雷帕霉素通过促进EB病毒裂解复制增强NaB/GCV抗胃癌效应的机制研究

Epstein-Barr virus (EBV) is the first discovered human tumor virus, and is associated with multiple malignancies including gastric carcinoma (GC), nasopharyngeal carcinoma (NPC), Burkitt’s lymphoma (BL) and Hodgkin's lymphoma (HL). To date, there is no efficacious drug for the treatment of EBV-positive tumors. Sodium butyrate (NaB), an inducer of EBV lytic cycle, in combination with the antiviral agent ganciclovir (GCV) could effectively inhibit the growth of EBV-associated tumors. However, the short half-life of NaB in vivo has limited the practical clinical application of NaB/GCV in cancer therapy. Our previous study demonstrated that rapamycin (Rapa) could promote NaB-mediated EBV lytic activation, and enhanced the killing effect of NaB/GCV on tumor cells. Based on our preliminary study, this application will focus on the study of EBV-associated gastric carcinoma (EBVaGC), and is proposed including the following three parts. The first part is to confirm the promotive role of Rapa in NaB-mediated EBV lytic replication by detecting expression levels of EBV lytic proteins and the level of viral DNA replication. The second part is to reveal the molecular mechanism underlying the regulation of NaB-mediated EBV lytic replication by Rapa via analyzing the activity and methylation profiling of EBV immediate-early gene promoters. The third part is to clarify the enhancement of the antitumor efficacy of NaB/GCV by Rapa through establishing an EBVaGC mouse model. This project will provide theoretical evidence for the targeted treatment of EBV-associated tumors.

EB病毒是首个发现的肿瘤相关病毒，与胃癌、鼻咽癌和淋巴瘤等多种恶性肿瘤的发生密切相关。目前尚无有效治疗EB病毒相关肿瘤的药物。EB病毒活化剂丁酸钠（NaB）与抗病毒药物更昔洛韦（GCV）联合用药能够有效抑制病毒相关肿瘤的生长。然而，NaB在体内的半衰期较短，限制了NaB/GCV在EB病毒相关肿瘤治疗中的应用。我们在前期研究中发现雷帕霉素能够促进NaB介导的EB病毒裂解活化，并增强NaB/GCV对肿瘤细胞的杀伤作用。本项目以EB病毒阳性胃癌细胞为研究对象，通过检测EB病毒裂解蛋白表达水平与病毒DNA复制水平，明确雷帕霉素对NaB介导的EB病毒裂解复制的促进作用；通过分析EB病毒立早基因启动子的活性与甲基化状态，揭示雷帕霉素调控NaB介导的EB病毒裂解复制的分子机制；通过监测荷瘤小鼠的肿瘤发展，阐明雷帕霉素对NaB/GCV抗肿瘤效应的增强作用，为EB病毒相关肿瘤的靶向治疗提供重要的理论依据。

EB病毒是一种感染率高且具有致癌性的疱疹病毒，EB病毒潜伏感染与肿瘤的发生和发展密切相关。本研究项目以EB病毒阳性胃癌细胞为研究对象，验证雷帕霉素对丁酸钠（NaB）介导的EB病毒裂解复制的调控作用，阐明雷帕霉素促进EB病毒裂解复制的分子机制，揭示雷帕霉素与NaB/更昔洛韦（GCV）联用在EB病毒相关胃癌发展中的作用。研究进展如下：.一、雷帕霉素对NaB介导的EB病毒裂解复制的调控作用。（1）Western杂交结果显示，雷帕霉素增强NaB对EB病毒裂解蛋白Zta，Rta，TK，EAD和DNase表达的调控作用。（2）Real-time PCR检测结果显示，雷帕霉素能够显著提高SNU-719细胞内和培养上清中EB病毒DNA拷贝数。（3）γ放射免疫分析结果显示，雷帕霉素能够显著提高胸苷激酶（TK）特异性底物125I-FIAU在SNU-719细胞内的聚集。.二、雷帕霉素调控EB病毒裂解活化的分子机制。（1）Real-time PCR检测结果显示，雷帕霉素能够提高EB病毒立早基因BZLF1和BRLF1的mRNA表达水平。（2）荧光素酶活性分析实验结果表明，雷帕霉素能够增强Zta启动子（Zp）和Rta启动子（Rp）的转录活性。（3）亚硫酸氢钠测序结果显示，雷帕霉素能够增强NaB对Zp和Rp甲基化的抑制作用。（4）通过染色质免疫共沉淀检测，发现雷帕霉素能够增强转录因子Sp1与Zp和Rp的结合活性。（5）鉴定了参与雷帕霉素调控EB病毒裂解活化的ATM/p53信号通路，该通路通过诱导Sp1的活化，促进EB病毒立早蛋白Zta表达。.三、雷帕霉素在EB病毒相关胃癌发展中的作用。（1）雷帕霉素与NaB/GCV联用能够抑制SNU-719细胞增殖，促进SNU-719细胞凋亡，并提高细胞内ROS水平。（2）雷帕霉素与NaB/GCV联用能够抑制小鼠肿瘤的生长，上调肿瘤组织中裂解基因（BZLF1和BRLF1）的mRNA表达水平，并提高肿瘤组织中EB病毒DNA拷贝数。（3）揭示雷帕霉素抑制EB病毒相关胃癌发展的作用机制，雷帕霉素与NaB/GCV联用能够上调小鼠肿瘤组织中凋亡相关蛋白（PARP剪切体和caspase-3剪切体）的表达。这一结果表明，雷帕霉素与NaB/GCV联用通过诱导肿瘤细胞凋亡，从而抑制小鼠肿瘤的生长。