TMEM176A基因启动子区在食管癌中的异常甲基化改变
发布时间：2018-08-31 12:17
【摘要】：背景食管癌是世界上最常见的恶性肿瘤之一,在癌症相关死亡率中位于第六位。食管腺癌和鳞状细胞癌是食管癌最为常见的两种类型,食管癌的发病具有明显的地区差异性,男女发病无明显差异,其中,腺癌在西方国家比较常见,鳞状细胞癌主要见于亚洲地区。在美国,每年估计有17040例的新发食管癌患者,另有15070人死于食管癌。2012年,食管癌在我国的发病率为18.6%,同时,我国食管癌的发生率是美国的20-30倍。食管癌的形成是一个多阶段的发展过程,包括：基底细胞的过度增生、异型增生、原位癌、进展期癌。许多的遗传学改变与表观遗传学变异参与到了食管癌的发生过程中,特别是抑癌基因的DNA高甲基化在食管癌中频繁发生。跨膜蛋白176 (transmembrane protein 176, TMEM176)是与跨膜蛋白4A家族相关的蛋白,存在于哺乳动物和斑马鱼类,TMEM176A是其中的一种亚型,位于7q36.1,在多个肿瘤中发现该区域存在等位基因的缺失。已有研究报道在乳腺癌中发现TMEM176A的异常DNA甲基化,但TMEM176A基因在食管癌中的甲基化状态及功能尚不清楚。目的探讨TMEM176A基因在食管癌中的甲基化情况及食管癌发生发展中的作用,为食管癌的早期诊断及治疗奠定基础。方法在我们前期研究中,通过高通量测序技术在结肠癌中发现TMEM176A基因表达缺失,因此我们采用半定量RT-PCR与甲基化特异性PCR的方法对TMEM176A基因在结肠癌细胞系中的表达及甲基化情况进行初步的筛选,发现TMEM176A在结肠癌中频发发生甲基化而导致其表达缺失。然后应用上述方法在食管癌细胞系中探讨TMEM176A基因的表达及甲基化情况,采用硫化测序证实甲基化特异性PCR的结果,并应用5-AZA处理因甲基化而缺失表达的细胞,探讨甲基化对TMEM176A表达的调控,通过在103例原发性食管癌中检测TMEM176A基因的甲基化情况,探讨TMEM176A甲基化作为食管癌诊断标志物的可能性。同时,我们还将克隆TMEM176A基因,在食管癌细胞系中探讨TMEM176A的作用及作用机制。结果我们发现TMEM176A基因在结肠癌中频发甲基化且其表达受启动子区甲基化的调控。TMEM176A在食管癌细胞系TE1、TE3、TE13、KYSE140、 KYSE180、KYSE410、KYSE450、KYSE520、Seg1、YES2、Colo680中表达缺失,同时启动子区呈现完全甲基化状态；在BiCl细胞系中TMEM176A呈高表达,且启动子区呈现非甲基化状态。去甲基化药物5-AZA处理以后,TMEM176A在TE1、TE3、TE7、KYSE150、KYSE410、KYSE510细胞系中的表达恢复或增加。这些结果表明在食管癌细胞系中TMEM176A的表达受其启动子区甲基化的调控。TMEM176A在原发性食管癌中频繁发生启动子区的甲基化,甲基化发生率为61.2%(63/103),TMEM176A的高甲基化与食管癌的肿瘤大小、分期、分化程度、淋巴结转移没有明显的相关性(P0.05)。结论TMEM176A在食管癌细胞系中的表达受DNA甲基化的调控,在原发性食管癌中频繁发生高甲基化。其生物学功能及临床意义有待进一步研究。
[Abstract]:Background esophageal cancer is one of the most common malignant tumors in the world and ranks sixth in cancer-related mortality. Esophageal adenocarcinoma and squamous cell carcinoma are the two most common types of esophageal cancer. Squamous cell carcinoma mainly occurs in Asia. In the United States, there are an estimated 17040 new esophageal cancer patients and 15070 deaths from esophageal cancer each year. In 2012, the incidence of esophageal cancer in China was 18.6, and the incidence of esophageal cancer in China was 20-30 times higher than that in the United States. The formation of esophageal cancer is a multistage process, including: basal cell hypertrophy, dysplasia, carcinoma in situ, advanced cancer. Many genetic changes and epigenetic variations are involved in the development of esophageal cancer, especially the DNA hypermethylation of tumor suppressor genes occurs frequently in esophageal cancer. Transmembrane protein 176 (TMEM176) is associated with the transmembrane protein 4A family. TMEM176A is one of the subtypes in mammals and zebrafish, located at 7q36.1. The deletion of allele in this region was found in many tumors. Abnormal DNA methylation of TMEM176A has been reported in breast cancer, but the methylation status and function of TMEM176A gene in esophageal cancer are not clear. Objective to investigate the role of TMEM176A gene in the methylation of esophageal carcinoma and the development of esophageal carcinoma, so as to lay a foundation for early diagnosis and treatment of esophageal carcinoma. Methods in our previous study, high throughput sequencing technique was used to detect the loss of TMEM176A gene expression in colon cancer. So we used semi-quantitative RT-PCR and methylation-specific PCR to screen the expression and methylation of TMEM176A gene in colon cancer cell line. We found that TMEM176A methylation occurred frequently in colon cancer resulting in its loss of expression. Then, the expression and methylation of TMEM176A gene in esophageal cancer cell line were studied by using the above method. The results of methylation specific PCR were confirmed by sulfidation sequencing, and 5-AZA was applied to treat the cells lacking expression due to methylation. To investigate the regulation of methylation on the expression of TMEM176A, the possibility of TMEM176A methylation as a diagnostic marker for esophageal carcinoma was explored by detecting the methylation of TMEM176A gene in 103 cases of primary esophageal carcinoma. At the same time, we will clone TMEM176A gene and explore the role and mechanism of TMEM176A in esophageal cancer cell line. Results We found that TMEM176A gene was frequently methylated in colon cancer and its expression was regulated by methylation of promoter region. TMEM176A was absent in esophageal cancer cell line TE1,TE3,TE13,KYSE140, KYSE180,KYSE410,KYSE450,KYSE520,Seg1,YES2,Colo680 and the promoter region was completely methylated. The expression of TMEM176A was high in BiCl cell line and the promoter was demethylated. The expression of TMEM176A in TE1,TE3,TE7,KYSE150,KYSE410,KYSE510 cells recovered or increased after 5-AZA treatment. These results suggest that the expression of TMEM176A in esophageal cancer cell lines is regulated by the methylation of its promoter region. TMEM176A is frequently methylated in the promoter region of primary esophageal carcinoma, and the incidence of methylation is 61.2% (63 / 103) hypermethylation of TMEM176A and the tumor size of esophageal carcinoma. There was no significant correlation among stages, differentiation and lymph node metastasis (P0.05). Conclusion the expression of TMEM176A in esophageal carcinoma cell line is regulated by DNA methylation and hypermethylation occurs frequently in primary esophageal carcinoma. Its biological function and clinical significance need further study.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.1
本文编号：2214961
[Abstract]:Background esophageal cancer is one of the most common malignant tumors in the world and ranks sixth in cancer-related mortality. Esophageal adenocarcinoma and squamous cell carcinoma are the two most common types of esophageal cancer. Squamous cell carcinoma mainly occurs in Asia. In the United States, there are an estimated 17040 new esophageal cancer patients and 15070 deaths from esophageal cancer each year. In 2012, the incidence of esophageal cancer in China was 18.6, and the incidence of esophageal cancer in China was 20-30 times higher than that in the United States. The formation of esophageal cancer is a multistage process, including: basal cell hypertrophy, dysplasia, carcinoma in situ, advanced cancer. Many genetic changes and epigenetic variations are involved in the development of esophageal cancer, especially the DNA hypermethylation of tumor suppressor genes occurs frequently in esophageal cancer. Transmembrane protein 176 (TMEM176) is associated with the transmembrane protein 4A family. TMEM176A is one of the subtypes in mammals and zebrafish, located at 7q36.1. The deletion of allele in this region was found in many tumors. Abnormal DNA methylation of TMEM176A has been reported in breast cancer, but the methylation status and function of TMEM176A gene in esophageal cancer are not clear. Objective to investigate the role of TMEM176A gene in the methylation of esophageal carcinoma and the development of esophageal carcinoma, so as to lay a foundation for early diagnosis and treatment of esophageal carcinoma. Methods in our previous study, high throughput sequencing technique was used to detect the loss of TMEM176A gene expression in colon cancer. So we used semi-quantitative RT-PCR and methylation-specific PCR to screen the expression and methylation of TMEM176A gene in colon cancer cell line. We found that TMEM176A methylation occurred frequently in colon cancer resulting in its loss of expression. Then, the expression and methylation of TMEM176A gene in esophageal cancer cell line were studied by using the above method. The results of methylation specific PCR were confirmed by sulfidation sequencing, and 5-AZA was applied to treat the cells lacking expression due to methylation. To investigate the regulation of methylation on the expression of TMEM176A, the possibility of TMEM176A methylation as a diagnostic marker for esophageal carcinoma was explored by detecting the methylation of TMEM176A gene in 103 cases of primary esophageal carcinoma. At the same time, we will clone TMEM176A gene and explore the role and mechanism of TMEM176A in esophageal cancer cell line. Results We found that TMEM176A gene was frequently methylated in colon cancer and its expression was regulated by methylation of promoter region. TMEM176A was absent in esophageal cancer cell line TE1,TE3,TE13,KYSE140, KYSE180,KYSE410,KYSE450,KYSE520,Seg1,YES2,Colo680 and the promoter region was completely methylated. The expression of TMEM176A was high in BiCl cell line and the promoter was demethylated. The expression of TMEM176A in TE1,TE3,TE7,KYSE150,KYSE410,KYSE510 cells recovered or increased after 5-AZA treatment. These results suggest that the expression of TMEM176A in esophageal cancer cell lines is regulated by the methylation of its promoter region. TMEM176A is frequently methylated in the promoter region of primary esophageal carcinoma, and the incidence of methylation is 61.2% (63 / 103) hypermethylation of TMEM176A and the tumor size of esophageal carcinoma. There was no significant correlation among stages, differentiation and lymph node metastasis (P0.05). Conclusion the expression of TMEM176A in esophageal carcinoma cell line is regulated by DNA methylation and hypermethylation occurs frequently in primary esophageal carcinoma. Its biological function and clinical significance need further study.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.1
【相似文献】
相关博士学位论文 前1条
1 张游;TMEM176A基因启动子区在食管癌中的异常甲基化改变[D];中国人民解放军医学院;2015年
，本文编号：2214961
本文链接：https://www.wllwen.com/yixuelunwen/zlx/2214961.html
