TMEM176A基因啟動子區(qū)在食管癌中的異常甲基化改變
發(fā)布時間：2018-08-31 12:17
【摘要】：背景食管癌是世界上最常見的惡性腫瘤之一,在癌癥相關死亡率中位于第六位。食管腺癌和鱗狀細胞癌是食管癌最為常見的兩種類型,食管癌的發(fā)病具有明顯的地區(qū)差異性,男女發(fā)病無明顯差異,其中,腺癌在西方國家比較常見,鱗狀細胞癌主要見于亞洲地區(qū)。在美國,每年估計有17040例的新發(fā)食管癌患者,另有15070人死于食管癌。2012年,食管癌在我國的發(fā)病率為18.6%,同時,我國食管癌的發(fā)生率是美國的20-30倍。食管癌的形成是一個多階段的發(fā)展過程,包括：基底細胞的過度增生、異型增生、原位癌、進展期癌。許多的遺傳學改變與表觀遺傳學變異參與到了食管癌的發(fā)生過程中,特別是抑癌基因的DNA高甲基化在食管癌中頻繁發(fā)生�？缒さ鞍�176 (transmembrane protein 176, TMEM176)是與跨膜蛋白4A家族相關的蛋白,存在于哺乳動物和斑馬魚類,TMEM176A是其中的一種亞型,位于7q36.1,在多個腫瘤中發(fā)現(xiàn)該區(qū)域存在等位基因的缺失。已有研究報道在乳腺癌中發(fā)現(xiàn)TMEM176A的異常DNA甲基化,但TMEM176A基因在食管癌中的甲基化狀態(tài)及功能尚不清楚。目的探討TMEM176A基因在食管癌中的甲基化情況及食管癌發(fā)生發(fā)展中的作用,為食管癌的早期診斷及治療奠定基礎。方法在我們前期研究中,通過高通量測序技術在結(jié)腸癌中發(fā)現(xiàn)TMEM176A基因表達缺失,因此我們采用半定量RT-PCR與甲基化特異性PCR的方法對TMEM176A基因在結(jié)腸癌細胞系中的表達及甲基化情況進行初步的篩選,發(fā)現(xiàn)TMEM176A在結(jié)腸癌中頻發(fā)發(fā)生甲基化而導致其表達缺失。然后應用上述方法在食管癌細胞系中探討TMEM176A基因的表達及甲基化情況,采用硫化測序證實甲基化特異性PCR的結(jié)果,并應用5-AZA處理因甲基化而缺失表達的細胞,探討甲基化對TMEM176A表達的調(diào)控,通過在103例原發(fā)性食管癌中檢測TMEM176A基因的甲基化情況,探討TMEM176A甲基化作為食管癌診斷標志物的可能性。同時,我們還將克隆TMEM176A基因,在食管癌細胞系中探討TMEM176A的作用及作用機制。結(jié)果我們發(fā)現(xiàn)TMEM176A基因在結(jié)腸癌中頻發(fā)甲基化且其表達受啟動子區(qū)甲基化的調(diào)控。TMEM176A在食管癌細胞系TE1、TE3、TE13、KYSE140、 KYSE180、KYSE410、KYSE450、KYSE520、Seg1、YES2、Colo680中表達缺失,同時啟動子區(qū)呈現(xiàn)完全甲基化狀態(tài)；在BiCl細胞系中TMEM176A呈高表達,且啟動子區(qū)呈現(xiàn)非甲基化狀態(tài)。去甲基化藥物5-AZA處理以后,TMEM176A在TE1、TE3、TE7、KYSE150、KYSE410、KYSE510細胞系中的表達恢復或增加。這些結(jié)果表明在食管癌細胞系中TMEM176A的表達受其啟動子區(qū)甲基化的調(diào)控。TMEM176A在原發(fā)性食管癌中頻繁發(fā)生啟動子區(qū)的甲基化,甲基化發(fā)生率為61.2%(63/103),TMEM176A的高甲基化與食管癌的腫瘤大小、分期、分化程度、淋巴結(jié)轉(zhuǎn)移沒有明顯的相關性(P0.05)。結(jié)論TMEM176A在食管癌細胞系中的表達受DNA甲基化的調(diào)控,在原發(fā)性食管癌中頻繁發(fā)生高甲基化。其生物學功能及臨床意義有待進一步研究。
[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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.1
本文編號：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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.1
【相似文獻】
相關博士學位論文 前1條
1 張游;TMEM176A基因啟動子區(qū)在食管癌中的異常甲基化改變[D];中國人民解放軍醫(yī)學院;2015年
，本文編號：2214961
本文鏈接：http://sikaile.net/yixuelunwen/zlx/2214961.html
最近更新
教材專著
