亡率显著高于单独用药。4 5-Aza-CdR使四株上皮性卵巢癌细胞中异常甲基化基因呈去甲基化状态，使其中部分基因，包括CAOV3中p16基因，OVCAR3中RASSF1A和MGMT基因，A2780中BRCA1基因，以及HO-8910中p16和MGMT基因去甲基化伴随mRNA表达及蛋白表达上调。5 NaB使OVCAR3中RASSF1A基因、CAOV3及HO-8910中p16基因甲基化减弱，伴随mRNA表达及蛋白表达部分上调。6 5-Aza-CdR与NaB联合用药使上述异常甲基化基因呈完全去甲基化状态，诱导A2780中hMLH1 mRNA及蛋白重新表达，使OVCAR3中RASSF1A基因、HO-8910及CAOV3中p16基因mRNA及蛋白表达较单独用药上调。结论1上皮性卵巢癌组织中RASSF1A、BRCA1、p16、hMLH1及MGMT基因启动子区异常甲基化与其发生发展相关。2上皮性卵巢癌组织存在MS1，MSI-H与非浆液性癌类型，尤其是粘液性上皮性卵巢癌发生发展密切相关。上皮性卵巢癌中MSI-H机制部分在于hMLH1基因启动子区异常甲基化。3 5-Aza-CdR能逆转RASSF1A、BRCA1、p16、hMLH1及MGMT基因异常甲基化状态，调控其中部分基因的表达。4 NaB与5-Aza-CdR对上述基因去甲基化及表达调控具有协同作用。5 5-Aza-CdR与NaB具有协同抑制上皮性卵巢癌细胞增殖的作用，可能与阻滞细胞周期、诱导凋亡及上调RASSF1A、p16及BRCAl基因的表达有关。更多还原

【Abstract】 Research on Abnormal Methylation of Several Tumor Suppressor Genes in Epithelial Ovarian CarcinomaObjective and significanceCarcinogenesis of epithelial ovarian carcinoma is a stepwise process of accumulation of epigenetic and genetic abnormalities that can lead to cellular dysfunction. Study on abnormal hypermethylation of DNA promoter region is a focus in this area. Epigenetics is defined as modifications of the genome, heritable during cell division, that do not involve a change in the DNA sequence. Epigenetics includes DNA methylation、histone modification、chromatin remodeling and RNA interference, which play important roles in gene expressing regulation. As a main part of epigenetics, DNA hypermethylation is the third way of tumor suppressor gene (TSG) silencing besides genetic gene allelic loss and somatic mutation, which has been associated closely with a wide variety of human cancers. Increasing evidence suggests that many kinds of genes may be hypermethylated in human malignant tumors, including those participate cell cycle control、cell differentiation, apoptosis, metastasis and angiogenesis. DNA methylation and histone acetylation are the two most widely studied epigenetic changes which regulate gene expression. DNA hypomethylation and hyperacetylation can up-regulate gene expression, and DNA hypermethylation and hypoacetylation can down-regulate gene expression. DNA methylation and histone acetylation interplay closely.Ovarian carcinoma is one of the most common gynecologic cancers, and abnormal DNA methylation plays important roles in the course of carcinogenesis and development in epithelial ovarian cancer. Promoter hypermethylation of several TSGs plays crucial roles in carcinogenesis of epithelial ovarian cancer. As suggested, DNA demethlylated agents and histone deacetylase inhibitors can be used in treatment of malignant tumors, including epithelial ovarian carcinoma. Deeply research on hypermethylation of TSGs will not only contribute to further understanding on mechanisms of tumorigenesis, but also promote clinical diagnosis and treatment in epithelial ovarian carcinoma.Materials and methods1 Materials63 surgical resected specimens of sporadic primary epithelial ovarian carcinoma、41 metastatic tissues of pelvic and abdomen cavity、10 adjacent non-cancerous ovarian tissues and 20 normal ovarian tissues were gathered from both Gynecologic Department of the First Affiliated Hospital of China Medical University and Tumor Specific Hospital of Liao Ning Province. Epithelial ovarian cancer lines CAOV3 and OVCaR3 were provided by Cell Biologic Department of China Medical University, HO-8910 by Tumor Research Institute of Shanghai, A2780 by Haematologic Disease Research Institute of Tianjin; Trizol Reagent by GIBCOL BRL company, demethylating agent 5-aza-2’-deoxycitydine(5-Aza-CdR) and histone deacetylase inhibitors(HDACIs) sodium butyrate(NaB) were the product of Sigma; RT-PCR kit was the product of TaKaRa Company; Wizard DNA Clean-up was bought from Promega company, RPMI 1640 culture medium was the product of Hyclone company, RASSF1A goat polyclone、BRCA1 polyclone and P16 mouse monoclone antibody from Santa cruz, hMLH1 rabbit and MGMT mouse monoclone antibody from Lab Vision Neomarkers.2 MethodsMethylation-specific PCP (MSP) was used to detect promoter methylation state. MTT assay, flow cytometry, and Annexin V-FITC staining were performed to analyze the cell proliferation activity, cell cycle, and apoptotic rate. RT-PCR and Western blot were used to detect expression of TSG at mRNA and protein level. Microsatellite instability was detected by PCR- polyacrylamide gel-silver stain method.ResultsSectionⅠPromoter hypermethylation and protein expression of several tumor suppressor genes in epithelial ovarian carcinoma1 Promoter hypermethylation of RASSF1A、BRCA1、p16、hMLH1 and MGMT was detected in epithelial ovarian cancer tissues and metastatic sites, the frequency was 49.2%、25.4%、20.6%、15.9% and 11.1% in ovarian cancer tissues and 58.5%、26.8%、22.0%、12.2% and 9.8% in metastatic sites respectively, which was significantly higher than that in normal ovarian tissues in RASSF1A, BRCA1 and p16 (P＜0.05).2 The frequency of protein expression ofRASSF1A、BRCA1、p16、hMLH1 and MGMT was 31.7%、28.6%、23.8%、27.0% and 44.4% in epithelial ovarian cancer tissues and 29.3%、31.7%、22.0%、24.4% and 48.8% in metastatic sites, which was significantly lower than that in normal ovarian tissues and adjacent non-cancerous ovarian tissues(P＜0.05). The protein expression was related with methylation state of promoter region, which suggested that promoter hypermethylation was one of the reasons that TSG lost expression.3 The frequency of promoter hypermethylation of RASSF1A was significantly lower in epithelial ovarian cancers of stageⅠandⅡthan that in stageⅢandⅣ(P＜0.01), and so was that in both well and moderately differentiated cancers than that in poorly differentiated ones(P＜0.01).4 CIMP was detected in epithelial ovarian carcinoma, and no statistical difference was found between different tumor stages, histological types and differentiated grades when compared with CIMP-negative cancers(P＞0.05). 5 There may exists two different kinds of epithelial ovarian carcinoma, one characterized with hypermethylation in RASSF1A and hMLH1, the other with BRCA1 and p16, maybe including MGMT hypermethylation.SectionⅡMSI and promoter hypermethylation of TSGs in epithelial ovarian carcinoma1 MSI was detected in epithelial ovarian carcinoma, and the frequency of MSI-H was 22.2% and 19.5% in ovarian cancer tissues and metastatic sites, and no statistical difference was found between them(P＞0.05).2 The frequency of MSI-H was significantly higher in mucinous type (38.9%) than that in serous (8.8%) epithelial ovarian cancer(P＜0.05), and so is that in non-serous type(37.9%) (including mucinous, endometrium and clear cell type) than that in serous one (P＜0.01).3 The methylation state of promoter region of hMLH1 and accordant lost expression at protein level correlate with MSI-H(P＜0.01).SectionⅢStudy on regulating effect of synergy of 5-Aza-CdR and NaB on methylation state and expression of TSGs in epithelial ovarian cancer cell lines1 5-Aza-CdR or NaB displayed a growth inhibitory effect on epithelial ovarian cancer cell line CAOV3、OVCaR3、HO-8910 and A2780, and the effect was enhanced when used combinantly(P＜0.05).2 Cell cycle was blocked at G1 phrase after four epithelial ovarian cancer cell lines were exposed to 5-Aza-CdR or NaB, and the effect was enhanced when used combinantly(P＜0.05). 3 The apoptosis rate increased significantly after four epithelial ovarian cancer cell lines were exposed to 5-Aza-CdR or NaB, and the effect was enhanced when used combinantly(P＜0.05).4 After treated with 5-Aza-CdR, TSGs in four epithelial ovarian cancer cell lines displayed different degree of demethylation effect, accompanied by up-regulation of part of TSGs, including p16 in CAOV3、RASSF1A and MGMT in OVCAR3、BRCA1 in A2780, as well as p16 and MGMT in HO-8910 at mRNA and protein level.5 The methylation sate decreased after treated with NaB in RASSF1A in OVCAR3, p16 in CAOV3 and HO-8910, accompanied by up-regulation at mRNA and protein level.6 The methylated TSGs displayed complete demethylation state after treated with 5-Aza-CdR and NaB together, which induced re-exprssion of hMLH1 in A2780 at both mRNA and protein level, up-regulation of RASSF1A in OVCAR3, p16 in HO-8910 and CAOV3.Conclusions1 Promoter hypermethylation of RASSF1A、BRCA1、p16、hMLH1 and MGMT correlates with tumorigenesis and development of epithelial ovarian carcinoma.2 MSI exists in epithelial ovarian carcinoma, and MSI-H correlates with tumorigenesis and development of non-serous type epithelial ovarian carcinoma, especially mucinous type. The mechanism of MSI-H in epithelial ovarian carcinoma lies partly in promoter hypermethylation of hMLH1 gene.3 5-Aza-CdR can reverse the abnormal hypermethylation state of RASSF1A、BRCA1、p16、hMLH1 and MGMT in human epithelial ovarian cancer cell lines and regulate the expression of some of the TSGs. 4 NaB coordinate with 5-Aza-CdR in inducing demethylation and regulating gene expression.5 5-Aza-CdR and NaB can inhibit the growth of epithelial ovarian cancer cells synergistically, which may be due to blocking cell cycle、inducing apoptosis and up-regulation of RASSF1A、p16 and BRCA1.更多还原