胃癌组织中ERCC1基因的高甲基化及DNMT1的表达

发布时间：2018-08-22 20:49

【摘要】：目的:通过对胃癌及癌旁组织基因表达情况的检测,分析ERCC1(人切除修复交叉互补基因1)基因启动子区甲基化的状况及DNMT1(DNA甲基转移酶1)基因的表达在胃癌演变过程中的作用。在消化系统恶性肿瘤中,胃癌是主要的致死疾病之一,大部分患者诊断时已经为晚期,所以胃癌的早期诊断尤为重要。胃癌产生与演变是一个复杂的病变过程,它受到多因素的共同调控,其中癌基因的非正常激活与抑癌基因转录功能的失活是两个极其重要的原因,表观遗传学层面上的改变在病变过程中发挥着重要的功能,最普遍的表观遗传学改变就是DNA甲基化。方法:(1)青岛市市立医院2014年1月至2014年10月经普外科切除的新鲜肿瘤标本共60例,经病理组织学诊断为为原发性胃癌,术前未行抗肿瘤治疗,比如放疗化疗,拥有完整的临床资料。癌组织取自癌病变区域的中心,并取距离癌组织至少5cm区域组织作为癌旁组织。分离收集到的标本组织中的DNA,然后使用亚硫酸钠修饰方法对所得的DNA进行装饰,采用聚合酶增链式反应技术对上述DNA进行扩大增殖。使用MSP方法对胃癌组织及癌旁组织ERCC1基因甲基化情况进行测验,并检验ERCC1和DNMT1表达水平。分析探讨在肿瘤演变过程中两个基因的所发挥的功效。(2)对收集到的60例标本组织中的RNA进行分离,然后进行逆转录反应,形成单链DNA,接着应用PCR技术扩大增殖,用荧光定量逆转录多聚合酶链RT-PCR技术对基因ERCC1、DNMT1的m RNA表达水平进行检验。(3)用免疫组织化学方法检测癌组织和癌旁组织中的ERCC1及DNMT1蛋白表达水平状态,探究它们在胃癌演变过程中所发挥的功效。使用SPSS17.0软件对试验得到的数据进行解析,使用χ2检验方法于计数资料之间的比较中,组间的比较t检验进行处理;以P0.05作为评判标准,差异具有统计学意义。结果:(1)应用MSP反应检测出胃癌组织ERCC1启动子区甲基化率为68.3%明显高于正常组织23.3%,差异有统计学意义(P0.001)。(2)应用RT-PCR反应方法进行检测,在ERCC1表达下调的胃癌组织中DNMT1的表达高于ERCC1表达正常的组织,表达水平差别有统计学意义(P0.05)。(3)应用免疫组织化学方法检验,ERCC1在癌旁组织中的表达阳性率为93.3%,明显高于胃癌组织56.7%,差异有统计学意义(χ2=21.21,P0.001)。DNMT1在胃癌组织中的表达阳性率为71.7%,明显高于癌旁组织16.7%,差异有统计学意义(P0.001)。(4)胃癌组织中ERCC1基因启动子区甲基化状态与ERCC1 m RNA表达之间的关系,在41例ERCC1 m RNA阴性表达的胃癌组织中,检测出38例该基因启动子区甲基化,而在ERCC1 m RNA表达阳性的19例胃癌组织中,仅检测出3例基因启动子甲基化,差异有统计学意义(P0.001)。(5)胃癌组织中ERCC1与DNMT1蛋白表达之间的相关性分析,胃癌组织中的ERCC1与DNMT1蛋白的表达呈负相关(P0.001).结论:(1)在胃癌组织中,可发现ERCC1基因启动子区域发生高甲基化改变,并且同时伴有该基因蛋白的表达减低,和胃癌的发生存在一定的关系。(2)胃癌组织中ERCC1基因的m RNA表达低于癌旁组织,m RNA的低表达与ERCC1基因启动子区Cp G岛高甲基化有关。(3)胃癌组织中的ERCC1与DNMT1蛋白的表达呈负相关,胃癌组织中的DNMT1的m RNA及蛋白表达水平明显上升,此改变或与抑癌基因ERCCl发生高甲基化存在一定的联系,造成ERCC1 m RNA表达减少或缺失,基因蛋白表达同样降低。
[Abstract]:Objective: To analyze the promoter methylation of ERCC1 gene and the role of DNMT1 gene expression in the development of gastric cancer by detecting gene expression in gastric cancer and adjacent tissues. The generation and evolution of gastric cancer is a complicated pathological process, which is regulated by many factors. Abnormal activation of oncogenes and inactivation of transcriptional function of tumor suppressor genes are two extremely important reasons. Epigenetic changes are found in gastric cancer. DNA methylation is the most common epigenetic change that plays an important role in the pathogenesis of gastric cancer. Methods: (1) A total of 60 fresh tumor specimens from Qingdao Municipal Hospital from January 2014 to October 2014 were surgically removed and diagnosed as primary gastric cancer by histopathology. Preoperative anti-tumor treatment, such as radiotherapy and chemotherapy, was completed. Cancer tissues were taken from the center of the lesion area and taken at least 5 cm away from the cancer tissue as adjacent tissues. DNA was isolated from the collected tissues and then decorated with sodium sulfite. The DNA was amplified and proliferated by polymerase chain reaction (PCR). Methods The methylation of ERCC1 gene in gastric cancer and adjacent tissues was tested, and the expression levels of ERCC1 and DNMT1 were examined. The expression of ERCC1 and DNMT1 was detected by fluorescence quantitative reverse transcription polymerase chain reaction (FQRT-PCR). (3) The expression of ERCC1 and DNMT1 proteins in cancer tissues and adjacent tissues was detected by immunohistochemistry to explore their effects on the development of gastric cancer. Results: (1) The methylation rate of ERCC1 promoter region in gastric cancer tissues detected by MSP reaction was 68.3% higher than that in normal tissues by 23.3%. The difference was statistically significant (P 0.001). (2) The expression of DNMT1 in gastric cancer tissues with down-regulation of ERCC1 was significantly higher than that in normal tissues (P 0.05). (3) The positive rate of ERCC1 expression in adjacent tissues was 93.3% by immunohistochemistry. The positive rate of DNMT1 expression in gastric cancer was 71.7%, which was significantly higher than that in adjacent tissues (P 0.001). (4) The relationship between the methylation status of ERCC1 gene promoter region and the expression of ERCC1 m RNA in gastric cancer tissues was negative in 41 cases. Methylation of the promoter region was detected in 38 gastric cancer tissues, but only 3 of 19 gastric cancer tissues with positive ERCC1 m RNA expression were detected in 3 cases (P 0.001). (5) Correlation analysis between the expression of ERCC1 and DNMT1 protein in gastric cancer tissues, ERCC1 and DNMT1 eggs in gastric cancer tissues. Conclusion: (1) Hypermethylation of the promoter region of ERCC1 gene can be found in gastric cancer tissues, and the expression of ERCC1 gene protein is decreased, which is associated with the occurrence of gastric cancer. (2) The expression of ERCC1 gene in gastric cancer tissues is lower than that in adjacent tissues, and the expression of M RNA is lower than that in adjacent tissues. (3) The expression of DNMT1 protein was negatively correlated with ERCC1 in gastric cancer tissues. The expression of DNMT1 m RNA and protein was significantly increased in gastric cancer tissues. This change may be related to the hypermethylation of tumor suppressor gene ERCCl, resulting in the decrease or deletion of ERCC1 m RNA expression and gene protein surface. The same has been reduced.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.2

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