RSAD2抗EB病毒复制及其促进鼻咽癌细胞侵袭转移的分子机制

The recurrence and metastasis of nasopharyngeal carcinoma (NPC) is always responsible for poor outcome and patients’ death, yet the mechanism for NPC remains unclear. RSAD2, also known as Viperin, is the interferon-induced cellular membrane factor responsible for the suppression of a variety of enveloped viruses, through the restriction of virions release from the host cell surface. Epstein-Barr virus (EBV) has been implicated as an etiological factor in NPC, a malignant epithelial cancer frequently occurred in South China. However, the mechanism for EBV infection and its evasion of innate immune remained to be elucidated. We found that the mRNA of RSAD2 was induced by EBV infection through comparing the transcriptomics of the NPC tumor tissues and normal nasopharyngeal tissues as well as nasopharyngeal epithelial cells with or without EBV infection. The mRNA and protein level of RSAD2 was induced by EBV infection. While over-expression of RSAD2 inhibited the replication of EBV. More than 90% of NPC patients in Southern area of China are EBV seropositive. RSAD2 protein expression was higher in the NPC tumor tissues compared with non-cancerous samples. In vitro experiments showed that over-expression of RSAD2 enhanced the metastasis of nasopharyngeal carcinoma. Taken together, these results suggested that RSAD2 suppressed the virus replication and involved in the pathology of NPC. In addition, EBV may evade the innate immune in nasopharyngeal epithelial cells. In this study, we plan to identify the EBV encoding genes responsible for the interaction with RSAD2 to elucidate the mechanism for the evasion of innate immune by EBV. This study will lead to enhance our understanding how EBV evades the innate immune of nasopharyngeal epithelial cells. In addition, the results will provide molecular evidence to explore novel antiviral interventions and novel therapeutic target for NPC.

RSAD2又叫Viperin，是干扰素诱导的抗病毒天然免疫分子，限制多种囊膜病毒的释放。EB病毒与鼻咽癌密切相关，其逃逸宿主天然免疫机制尚不清楚。通过分析EB病毒感染鼻咽上皮细胞前后表达谱芯片和鼻咽癌转录组数据，我们发现EB病毒感染能诱导RSAD2 mRNA表达。进一步发现过表达RSAD2抑制EB病毒复制；中国华南地区鼻咽癌高发且90%以上是EB病毒阳性，提示EB病毒具有逃逸RSAD2天然免疫机制；同时我们还发现RSAD2的表达促进鼻咽癌细胞转移；这些表明RSAD2不但具有抗EB病毒天然免疫功能，同时也参与鼻咽癌的转移，但其具体机制并不清楚。本项目拟通过过表达和knockdown体系、全基因表达谱芯片、免疫亲和沉淀、Transwell 侵袭实验和体内成瘤实验等技术研究RSAD2抗EB病毒、病毒逃逸天然免疫和促进鼻咽癌细胞转移的分子机制，以其为鼻咽癌的防治提供新的思路和靶点。

EB病毒(Epstein-Barr Virus，EBV)属于γ疱疹病毒科，是第一个被发现与人类肿瘤相关的DNA病毒，与鼻咽癌、10%胃癌和淋巴瘤等密切相关，其逃逸宿主的天然免疫机制尚不清楚。RSAD2又叫Viperin，是干扰素诱导的抗病毒天然免疫分子，限制多种囊膜病毒的释放。通过分析EB病毒感染鼻咽上皮细胞前后表达谱芯片和鼻咽癌转录组数据，发现EB病毒感染能诱导RSAD2 mRNA表达；进一步验证发现EB病毒促进RSAD2在转录和蛋白水平的表达；发现RASD2的表达抑制EB病毒的复制以及EB病毒相关裂解基因的表达。通过免疫亲和沉淀、SDS-PAGE和质谱等技术发现可能与RSAD2潜在互作蛋白包括Prohibitin, Electron transfer flavoprotein, RPS4X protein等。发现RSAD2的表达受到EB病毒基因LMP2A的调控，并且发现LMP2A促进鼻咽癌细胞侵袭的能力可能部分依赖RSAD2的表达，干扰RSAD2的表达后对肿瘤细胞的增殖没有影响。进一步采用RNA-Seq测序技术发现RSAD2能够激活神经活动配体-受体相互作用，细胞因子-细胞因子受体相互作用和补体途径等信号通路，干扰RSAD2的表达后促进CXCL2和CXCL9的表达，抑制IL-8的表达。通过对RSAD2在EB病毒感染和鼻咽癌发生发展过程中的深入研究，有助于阐明抗病毒分子RSAD2促进肿瘤转移新的生物学功能，同时将为EB病毒LMP2A通过调控天然免疫分子RSAD2促进鼻咽癌的发生提供证据，为鼻咽癌的防治提供新的治疗手段和靶点。同时结合单细胞拉曼技术、表观遗传和质谱等手段，从单细胞层面、蛋白翻译后修饰和氨基酸代谢角度阐明EB病毒在鼻咽癌发生发展中的作用机制，发现通过单细胞拉曼图谱指纹结合机器学习可准确区分是否感染EB病毒的癌细胞；发现蛋白的琥珀酰化、巴豆酰化修饰随着EB病毒的急性感染逐渐减弱；发现谷氨酸代谢在鼻咽癌发生发展过程中发挥重要作用，这些研究为EB病毒相关的肿瘤抗病毒防治提供新的理论依据和靶点。