食管鳞状细胞癌差异甲基化片段的筛选及相关基因的鉴定

【作者】 赵保健；

【导师】 马旭； 吴尔若； 贾孟春；

【作者基本信息】 中国协和医科大学 ， 遗传学， 2008， 博士

【摘要】 食管癌是世界上最常见的10种恶性肿瘤之一,全世界每年约有41万新发病例并且大多发生于发展中国家,同时每年约有33万人死于食管癌。我国是食管癌的高发地区,食管癌占据恶性肿瘤发病率的第6位,死亡率为第4位。每年因食管癌死亡者约15万人,占全部恶性肿瘤死亡的15%左右。食管癌死亡率高的原因在于很难早期诊断,而早期食管癌患者预后较好,经治疗后5年生存率可达到90%以上;晚期患者预后很差,5年生存率仪为10%左右。因此,进一步深入研究食管癌的发病机理,发现新的早期诊断肿瘤标志物和发展新的治疗措施对于食管癌的防治十分重要。食管癌的发生与多种因素有关,外界环境因素的长期刺激在食管癌的发生发展过程中具有重要地位。近年来对肿瘤表观遗传学如基因启动子区的甲基化的研究发现,肿瘤相关基因的甲基化常常是肿瘤发生过程中的一个早期事件,是对外界环境长期刺激发生的早期而稳定的变化,肿瘤特异的甲基化基因多为细胞发育和代谢过程中某些通路的重要基因,并且这些基因的表观沉默可以被某些药物逆转。与其他肿瘤的研究相比,对食管鳞状细胞癌相关基因甲基化的研究比较少,有很大的研究空间。囚此,寻找和研究食管鳞状细胞癌相关的甲基化基因,在阐明肿瘤的发生机制、预测肿瘤的发生发展和肿瘤的治疗方面具有重要的研究意义和应用前景。研究目的:寻找食管鳞状细胞癌相关的异常甲基化的基因,并对筛选到的基因在肿瘤发生过程中的作用机制进行研究,为进一步研究食管鳞状细胞癌的早期诊断和治疗奠定基础。研究方法:首先利用甲基化敏感随机扩增PCR(methylation-sensitive AP-PCR,MS AP-PCR)技术,对部分食管鳞状细胞癌肿瘤组织和配对的癌旁组织进行筛选差异甲基化片段,克隆测序后进行生物信息学分析。利用亚硫酸盐修饰后基因组测序、RT-PCR和实时荧光定量PCR技术对筛选到的有意义的片段在肿瘤组织和细胞系中进行研究,分析这些片段在肿瘤发生发展中的意义。结果:1.食管鳞状细胞癌差异甲基化片段的筛选应用MS AP-PCR技术对食管鳞状细胞癌肿瘤组织和配对的癌旁组织进行全基因组甲基化分析,获得了食管鳞状细胞癌的甲基化差异图谱。最终筛选到12个差异表达的高甲基化片段(hypermethylated DNA fragments,HMDF),7个片段符合CpG岛特征。在符合CpG岛特征的片段中有5个定位于已知基因5′区域。生物信息学分析表明这7个片段均含有许多转录因子结合位点。2.食管鳞状细胞痛中差异甲基化片断的进一步鉴定和分析2.1启动子区呈异常高甲基化的基因2.1.1 TPEF基因的异常甲基化对22例食管癌患者进行了基因组和表达水平的检测,发现22例患者中有12例患者的肿瘤中发生了TPEF基因启动子区的高甲基化(54.5%),并伴随着表达的下调。细胞系实验发现在加入去甲基化药物5′-AZA作用后,随着启动子区甲基化水平的降低,TPEF基因的表达有明显的表达回复。2.1.2 EDNRB基因的异常甲基化对21例食管癌患者进行了基因组和表达水平的检测,发现21例患者中有5例肿瘤组织中发生了EDNRB基因启动子区的高甲基化(23.8%),并伴随着表达的下调。细胞系实验发现在加入去甲基化药物5′-AZA作用后,EDNRB基因启动子区甲基化水平降低,并伴有EDNRB基因明显的表达回复。2.2.长片断甲基化区域在食管鳞状细胞癌中的初步研究发现高甲基化片段HMDF3(EDNRB)和HMDF12均定位于染色体13q22.3,中间区域长约1.5M,含有多个参与重要功能代谢的基因。细胞系实验表明,经去甲基化药物处理后一定程度上恢复了该区域内的大部分基因表达。结论:本论文应用MS AP-PCR的方法对食管鳞状细胞癌进行了全基因组甲基化分析,获得特异的DNA甲基化差异图谱,筛选到一些具有重要意义的片段。首次报道了TPEF基因与EDNRB基因的启动子区在食管鳞状细胞癌中呈异常高甲基化状态,从而影响了该基因的正常表达。此外首次对食管鳞状细胞癌中一个长片段区域的甲基化状态进行了研究。更多还原

【Abstract】 Esophageal carcinoma is one of the 10 most common malignant tumors in human, which accounts for 1.2 million new cases worldwide annually and most of them occurred in developmenting countries, meanwhile it claims about 330,000 lives worldwide each year. In China, esophageal cancer is the sixth most common cancer; meanwhile, it ranks as the fourth leading cause of cancer-related death, accounting for 150,000 deaths per year. The difficulty of early diagnosis contribute largely to the high mortality of esophageal cancer, for example, patients with early-stage esophageal cancer always have a good clinical outcome, whereas the overall 5-year survival is only about 10% in the late or advanced stage of ESCC. Therefore, further researches of screening new marker for early detection and developing new cancer treatment remain the best hope for a cure. Many factors were involved in the development of esophageal carcinoma, among them environment stimulus has been proved to be a high risk factor. During recent years, there has been a growing interest in the role of epigenetic changes in carcinogenesis, especially DNA cytosine methylation in gene promoter regions, which was the relatively early and stable reaction to long-term environment stimulus, therefore DNA methylation may be of great value in tumor early detection. Except that, as the methylated genes always play important factors in cell development and regulation and the epigenetic silencing can more or less be reversed by drug treatment, study of epigenetic modification shed light on new field in tumor therapy. Compared with numerous studies in other tumor types, it is being in the primary stage for ESCC epigenetic modification. Therefore, screening ESCC-related hypermethylated fragments and exploring their functions in tumorigenesis will significantly contribute to the further studies of tumor molecular mechanism, early detection measures and novel effective therapies.Objects: To screen novel tumor-related genes being aberrantly hypermethylated and study functions of those genes in the progression of ESCC, then go further to provide basement in the further research of early detection and effective therapeutic strategies in ESCC.Methods: Firstly, differentially methylated CpG islands were identified between several paired ESCC tumors and normal tissues by methylation-sensitive AP-PCR (MS AP-PCR) method, which were visualized as bands in the electrophoresis gels after silver staining, and then those fragments screened were isolated and subjected to bioinformatic analysis. Subsequently, the functions of those meaningful fragments were, in ESCC tumor tissues and cell lines, investigated with several methods including bisulfite-sequencing PCR(BSP), RT-PCR and Quantitative real time PCR (QRT-PCR), to explore their potential roles in the Carcinogenesis and Progression of ESCC.Result:(1) Identification of differentially methylated DNA fragments in ESCCDifferently methylated maps of ESCC were obtained by screening paired tissues with genome-wide methylation analysis method. Twelve hypermethylated DNA fragments (HMDF) were identified, and seven of them containing the GC-rich sequences conformed to the minimal criteria that define CpG islands. Within those seven fragments containing CpG islands, five of them were located in the 5’ region of known genes; meanwhile, all of them contain several transcript factor binding sites.(2) Further studies of meaningful fragments identified in ESCC2.1. Aberrant methylation of several corresponding genes in ESCC2.1.1 Aberrant promoter methylation of the TPEF geneMethylation status of TPEF gene promoter and expressions of TPEF transcript were detected in 22 ESCC samples, in which 12 (54.5%) of 22 ESCC tumors exhibited TPEF promoter hypermethylation accompanied by reduced expressions when compared with matched adjacent normal tissues. Studies on ESCC cell lines showed decreased methylation status of TPEF gene promoter and restored expressions of TPEF mRNA after treatment with the demethylating agent.2.1.2 Aberrant promoter methylation of the EDNRB geneMethylation status of EDNRB gene promoter and expressions of EDNRB transcripts were detected in 21 ESCC samples, in which 5 (23.8%) of 21 ESCC tumors exhibited EDNRB promoter hypermethylation and reduced expression occurred compared with matched adjacent normal tissues. Studies on ESCC cell lines showed decreased methylation status of EDNRB gene promoter and increased expression levels of EDNRB mRNA after treatment with the demethylating agent.2.2 The preliminary study of a large region being methylated in ESCCAn interesting result was that two HMDFs (HMDF 12 and HMDF3) are both located at chromosome 13q22.3, the regions between them spanned about 1.5Mb and contained several important genes. Studies on ESCC cell lines showed that the mRNA levels of almost all genes located at the region were increased in different degrees after treatment with the demethylating agent.Conclusions:In our study, ESCC special genome-wide different DNA methylation profiles and meaningful hypermethyled fragments were obtained by MS AP-PCR, and it was the first report about aberrant hypermethylation of TPEF and EDNRB genes in ESCC. Meanwhile, we firstly explored a large hypermethylated region in ESCC, genes located in which were epigenetic silenced and mRNA expressions could be restored by treatment with the demethylating agent.更多还原