【作者】 郑峰；

【导师】 伊力亚尔·夏合丁；

【作者基本信息】 新疆医科大学 ， 外科学， 2013， 博士

【摘要】 目的：食管癌是较常见的恶性肿瘤之一，我国食管癌的发病率和死亡率居世界第一，每年约有16～20万人死于此病。新疆是食管癌高发区之一，其中哈萨克族发病率最高，比其他少数民族高2～3倍，病死率在全国少数民族中占第一位。食管癌作为新疆的常见病和多发病，已严重影响了本地区各族人民的身体健康。哈萨克族生活习惯及居住环境特殊，是较好的研究人群。利用新疆哈萨克族食管癌疾病资源，开展食管癌的基础研究，建立肿瘤分子标志物体系，结合临床研究来明确食管癌发病原因，对于新疆食管癌的预防、诊断及临床治疗具有重大意义。食管癌的病因复杂，目前普遍认为食管癌是多因素作用、多基因参与、多阶段发展的疾病，然而其发生和发展的确切机制仍不太清楚。在对哈萨克族食管癌高发区流行病学调查工作中，发现哈萨克族食管癌具有家族聚集现象，并且遗传免疫因素在食管癌的发生中起着一定的作用。大量研究表明，人类白细胞抗原Ⅰ类分子（human leukocyte antigenclass Ⅰ，HLA-Ⅰ）表达低下或缺失，可导致肿瘤细胞逃逸宿主免疫监视，从而与肿瘤发生密切相关。HLA-Ⅰ类分子是机体免疫应答信息产生和传递的基础，主要功能是将细胞内源性抗原肽呈递到细胞表面，实现CD8+T细胞对自身抗原的识别和免疫监视。而此内源性抗原肽呈递过程必须在抗原呈递元件（antigen processing machinery，APM）的协助下才能完成。APM成员蛋白是一组抗原呈递相关蛋白，负责协助HLA-Ⅰ类分子组装、负载和提呈内源性抗原肽，介导抗肿瘤的T细胞免疫功能。APM成员蛋白表达下调可影响正常的抗原呈递过程，使肿瘤逃避免疫监视而增加肿瘤发生和发展的风险。DNA甲基化是肿瘤表观遗传学调控的一个重要方式，肿瘤细胞基因组整体水平低甲基化和启动子区高甲基化是已被公认的肿瘤表观遗传性机制。APM基因启动子区的异常甲基化可引发基因表达沉默，导致功能蛋白的表达下调或缺失，从而促进肿瘤的发生和发展，在宫颈癌、膀胱癌、头、颈部肿瘤中已有报道，但有关食管癌的研究很少。本研究拟从表观遗传学角度，以HLA-Ⅰ和APM成员基因为候选基因，利用新疆哈萨克族食管癌病变资源，从基因表达调控层次，分析APM成员基因启动子区异常甲基化与哈萨克族食管鳞癌发生之间的关系。方法：（1）采用亚硫酸氢盐测序PCR（bisulfate sequeneing PCR，BSP）法检测人食管鳞癌ECa109细胞DNA中基因启动子区甲基化位点。利用在线数据库提供的人类基因组计划资源，对HLA-B、TAP1、TAP2、LMP2、LMP7、ERAP1，Tapasin和ERp57等8种基因启动子区设计特异性PCR引物，经基因启动子区目的CpG岛片段的PCR扩增、克隆和测序，获得基因启动子区甲基化相关的序列和CpG位点信息。（2）收集新鲜哈萨克族食管癌及癌旁正常食管组织标本50例，提取组织DNA。选取前一步研究中筛选出来的HLA-B、TAP2、LMP7、Tapasin和ERp57等5种基因，利用MassARRAY甲基化检测技术，对候选基因启动子区CpG岛甲基化水平进行定量分析，明确食管癌和癌旁正常组织中总体及单个CpG位点甲基化水平的差异。（3）采用免疫组织化学SP法，检测LMP7、Tapasin、ERp57和HLA-Ⅰ蛋白在食管癌组织中的表达水平。使用SPSS16.0统计分析软件包对数据进行处理，采用非参数秩和检验分析以上4种蛋白表达水平与食管癌患者临床病理特征的关系。采用Spearman等级相关分析检验LMP7、Tapasin、ERp57与HLA-Ⅰ蛋白表达水平的相关性，采用方差分析检验LMP7、Tapasin、ERp57基因甲基化和蛋白表达水平的关系。P值取双侧，以a=0.05为临界值。结果：（1）在人食管鳞癌ECa109细胞DNA中，HLA-B、TAP2、LMP7、Tapasin和ERp57等5种基因启动子区的目的CpG岛均发生了不同程度的甲基化，发生甲基化的CpG位点占所有CpG位点的比例分别为10/14、8/8、2/22、5/12和18/18。而TAP1、LMP2、ERAP1等3种基因启动子区没有发现甲基化位点。（2）根据MassARRAY技术对50例哈萨克族食管癌及癌旁正常组织中的甲基化定量检测结果，发现LMP7、Tapasin和ERp57基因在食管癌中的甲基化水平高于癌旁组织（P＜0.05），HLA-B和TAP2基因在食管癌和癌旁组织中的甲基化水平无差异（P＞0.05）。进行单个CpG位点甲基化水平分析显示：LMP7基因启动子区CpG₅、CpG₉、CpG₂0、CpG₂1、CpG₂2位点；Tapasin基因启动子区CpG₁、CpG₆位点；ERp57基因启动子区CpG₁位点的甲基化水平在食管癌和癌旁正常组织之间存在统计学差异（P＜0.05）。对3种基因甲基化水平与肿瘤临床病理特征进行分析，发现LMP7和ERp57基因甲基化与食管癌患者TNM分期有关，Ⅰ期+ⅠⅠ期组的甲基化水平高于ⅠⅠⅠ期+ⅠV期组；LMP7、ERp57基因甲基化与肿瘤分化程度有关，中、低分化组的甲基化水平高于高分化组；ERp57基因甲基化与肿瘤的血管侵袭性有关，侵袭血管生长组的甲基化水平高于不侵袭血管生长组，以上差异均有统计学意义（P＜0.05）。（3）免疫组化结果显示：食管癌组织中HLA-Ⅰ、Tapasin、LMP7和Erp57蛋白均有明显的表达下调或缺失，与癌旁正常组织相比有统计学意义（P＜0.05）。HLA-Ⅰ蛋白表达下调与肿瘤分化程度、肿瘤侵润深度密切相关；LMP7蛋白表达下调与肿瘤分化程度、肿瘤侵润深度、肿瘤血管侵袭性生长、淋巴结转移、TNM分期密切相关；Tapasin蛋白表达下调与肿瘤分化程度、肿瘤侵润深度密切相关；ERp57蛋白表达下调与淋巴结转移、肿瘤血管侵袭性生长、肿瘤侵润深度、TNM分期密切相关，以上差异均有统计学意义（P＜0.05）。在食管癌组织中，HLA-Ⅰ与LMP7、Tapasin、ERp57蛋白表达水平呈正相关（r值分别为0.823、0.721、0.378，P值均＜0.05）。LMP7、ERp57基因在不同甲基化水平之间的蛋白表达水平差异有统计学意义（P＜0.05），随着基因启动子区甲基化水平的增高，相应蛋白的表达水平降低。而Tapasin基因在不同甲基化水平食管癌组织中的蛋白表达水平没有统计学差异（P＞0.05）。结论：（1）在人食管鳞癌ECa109细胞中，HLA-B、TAP2、LMP7、Tapasin和ERp57等5种基因启动子区的CpG岛均发生了不同程度的CpG位点甲基化，可能是导致人食管鳞状上皮细胞癌变的重要原因。（2）在新疆哈萨克族食管癌组织标本中，LMP7、Tapasin和ERp57基因甲基化水平异常增高，并与肿瘤的临床病理特征密切相关，是导致哈萨克族食管癌发生的高危因素。LMP7基因启动子区的CpG₅、CpG₉、CpG₂0、CpG₂1、CpG₂2位点；Tapasin基因启动子区的CpG₁、CpG₆位点；ERp57基因启动子区的CpG₁位点与基因的甲基化密切相关，可能是调控相应基因启动子区甲基化的关键位点。（3）在新疆哈萨克族食管癌组织标本中，HLA-Ⅰ、LMP7、Tapasin和Erp57蛋白出现明显的表达下调和缺失，并与食管癌的临床病理特征密切相关，说明HLA-Ⅰ及APM成员蛋白协同参与了食管鳞状上皮的免疫应答，可以防止食管癌的发生，对机体有保护作用。APM成员LMP7、ERp57基因启动子区甲基化，可以抑制基因蛋白表达，影响HLA-Ⅰ分子的组装和呈递，造成HLA-Ⅰ在肿瘤细胞表面的下调或缺失，使肿瘤细胞逃避了机体的免疫监视和杀伤，导致了肿瘤的发生，可能是新疆哈萨克族食管癌发生的表观遗传学机制。更多还原

【Abstract】 Aim of study: Esophageal cancer is one of the most common malignancies. Thereare about16²0million people died of the disease each year in china, morbidity andmortality ranking the first in the world. Xinjiang is one of the high-risk areas foresophageal squamous cell carcinoma （ESCC）, in which Kazakh people have the highestincidence of2to3times higher than other minorities. The mortality of Kazakh ESCCoccupied the first place in all minorities and was2.3times higher than the nationalaverage rate. As a common and frequently occurring disease in Xinjiang, ESCC hasseriously affected the quality of life in people who live in the region. Because kazakhpeople live in a distinctive environment and have unique living habits, so the populationis a good object for study. To investigate the reason of ESCC and establish tumormolecular marker system is very important for diagnosis, clinical treatment andprevention of ESCC in Kazakh population. Taking Xinjiang Kazakh ESCC resources tocarry out basic research of ESCC is of great significance. Epidemiological studiesrevealed that the incidence of ESCC was associated with multiple factors, many geneswere involved in the different carcinoma development stage. Investigations found thatthere was a familial aggression phenomenon in high-incidence people and the immunefactors played a important role in the incidence of ESCC. However, the definitemechanism is still not clear. Numerous studies show that low expression or absence ofhuman leukocyte antigen class I （HLA-I） can cause tumor cells escaping the host immunesurveillance, which is closely related with tumor development. Human leukocyte antigenclass I is the basis of the immune response system generation and transmission. It’sprimary function is deriving antigen peptides from tumor cells and presenting it to cytotoxic T lymphocytes, by which realizing self antigens recognition and immune surveillance.This endogenous antigen peptide presenting process must be assisted by antigenprocessing machinery （APM） components to achieve the normal function of the HLA-I.Antigen processing machinery components are a group of antigen presenting proteinsresponsible for assisting the HLA-I molecular assembly, loading and presentingendogenous antigen peptide. Defects in antigen processing machinery componentsexpression may provide tumor cells with a mechanism to escape from recognition anddestruction by HLA class I antigen-restricted tumor antigen-specific cytotoxic T cells.DNA methylation is an important mechanism in silencing the expression of genes. Thefunctional relevance and potential clinical significance of these epigenetic alterationshave been addressed. More recently, epigenetic events associated with tumordevelopment and progression have been found to underlie changes in HLA antigen, APMcomponent, co-stimulatory molecule expression in malignant cells, including cervicalcancer, bladder cancer, head and neck cancer. However, expression and functionalproperties of HLA-I antigens and APM components in malignant cells of ESCC havebeen investigated in a limited extent. To overcome the limitation in the present study, wehave selected Xinjiang Kazakh ESCC species, investigated the methylation level ofhuman leukocyte antigen class I and antigen-processing machinery components （TAP1,TAP2, LMP2, LMP7, ERAP1, Tapasin, and ERp57） as well as functional proteinexpression in25primary esophageal squamous cell carcinomas and in25matchedhealthy esophageal tissues. We want to find out the association of APM componentsmethylation and development of ESCC in Xinjiang Kazakh people through epigeneticangle. Methods:（1）ECa109cells were treated with bisulfate sequeneing PCR （BSP）analysis to evaluate the CpG island methylation status of HLA-B, TAP1, TAP2, LMP2,LMP7, ERAP1, Tapasin and ERp57gene. Using the resources of the online database ofthe human genome project, genetic information was obtained from the genbank database,and specific PCR primers of CpG island fragment were designed through specializedsoftware scanning gene promoter regions. The methylation status of specific target geneswas gained by PCR amplification, cloning and sequencing.（2）50cases of fresh KazakhESCC species and matched healthy esophageal tissues were collected and the DNA wasextracted. HLA-B TAP2LMP7Tapasin and ERp57genes were choosed as candidategenes screened out from the previous step study. MassARRAY technology was employedto analyze the methylation levels of methylated CpG sites in the gene promoter fragmentsbetween ESCC and normal control.（3）The HLA-I, LMP7, Tapasin, and ERp57antigens were detected by immunohistochemical SP method in50cases of esophageal species.The association between the downregulated expression of HLA-I, LMP7, Tapasin,ERp57antigens and clinicopathological features of ESCC was analyzed by theKruskal-waillis test. Correlation of LMP7, Tapasin, ERp57genes promoter methylationlevels with protein expression levels was identified by the Spearman rank correlationcoefficient. The data was processed by SPSS16.0statistical analysis software,“P” valuewith bilateral, signincance at a=0.05. Results:（1） CpG islands in promoter region werehypermethylated in the Eca109cells, methylated CpG sites were found to differentdegrees in HLA-B, TAP2, LMP7, Tapasin and Rp57genes. The proportion of methylatedCpG sites in all CpG sites of each gene were10/14,8/8,2/22,5/12,18/18respectively.No methylated CpG site was found in TAP1, LMP2, ERAP1gene promoterregions.（2）According to MassARRAY methylation level detecting, the methylationlevels of LMP7,Tapasin and ERp57genes in ESCC species were higher than matchedhealthy esophageal tissues （P＜0.05）, while there were no significant difference inHLA-B and TAP2genes （P＞0.05）. Single CpG sites methylation level analysis showedthat: CpG₅, CpG₉, CpG₂0, CpG₂1, CpG₂2in the LMP7gene promoter region;CpG₁, CpG₆in the Tapasin gene promoter region; CpG₁in the ERp57gene promoterregion were statistically different between ESCC and adjacent normal tissues. Analysisbetween LMP7, Tapasin, ERp57gene methylation levels and clinicopathological featuresfound that: LMP7, ERp57genes promoter methylation levels in stage I and stage II grouppatients are higher than stage III and stage IV group; LMP7, ERp57genes methylationlevels in moderately and poorly differientied carcinomas are higher than welldifferientied tissues; ERp57gene methylation level in tumor vascular invasion group ishigher than without vascular invasion group （P＜0.05, respectively）.（3）The expressionof HLA-I, LMP7, Tapasin and ERp57antigens were reduced in tumor lesions comparedwith matched healthy tissues, the difference was statistically significant respectively（P＜0.05）. Downregulated expression of HLA-I is closely associated with tumordifferentiation, depth of tumor invasion; Downregulated expression of LMP7is closelyassociated with tumor differentiation, lymph node metastasis, depth of tumor invasion,vascular invasion, TNM staging; Downregulated expression of Tapasin is closelyassociated with tumor differentiation, depth of tumor invasion; Downregulatedexpression of Erp57is closely associated with lymph node metastasis, depth of tumorinvasion, vascular invasion, TNM staging （P＜0.05, respectively）. Analysis by theSpearman rank correlation coefficient showed that LMP7, Tapasin and ERp57protein expression level were significantly correlated with that of HLA-I （r=0.823, r=0.721andr=0.378, P＜0.05, respectively）. The methylation levels of LMP7and ERp57genepromoters were negatively correlated with protein expression levels （P＜0.05,respectively）. Conclusion:（1）The CpG islands in promoter fragments of HLA-B, TAP2,LMP7, Tapasin and Rp57genes existed different degrees of mehthylation in Eca109cellline. This may be specific epigenetic changes in esophageal squamous cell carcinoma. Itprovides a important mechanism for esophageal cancer development.（2）In XinjiangKazakh ESCC tissue specimens, the methylation levels of LMP7, Tapasin and ERp57genes enhanced abnormally and are closely associated with clinicopathological features.It should be noted as high risk factors of ESCC development in Xinjiang Kazakhpopulation. Aberrant hypermethylation of CpG islands （CpG₅, CpG₉, CpG₂0,CpG₂1, CpG₂2in the LMP7gene promoter region; CpG₁, CpG₆in the Tapasingene promoter region; CpG₁in the ERp57gene promoter region） may be the key sitesof regulating the gene methylation. These CpG sites can affect multiple cellular functionsby silencing transcriptional process.（3）HLA-I, LMP7, Tapasin and ERp57antigens playthe critical role in tumor cell immune surveillance and killing, they can prevent theoccurrence of esophageal cancer and produce a protective effect on the body. Themethylated CpG sites in LMP7and ERp57gene promoter can affect the level of proteinexpression, which play a significant role in hindering HLA-I molecules assembly andpresenting, the latter changes result in the HLA-I down or missing in the surface of tumorcells. The whole process cause tumor cells escape from body’s immune surveillance andlead to carcinoma development. This maybe is the epigenetic mechanism of XinjiangKazakh ESCC.更多还原