EBV相關(guān)胃癌及鼻咽癌中病毒潛伏期基因啟動子甲基化狀態(tài)的研究
發(fā)布時間：2018-10-31 21:07
【摘要】： EB病毒(Epstein-Barr virus, EBV)是重要的DNA腫瘤病毒,屬人類皰疹病毒科γ皰疹病毒亞科成員,與人類多種惡性腫瘤密切相關(guān)。EBV相關(guān)胃癌(EBV-associated gastric carcinoma, EBVaGC)及鼻咽癌組織中病毒基因表現(xiàn)為限制性表達,可能與病毒利用表觀遺傳機制,尤其是利用DNA甲基化來調(diào)控病毒基因啟動子的活性。研究表明,EBV相關(guān)腫瘤組織中病毒可借甲基化沉默部分基因,導致相關(guān)蛋白表達水平的降低,因而對宿主免疫系統(tǒng)的刺激也減弱,病毒借此來逃避宿主免疫攻擊,同時也提示針對病毒基因表觀改變的靶向去甲基化作用有可能成為治療EBV陽性腫瘤的新方法。 目的研究分析青島地區(qū)EBVaGC及鼻咽癌組織中EBV主要潛伏期編碼基因啟動子甲基化狀態(tài)。 方法選擇32例EBVaGC及20例EBV陽性鼻咽癌(nasopharyngeal carcinoma,NPC)作為研究對象,以EBV陽性細胞系Raji, B95-8和Akata作為對照,采用甲基化特異性PCR (Methylation-Specific PCR, MSP)及亞硫酸氫鹽-基因組測序法(Bisulfite genomic sequencing, BGS)檢測分析其EBV核抗原編碼基因啟動子Cp,Wp和Qp以及EBV潛伏膜蛋白LMP1和LMP2A編碼基因啟動子的甲基化狀態(tài)。 結(jié)果①EBV陽性細胞系中EBV編碼基因啟動子甲基化狀態(tài)表現(xiàn)為：B95-8細胞系Cp未發(fā)生甲基化,Raji細胞系Cp呈高甲基化,但在Akata細胞系同時檢測到甲基化和未甲基化的Cp。Raji和Akata細胞系Wp呈高甲基化,但在B95-8細胞系中則表現(xiàn)為部分未甲基。3種細胞系中均未檢測到Qp、LMPlp和LMP2Ap的甲基化。②EBVaGC及鼻咽癌腫瘤標本中EBV核抗原編碼基因啟動子的甲基化狀態(tài)與腫瘤細胞的潛伏感染類型基本一致：Cp和Wp呈高甲基化狀態(tài),而Qp未發(fā)生甲基化。③EBVaGC和EBV陽性鼻咽癌組織中LMP1和LMP2A編碼基因啟動子甲基化狀態(tài)有差別,二者在EBVaGC組織中的甲基化程度明顯高于EBV陽性鼻咽癌(LMP1：χ2=19.1462, P0.0001; LMP2Ap:χ2=11.0139, P=0.0009)。 結(jié)論EBV相關(guān)胃癌和EBV陽性鼻咽癌組織中病毒潛伏期基因啟動子的甲基化狀態(tài)不同,甲基化是調(diào)控EBV潛伏期基因啟動子活動的重要機制之一。
[Abstract]:EB virus (Epstein-Barr virus, EBV) is an important DNA tumor virus, belonging to the subfamily of human herpesviridae 緯 herpesvirus. EBV associated gastric cancer (EBV-associated gastric carcinoma,) is closely related to various human malignant tumors. EBVaGC) and nasopharyngeal carcinoma (NPC) tissues, which may be associated with the use of epigenetic mechanisms, especially the use of DNA methylation to regulate the activity of viral gene promoters. Studies have shown that viruses in tumor tissues associated with EBV can silence some genes by methylation, leading to a decrease in the level of expression of related proteins, which weakens the stimulation of the host immune system, thereby evading the host immune attack. It is also suggested that the targeted demethylation targeting the epigenetic changes of virus genes may be a new method for the treatment of EBV positive tumors. Objective to study the methylation status of promoter of EBV major latent gene in EBVaGC and nasopharyngeal carcinoma tissues in Qingdao. Methods 32 cases of EBVaGC and 20 cases of EBV positive nasopharyngeal carcinoma (nasopharyngeal carcinoma,NPC) were studied. The EBV positive cell lines Raji, B95-8 and Akata were used as control, and the methylation specific PCR (Methylation-Specific PCR,) was used. MSP) and (Bisulfite genomic sequencing, BGS) were used to detect the methylation status of the promoter of EBV nuclear antigen encoding gene Cp,Wp and Qp, as well as EBV latent membrane protein LMP1 and LMP2A gene promoter. Results the methylation status of the promoter of EBV coding gene in 1EBV positive cell lines was as follows: no methylation occurred in B95-8 cell line Cp, but hypermethylation in Raji cell line Cp. However, hypermethylation of both methylated and unmethylated Cp.Raji and Akata Wp was detected in Akata cell line, but Qp, was not detected in B95-8 cell line. Methylation of LMPlp and LMP2Ap. The methylation status of EBV nucleoantigen gene promoter in 2EBVaGC and nasopharyngeal carcinoma samples was basically consistent with the latent infection type of tumor cells: Cp and Wp were hypermethylated. However, there was no methylation in Qp. The methylation status of LMP1 and LMP2A gene promoter in 3EBVaGC and EBV positive nasopharyngeal carcinoma tissues was significantly higher than that in EBV positive nasopharyngeal carcinoma tissues (LMP1: 蠂 2, 19.1462, P 0.0001). LMP2Ap: 蠂 2: 11.0139, P = 0.0009). Conclusion the methylation status of viral latent gene promoter in EBV associated gastric cancer and EBV positive nasopharyngeal carcinoma is different. Methylation is one of the important mechanisms that regulate the promoter activity of EBV latency gene.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R735.2;R739.63
本文編號：2303553
[Abstract]:EB virus (Epstein-Barr virus, EBV) is an important DNA tumor virus, belonging to the subfamily of human herpesviridae 緯 herpesvirus. EBV associated gastric cancer (EBV-associated gastric carcinoma,) is closely related to various human malignant tumors. EBVaGC) and nasopharyngeal carcinoma (NPC) tissues, which may be associated with the use of epigenetic mechanisms, especially the use of DNA methylation to regulate the activity of viral gene promoters. Studies have shown that viruses in tumor tissues associated with EBV can silence some genes by methylation, leading to a decrease in the level of expression of related proteins, which weakens the stimulation of the host immune system, thereby evading the host immune attack. It is also suggested that the targeted demethylation targeting the epigenetic changes of virus genes may be a new method for the treatment of EBV positive tumors. Objective to study the methylation status of promoter of EBV major latent gene in EBVaGC and nasopharyngeal carcinoma tissues in Qingdao. Methods 32 cases of EBVaGC and 20 cases of EBV positive nasopharyngeal carcinoma (nasopharyngeal carcinoma,NPC) were studied. The EBV positive cell lines Raji, B95-8 and Akata were used as control, and the methylation specific PCR (Methylation-Specific PCR,) was used. MSP) and (Bisulfite genomic sequencing, BGS) were used to detect the methylation status of the promoter of EBV nuclear antigen encoding gene Cp,Wp and Qp, as well as EBV latent membrane protein LMP1 and LMP2A gene promoter. Results the methylation status of the promoter of EBV coding gene in 1EBV positive cell lines was as follows: no methylation occurred in B95-8 cell line Cp, but hypermethylation in Raji cell line Cp. However, hypermethylation of both methylated and unmethylated Cp.Raji and Akata Wp was detected in Akata cell line, but Qp, was not detected in B95-8 cell line. Methylation of LMPlp and LMP2Ap. The methylation status of EBV nucleoantigen gene promoter in 2EBVaGC and nasopharyngeal carcinoma samples was basically consistent with the latent infection type of tumor cells: Cp and Wp were hypermethylated. However, there was no methylation in Qp. The methylation status of LMP1 and LMP2A gene promoter in 3EBVaGC and EBV positive nasopharyngeal carcinoma tissues was significantly higher than that in EBV positive nasopharyngeal carcinoma tissues (LMP1: 蠂 2, 19.1462, P 0.0001). LMP2Ap: 蠂 2: 11.0139, P = 0.0009). Conclusion the methylation status of viral latent gene promoter in EBV associated gastric cancer and EBV positive nasopharyngeal carcinoma is different. Methylation is one of the important mechanisms that regulate the promoter activity of EBV latency gene.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R735.2;R739.63
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