【作者】 李林蔚；

【导师】 陆士新；

【作者基本信息】 中国协和医科大学 ， 肿瘤学， 2008， 博士

【摘要】 食管癌是严重危害人类健康的常见消化道恶性肿瘤之一,其发病率在世界上位居恶性肿瘤的第七位,死亡率位于第六位。中国是食管癌的高发区,诊断的食管癌中90%是食管鳞状细胞癌(ESCC)。目前关于ESCC发生的机制还不清楚,本研究旨在探讨食管癌相关基因4(ECRG4)在ESCC发生中的作用。ECRG4是我室克隆和鉴定的新基因,并在Genbank(AF 268198)登记。激光扫描共聚焦显微镜成像显示,内源性和外源性ECRG4蛋白主要定位在细胞质和细胞膜。Western blot方法在细胞无血清培养液中检测到ECRG4蛋白存在,提示ECRG4可能是分泌蛋白。我们制作了特异识别ECRG4蛋白的兔多克隆抗体,通过ESCC组织芯片以免疫组织化学和Western blot方法,分析了130对食管癌与癌旁组织,发现在68.5%(89/130)的ESCC组织中ECRG4蛋白表达下调。抑癌基因在肿瘤组织表达下调常常是由于其启动子高甲基化,而我们用去甲基化药物5-aza-2’-deoxycytidine处理可以恢复EC9706细胞ECRG4 mRNA的表达,说明启动子高甲基化可能是ECRG4在ESCC转录失活的主要机制。ECRG4蛋白表达下调与ESCC局部淋巴结转移、原发肿瘤大小和临床病理分期成正相关(P＜0.05)。ECRG4蛋白表达与ESCC病人的术后生存时间成正相关,是ESCC的独立预后指标(P＜0.05)。ECRG4基因转染到EC9706细胞能抑制细胞增殖、平板克隆形成率、软琼脂克隆形成率、细胞周期进展和裸鼠移植瘤生长(P＜0.05)。纯化的重组ECRG4蛋白可以体外抑制EC9706细胞的增殖,抑制率随着ECRG4蛋白浓度增加而升高,成剂量-效应关系(P＜0.05)。ECRG4基因转染到EC9706细胞,可以抑制EC9706细胞NF-κB的表达和核转位,以及NF-κB靶基因COX-2的表达。我们的研究结果表明,ECRG4是ESCC的候选抑癌基因,ECRG4蛋白是预测ESCC预后的候选标志物。启动子高甲基化可能是ECRG4在ESCC转录失活的主要机制。ECRG4可能通过参与NF-κB通路调控COX-2的表达来发挥其抑癌功能。ECRG4重组蛋白可以作为ESCC的潜在治疗药物。更多还原

【Abstract】 Esophageal cancer is the sixth most frequent cause of deaths from cancer worldwide, and occurs at very high frequency in certain areas of China. Esophageal squamous cell carcinoma (ESCC) is the most prevalent type which accounts for～90% of esophageal carcinomas diagnosed in China. Until now, the underlying molecular mechanism for ESCC is still unclear. The present study is to investigate the function of esophageal cancer related gene 4 (ECRG4) in ESCC.The ECRG4 gene was identified and cloned in our laboratory, and then it was registered at Genbank (AF325503). The images of laser-scanning confocal microscopy analysis indicated that endogenous and exogenous ECRG4 proteins were localized in the cytoplasm and cell membrane. We also found ECRG4 protein in cellular medium by Western blot, indicting ECRG4 is a secreted protein. We have prepared anti-ECRG4 rabbit polyclonal antibody and found that the expression of ECRG4 protein was down-regulated in 68.5% (89/130) ESCC samples using tissue microarray and analysis of immunohistochemical staining and Western blot. The expression of tumor suppressor gene is often down-regulated in tumor tissues due to the hypermethylation in the gene promoter, and treatment with the DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine restored ECRG4 mRNA expression in EC9706 cell line, it showed that promoter hypermethylation may be one main mechanism leading to the silencing of ECRG4 in ESCC. ECRG4 protein down-regulation was significantly associated with regional lymph node metastasis, primary tumor volume and tumor stage in ESCC (P < 0.05). ESCC patients with high ECRG4 expression had longer overall survival than those with low ECRG4 expression by Kaplan-Meier survival analysis (P < 0.05). ECRG4 protein was an independent prognostic factor for ESCC by multivariable Cox proportional hazards regression analysis (P < 0.05).The transfection of ECRG4 gene in EC9706 cell line inhibited cell proliferation, colony formation, anchorage-independent growth, cell cycle progression and tumor growth in vivo (P < 0.05). Purified recombinant ECRG4 protein could inhibit EC9706 cells proliferation in vitro exhibiting dose-effect relationship (P < 0.05). The transfection of ECRG4 gene inhibited NF-κB expression and nuclear translocation, and inhibited NF-κB target gene COX-2 expression in EC9706 cell line.In conclusion, ECRG4 is a novel candidate tumor suppressor in ESCC, and ECRG4 protein is a candidate prognostic marker for ESCC. Aberrant promoter hypermethylation is one of the major mechanisms leading to the silencing of ECRG4 gene in ESCC. ECRG4 might induce COX-2 down-regulation through NF-κB pathway to inhibit tumor growth in ESCC. The recombinant ECRG4 protein is a prospective candidate of anti-tumor drugs for ESCC.更多还原