【作者】 杨晓亚；

【导师】 白云；

【作者基本信息】 第三军医大学 ， 免疫学， 2008， 博士

【摘要】 食管癌是人类九种最常见的恶性肿瘤之一,全世界每年新增病例30万,其中70 %的病例发生在我国,并且其发病率仍呈上升趋势。食管癌是高致死性的疾病,术后5年生存率仅20 % ~ 30 %。因此,研究食管癌的发病机制,寻找新的诊断和治疗靶点,已经成为提高其预防和治疗效果的当务之急。肿瘤是一个多基因参与的与细胞周期紊乱和凋亡受阻密切相关的疾病。Survivin是凋亡抑制蛋白(IAP)家族的成员之一,不仅抑制凋亡,也参与细胞有丝分裂,在细胞凋亡和细胞周期的基因调控中发挥重要作用。正常情况下,survivin表达于胚胎和发育的胎儿组织细胞,不表达于终末分化的组织细胞。而在大多数人类肿瘤中,survivin在肿瘤细胞重新获得高表达,但在相应的癌旁组织中无表达。已有大量的研究证实,肿瘤细胞高表达survivin在肿瘤发生、发展过程中起着重要的作用,其表达水平同多种肿瘤的恶性程度、对放、化疗的抵抗性、以及肿瘤的不良预后呈正相关。因此,survivin已被用作新的肿瘤标志物,成为肿瘤诊断和治疗的研究热点。由于survivin在肿瘤组织中异常高表达的独特方式,探讨其表达调控机制,以用于肿瘤的治疗就成为人们关注的一个重要问题。研究表明,肿瘤组织中survivin的表达调控主要发生在转录水平。已有研究报道了多种转录因子,包括:Sp1、Sp3、Egr-1、Stat3和Rb/E2F、p53等参与不同细胞survivin mRNA的转录调控,survivin在肿瘤组织中的高表达有可能由这些转录因子的异常所引起。在参与到survivin转录调控的转录因子中,野生型p53对survivin表达的负调节作用近年来很受关注。Survivin启动子区含有p53转录因子的结合位点,野生型p53可直接结合survivin启动子进而抑制survivin启动子的活性,p53突变之后则失去了野生型p53的功能。在以往的研究中,尚未有涉及食管癌肿瘤组织中survivin表达与p53突变关系的报道,因此,进一步探讨食管癌组织survivin表达是否受野生型p53调节,对明确食管癌中survivin高表达的调节机制具有重要的价值。单核苷酸多态(single nucleotide polymorphism,SNP)被认为是一种稳定遗传的早期突变,与疾病有着稳定的相关性。研究表明在survivin的启动子和编码区域均发现有SNPs变异,这些SNPs位点可能与survivin的表达调控相关,从而与机体对食管癌的易感性相关联。在本研究中,我们利用公布的SNP数据(http://pga.gs.washington.edu、http://www.ncbi.nlm.nih.gov)结合文献检索与生物信息学分析选取了survivin基因启动子区五个多态性位点(-31G/C、-141G/C、-241T/C、-625G/C、-644T/C),首先了解这些多态位点在重庆地区正常人群中的频率分布,比较与其他公布的人种数据的差异。在此基础上,利用我们已经建立的食管癌临床资料和DNA样本数据库,采用病例对照的设计方法,进一步检测上述多态位点在食管癌人群中的基因型和等位基因频率,并从单位点分析、单倍型分析等方面,探讨survivin启动子多态性与食管癌患病风险之间的关联,分析survivin启动子区的多态位点与食管癌组织中survivin表达的相关性,筛查与食管癌survivin基因高表达相关联的SNP位点,为进一步研究分析其影响表达调控的分子机制奠定基础。DNA甲基化修饰是表观遗传修饰的主要方式,甲基化主要发生于基因启动子附近的CpG富集区域(又称CpG岛)。迄今为止,关于肿瘤癌基因异常激活和抑癌基因失活与该基因的启动子区域CpG岛甲基化的直接关系已有大量的报道。Survivn基因含有一个大的CpG岛,从启动子区一直延伸到第一外显子区域,提示其甲基化修饰可参与survivin基因的转录调控。本研究首先选择少量survivin mRNA表达状态有差异的食管癌及匹配的癌旁组织标本,检测survivin基因第一外显子区甲基化状态差异,以期对阐明食管癌组织survivin启动子甲基化状态是否参与其表达调节提供初步线索。综上所述,为了揭示survivin在食管癌肿瘤组织中异常表达的可能机制,综合目前针对survivin表达调控以及肿瘤组织中survivin异常表达机制的研究现状,本研究检测了食管癌中可能影响survivin表达的转录因子(p53突变),以及遗传学(启动子多态性)、表观遗传学(CpG岛甲基化)机制,通过初步研究,获得了以下结果:1.分析survivin mRNA表达与p53基因突变之间的关系。收集100例食管癌组织标本,利用RT-PCR检测了survivin mRNA的表达,总表达率为64 %;PCR-SSCP法检测了肿瘤组织中p53基因第5、6、7、8号外显子突变,总突变率为31 %。突变型p53组survivin mRNA阳性率为80.6 %,野生型p53组survivin mRNA阳性率只有56.5 %,两组survivin mRNA阳性率存在显著差异(p=0.020),突变型p53组survivin mRNA阳性率显著高于野生型p53组。2.选择survivin基因启动子区五个多态位点(-31G/C、-141G/C、-241T/C、-625G/C和-644T/C),利用PCR-RFLP或PCR-错配引物酶切的方法对重庆地区正常汉族人群进行基因分型。将所得数据与NCBI SNP数据库数据作比较,结果显示,-31G/C、-241T/C、-644T/C位点在重庆汉族人中的分布与NCBI中全球数据有显著差异。重庆汉族人-31G/C、-644T/C位点数据与NCBI亚洲人群的数据基本一致,与欧洲人和非洲人的数据分布相比差异显著。重庆汉族人-625G/C位点数据与亚洲人、非洲人群数据分布有显著差异。提示survivin启动子的多态性在不同种族间分布频率存在着差异。3.采用病例-对照的设计方法,分析了survivin启动子多态性及单倍型与食管癌患病风险之间的关系。结果显示:(1)-31G/C和-644T/C位点的等位基因型和基因型在对照组及肿瘤组之间的分布没有显著差异。而-625G/C位点与食管癌发病风险相关。Survivin启动子-625G/C多态位点的CC纯合基因型食管癌发病风险是另两种基因型的2.120倍(95%CI:1.247-3.603)。(2)以-31G/C、-625G/C和-644T/C三个位点为核心构建单倍型,单体型-644T--625C--31C在食管癌病人组中出现的概率是在对照组中出现概率的2.473倍(95%CI,1.532~3.994),拥有此单体型的病人患食管癌的风险较高。-644T--625G--31C为一个保护性的单体型(OR = 0.736),拥有此单体型的个体比较不容易患食管癌(95%CI,0.573~0.946)。单体型的分析结果与单个位点分析结果一致。4.分析了survivin启动子多态性与食管癌肿瘤组织survivin表达之间的关系,结果发现:-31G/C和-644T/C位点的等位基因型、基因型在survivin阳性表达组和survivin阴性表达组之间的分布并没有显著差异。而-625G/C位点等位基因型以及基因型分布在两组之间均有显著性差异(p值分别为0.024、0.033),提示-625C等位基因与肿瘤组织survivin的高表达相关。5.检测食管癌肿瘤组织中survivin基因甲基化状态。选取肿瘤组织有明显survivin表达而相应的癌旁组织检测不到survivin表达的4对肿瘤、癌旁组织标本作为研究对象,用亚硫酸盐修饰后测序的方法检测其survivin第一外显子的甲基化状态。初步结果显示, survivin阳性的肿瘤组织和survivin阴性的癌旁组织,其survivin基因第一外显子的甲基化状态相同,survivin第一外显子区所有的CpG位点都呈非甲基化状态。初步提示食管癌肿瘤组织中survivin的高表达可能并非由survivin基因甲基化失常而引起。由于本研究样本例数较少,阐明食管癌组织survivin启动子的甲基化状态是否参与其表达调节尚需更大样本的后续研究来进行。本研究的结果揭示了食管癌肿瘤组织中survivin异常高表达的部分机制,为食管癌发病分子机制的研究提供了线索,进而为食管癌的诊断提供预防和预后信息,以及给食管癌的治疗提供新的途径。更多还原

【Abstract】 Esophageal cancer is regarded as the ninth most common malignancy worldwide. Compared with other nations, there is higher incidence in China. Esophageal cancer is an extremely fatal disease. In spite of major advancement in cancer treatment, prognosis is still poor.Owing to recent advancements in tumor biology, apoptosis plays an important role in organ homeostasis, impairment of apoptosis facilitates the accumulation of gene mutations by prolonging the cell cycle span and promoting resistance to immune-based cytotoxicity, finally contributing to carcinogenesis. Survivin is an inhibitor of apoptosis protein, which blocks apoptosis induced by a variety of triggers. Survivin plays an important role in the development and progression of cancers. The expression of survivin is ubiquitous in fetal tissues, but is negligible in the majority of terminally differentiated adult tissues. In various cancers including esophageal cancer, survivin is significantly overexpressed. However, mechanism of survivin being re-expressed in cancers is still unclear and remains to be further studied.Many researches using RT-PCR showed a high level of survivin mRNA in cancer tissues compared with normal tissues, indicating that the survivin overexpression in cancers may arise predominately at the level of transcriptional regulation of the survivin gene. There are many transcription factors regulated survivin transcription in various cell types, including Sp1, Sp3, Egr-1, Stat3Rb, E2F and p53. Among these transcription factors, p53 is especially important in tumors. Like survivin, p53 is also a critical mediator of apoptosis and tumorigenesis. The p53 gene is mutated in various human cancers. In vitro transient transfection demonstrated that the expression of wild type p53 was associated with strong repression of the survivin promoter activity in various cell types, while mutant p53 wasn’t. Several clinical studies revealed that the p53 gene mutation might contribute to aberrant survivin expression in non-small cell lung cancers, breast cancers and laryngeal squamous cell carcinomas. While other clinical studies displayed different results. Kawasaki et al indicated that the survivin expression in colon cancer tissues had no relation with p53 mutation; Tanaka et al drawed the same conclusion in breast cancers. Whether the p53 gene mutation contributes to aberrant survivin expression in esophageal cancers or not is yet to be determined. In order to study the possible correlation between p53 and survivin in esophageal cancers, we compared survivin expression between the cancer tissues of wild type p53 and that of mutant p53.Single-nucleotide polymorphisms (SNP) are inheritable mutation and had steady correlation with various diseases. Several polymorphisms in survivin promoter have been identified previously. These SNPs might be correlated with the risk of tumors. The SNPs data of survivin gene in the NCBI (National Center for Biotechnology Information) SNP database are mainly submitted by European, American or Japanese, and the majority of SNPs have no information about the frequency distribution in different race. The SNP frequencies in various race are different, the SNPs’distribution in survivin gene region in Chinese Han nationality and the function of these SNPs have not been satisfactorily described yet. After searched the public SNP database (http://pga.gs.washington.edu, http://www.ncbi.nlm.nih.gov) and literatures, we identified five SNPs, -31G/C, -141G/C, -241T/C, -644T/C and -625G/C. In the present study, we compared these SNPs’distribution in Chinese Han nationality of Chongqing city with the frequency distribution in other race of people. Then we analyzed the relationship between the survivin promoter polymorphisms and the survivin expression of corresponding tumor tissues in order to detect whether the SNPs influence survivin expression or not. Moreover, we have compared the frequency of four survivin promoter polymorphisms in Chinese sporadic esophageal cancer patients and healthy controls in an attempt to identify a possible association between individual genetic variation and susceptibility to esophageal cancer.CpG methylation is thought to be critical for regulating transcription by the GC rich region around the transcription start site. Survivin has a large CpG island spanning from promoter to exon1 region, which increases the possibility that survivin overexpression in cancers may be the result of the dys-methylation status in neoplastic cells. However, Li and Altieri demonstrated that the CpG island in the survivin promoter was unmethylated in both normal and neoplastic tissues in breast, lung and colon cancers. In contrast, there were other researches reporting that demethylation of CpG sites in the survivin gene exon 1 was involved in the survivin expression in ovarian and oral cancers. These different data indicated that the methylation status of the human survivin promoter might depend on the different CpG sites reserchers detected. We are interested in the methylation status of survivin exon 1 region in esophageal cancer, so whether the CpG island in survivin gene is methylated in normal cells and unmethylated in esophageal cancer were studied in this research.In summary, our study is to investigate the mechanisms of survivin overexpression in esophageal cancer, the following data showed the contents and results of our study:1. Esophageal cancer with mutant p53 showed higher survivin gene expression. We measured survivin mRNA expression using RT-PCR in esophageal cancer and adjacent non-cancerous tissues of 100 patients. PCR-single-strand conformation polymorphism (SSCP) analysis was used to detect exons 5–8 mutations of the p53 gene in espohageal cancer tissues. To determine whether the loss of wild type p53 enhanced survivin gene expression in esophageal cancer tissue, the relationship between survivin mRNA expression and p53 gene mutation was analyzed. The ratio of survivin-positive tumors was significantly higher in tumors with mutant p53 than that in tumors with wild type p53 (P=0.020).2. Polymorphisms of survivin promoter were identified using the PCR-RFLP technique (-31G/C, -141G/C, -241T/C) or primer-introduced restriction analysis-PCR assay (-644T/C, -625G/C). We compared these SNPs’distribution in Chinese Han nationality of Chongqing city with the data in NCBI database about the frequency distribution in other race of people. We found significant differences in allele frequencies of -31G/C, -241T/C and -644T/C polymorphisms between Chinese Han nationality of Chongqing city and the NCBI data of global people. The allele frequencies of -31G/C and -644T/C polymorphisms in Chinese Han nationality of Chongqing city were equal to the NCBI’Asia data, but were different to the European and African data. There was significant difference in allele frequencies of -625G/C polymorphisms between Chinese Han nationality of Chongqing city and the NCBI data of Asia and African people.3. Polymorphisms of Survivin Promoter Are Associated with Risk of Esophageal Cancer. The frequencies of -644T/C and -31G/C polymorphisms were not significantly different between patients and controls. However, the significant differences in both allele and genotype frequencies between esophageal cancer patients and cancer-free controls were found in -625G/C polymorphism. Compared with the other genotype, the -625CC genotype was associated with significant elevated risk of esophageal cancer (OR=2.120, 95%CI: 1.247-3.603).4. We examined the combined effect of the survivin promoter polymorphisms, the haplotypes constructed of -644T/C, -625G/C, -31G/C revealed significant associations with esophageal cancer (global P = 0.0034). -644T--625C--31C was a risk haplotype for esophageal cancer (P=0.0001) and -644T?-625G?-31C, a protective haplotype (P = 0.0165).5. Survivin expression was associated with survivin promoter polymorphisms. The difference between the groups of different survivin expression level was statistically significant to the -625G/C polymorphism (P = 0.024), but not obvious for the -31G/C and -644T/C polymorphisms. The variant C-allele in the -625G/C SNP site was associated with a higher possibility of survivin expression in esophageal cancer patients. Furthermore, the patients with survivin-negative cancer tissues were more likely to exhibit the GG genotype (p=0.033).6. We investigated the methylation profiles of the survivin exon1 using BSP method in esophageal cancer and adjacent non-cancerous tissues of 4 patients. The cancer tissues we studied were survivin-positive and matched normal tissues were survivin-negative. After BSP, we didn’t find any cytosines in the PCR products’sequences. Therefore, the survivin exon 1 had no methylated CpG site. The survivin-negative non-cancerous tissues and the survivin-positive cancer tissues didn’t show different methylation status in the regions at least in the samples we studied.Our results revealed an association between p53 mutation and increased survivin expression in esophageal cancer. At the same time, we found the survivin promoter -625CC genotype was more likely to have survivin expression in cancer tissue. These provided substantial mechanism contributing to survivin up-regulation in esophageal cancer. Furthermore, our case-control study suggests a significant association between survivin promoter SNP and esophageal cancer. Moreover, we identified the high consistency of a common risk (-644T?-625C?-31C) and a protective (-644T?-625G?-31C) haplotype in survivin promoter linked with esophageal cancer. To our knowledge, the present study is the first report demonstrating that survivin expression is negatively regulated by p53 gene status in esophageal cancer patients. This is also the first research showing a significant relationship between the survivin promoter -625G/C polymorphism with its mRNA expression and the risk of esophageal cancer. Although the association was statistically significant with the small number of samples, this needs to be confirmed with a larger sample.更多还原