【作者】 林俊；

【导师】 金洪传；

【作者基本信息】 浙江大学 ， 肿瘤学， 2013， 硕士

【摘要】 目前的研究已经发现,胃癌的发生发展是一个多因素、多步骤的过程,细胞内信号通路的异常调节是重要的胃癌发病机制之一,是一个涉及多基因遗传学和表观遗传学改变的累积过程,包括癌基因的激活和抑癌基因的失活。在胃肠道肿瘤的这种表型演变过程中,表观遗传修饰尤其是DNA甲基化扮演着一个重要的角色。胃癌的发生发展与癌基因的激活和肿瘤抑制基因的失活直接相关。目前的研究认为,肿瘤抑癌基因的表观遗传调控失调在肿瘤的发生和发展过程中发挥着重要的作用。其中,DNA甲基化是最早发现的基因表观修饰方式之一,对其研究也最为广泛和深入。DNA甲基化常导致选择性基因的启动子区域过度甲基化和某些抑癌基因的表达沉默。新的甲基化基因的鉴定能够为肿瘤的监测、预防、诊断和治疗提供一种新的策略。致瘤信号如Ras/MAPK信号的异常激活在人癌症中非常常见。不仅基因发生变化,Ras/MAPK信号中抑制剂的表观沉默,也会造成Ras/MAPK信号的激活。最近膜联蛋白A6(ANXA6)已被证明能与Ras-GAP1相互作用,抑制人乳腺癌中的Ras激活。然而,它是否以及如何应用在人类癌症治疗仍然未知。方法和材料：1.挑选AGS、Kato Ⅲ、MKN28、MKN45、NCI-N87、SNU1细胞株。用RT PCR方法检测胃癌细胞ANXA6的表达。2.进一步利用MSP、BGS等方法分析ANXA6基因的表达调控机制,探讨ANXA6基因启动子CGI甲基化与胃癌组织中ANXA6基因下调有无相关性。3.采用Real-time PCR分析YY1表达变化前后的ANXA6表达情况,并进行DNA甲基化状态分析,研究ANXA6的表达调控机制。4.采用X2检验法分析具备ANXA6甲基化的患者特性关联性。用乘积极限法计算总存活概率,曲线间差异则用对数秩检验进行评估。另采用单变量Cox比例风险模型估算与ANXA6甲基化以及其他预测变量相关的死亡相对危险度,并建立多变量Cox模型来估算ANXA6甲基化的相对危险度,在年龄、性别、幽门螺旋杆菌感染、Lauren分型、肿瘤分化和TNM分期方面有作调整。结果：1. ANXA6在胃癌细胞中表达下调。在药物脱甲基后,它的表达升高。ANXA6启动子在胃癌细胞系(6/6)和原发性胃癌组织(29/156)中被甲基化。2.在胃癌细胞AGS、MKN45的YY1蛋白表达下降的过程中,RT-PCR结果显示ANXA6表达明显上升,同时DNA甲基化程度下降。YY1蛋白的沉寂促进了ANXA6启动子的脱甲基,提高了ANXA6表达水平。3.在胃癌病变组织中,ANXA6启动子甲基化的阳性率为18.6%,但ANXA6甲基化与肿瘤细胞分化程度,TNM分期等临床参数并无明显关联。无论卡普兰-迈耶曲线,还是Cox回归分析,都未能揭示ANXA6甲基化对于预测胃癌患者存活的重要作用。结论：胃癌中的ANXA6基因的甲基化水平上升伴随着表达下调；去甲基化处理后,ANXA6表达上调,表明甲基化可能是导致ANXA6基因表达下调的主要原因。YY1对于ANXA6的甲基化的形成或维持至关重要。本研究未发现ANXA6启动子甲基化与总胃癌患者存活数、分化与分期等临床参数的关联性,可能与研究样本偏小有关更多还原

【Abstract】 Recent studies have found that the oncogenesis and development of gastric cancer is a multi-step process involving multi-factors, in which, the abnormal regulation of intracellular signaling pathways is an important pathogenesis of gastric cancer. This regulation is a cumulative process involving multiple genetic and epigenetic gene changes, including the activation of oncogenes and the inactivation of antioncogenes. In the phenotypic evolution of gastroenteric tumor, the epigenetic modifications, particularly DNA methylation, play an important role. The oncogenesis and development is directly related to the activation of oncogenes and the inactivation of antioncogenes. In which, the DNA methylation a epigenetic modification that was firstly discovered, so it is most extensively and deeply researched. DNA methylation often leads to hypermethylation in promoter region of selective genes and silence of some antioncogenes. Current studies suggest that the epigenetic regulation disorder of antioncogenes plays an important role in tumor oncogenesis and development. The new identification of methylated genes can provide a new strategy for the monitoring, prevention, diagnosis and treatment of tumor.The abnormal activation of oncogenic signals such as Ras/MAPK signals was very common in human cancers.In addition to genetic changes, the epigenetic silence of inhibitors in Ras/MAPK signals will also cause Ras/MAPK signal activation. Recent studied showed that ANXA6can interacte with Ras-GAP1to inhibit the Ras activation in human breast cancer. However, it is unknown whether and how it assists in the treatment of human cancer.Methods and Materials:1. AGS, Kato Ⅲ, MKN28, MKN45, NCI-N87, SNU1cell lines were collected. The RT PCR method was used to determine the expression of ANXA6in gastric cancer cells.2. Then the MSP, BGS methods were used to further analyze the expression regulation mechanism of ANXA6gene, and to explore the absence or presence of the relevance between ANXA6gene promoter CGI hypermethylation and ANXA6downregulation in gastric cancer tissues.3. The Real-time PCR analysis was used to analyze the ANXA6expression before and after YY1expression changes, and to analyze the methylation status of DNA, to research the regulation mechanisms of ANXA6expression.4. The χ2test was used to analyze the characteristic relevance among patients with ANXA6methylation. The Kaplan Meier product-limit method was used to calculate the total probability of survival, the log-rank test was used to evaluate the difference between the curves. The univariate Cox proportional hazards model was used to estimate the relative risks of death of ANXA6methylation and other predictive variables, and the multivariate Cox model was built to estimate the relative risk of ANXA6methylation, so as to make some adjustments in age, gender, Helicobacter pylori infection, Lauren classification, tumor differentiation and TNM staging.Results:1. ANXA6was downregulated in gastric cancer cells. After drug demethylation, its expression was difficulty to be restored. ANXA6promoter was methylated in gastric cancer cell lines (6/6) and primary gastric cancer tissues (29/156).2. During expression decrease of YY1in gastric cancer cells AGS, MKN45, the RT-PCR results showed ANXA6expression increased significantly, while DNA methylation level decreased. The silence of YY1protein restored the ANXA6expression, and promoted ANXA6promoter demethylation.3. In gastric lesions, the positive rate of ANXA6promoter methylation was18.6%, but the ANXA6methylation was not related to tumor cell differentiation, TNM staging and other clinical parameters. Both Kaplan-Meier curves and Cox regression analysis were failed to reveal the important role of ANXA methylation for predicting the survival of patients with gastric cancer.Conclusion:ANXA6is downregulated while hypermethylation rises in gastric cancer; after demethylation treatment, ANXA6expression is restored, suggesting that the methylation may be the main reason leading to ANXA6downregulation. YY1is essential for the formation or maintenance of ANXA6methylation. In this experiment, the relevance between ANXA6promoter methylation and overall survival, differentiation and staging and other clinical parameters was not found, which may be due to the limited sample size and heterogeneous clinical data of cases.更多还原