EBV-LMP1与抑癌基因WWOX失活在鼻咽癌发生中的机制研究

Guangxi is the high-incidence area of nasopharyngeal carcinoma (NPC) throughout the country. Early in our project, we first found the decreased expression level of tumor suppressor gene WWOX in NPC tissue. Furthermore, the lower expression level of WWOX was found along with the NPC progression and was also associated with lymph node metastasis. Althouh we know that it is mainly due to the methylation in promoter of WWOX gene, the down-regulation mechanism is still not clear. As nasopharyngeal carcinoma is closely related to the EBV infection, and the methylation of tumor supressor gene can also be induced by EBV. Therefore, we intend to regard LMP1 (the main oncogene of EBV) as a breakthrough point. The technology of gene transfection, siRNA silencing and differential proteomics analysis are applied to clarify whether LMP1 has influence on gene promoter methylation of WWOX and discuss the mechanism that EBV induces WWOX inactivation. Furthermore, we will study whether the interaction between LMP1 and WWOX has influence on downstream genes to get knowledge of downstream regulation network and key control protein molecules, which contributes to selecting biomarker for eary diagnosis of NPC and providing new idea for development of new therapeutic targets.

广西是全国鼻咽癌（NPC）高发区。本课题前期研究首次发现抑癌基因WWOX在NPC组织中表达降低，且随患者病情进展而逐步下降，并与淋巴结转移相关。其下调的主要原因是由于启动子甲基化所致，但其机制仍未清楚。鼻咽癌与EBV感染密切相关，而EBV可诱导抑癌基因甲基化，本课题拟以EBV的主要致癌基因LMP1为切入点，采用基因转染技术、siRNA沉默技术及差异蛋白质组学分析，研究EBV主要致瘤成分LMP1对WWOX基因启动子甲基化的影响，探讨 EB病毒诱导WWOX失活的机制。进一步研究鼻咽癌LMP1与WWOX相互作用对下游基因的影响，阐明其下游调节网络途径及关键的调控蛋白质分子，为筛选早期诊断的生物标志物、研发新治疗靶标提供新思路。

广西是全国鼻咽癌（NPC）高发区。本课题组前期研究首次发现抑癌基因WWOX在NPC组织中表达降低，且随患者病情进展而逐步下降，并与淋巴结转移相关。本课题采用基因转染技术、siRNA沉默技术、人基因表达谱芯片及生物信息学分析、HCS细胞增殖筛选、细胞功能实验、鼻咽癌组织样本免疫组化和qPCR的方法，研究导致WWOX在鼻咽癌中表达下调的上游调控机制及下游相关调控蛋白质分子、调节网络，发现LMP1通过AKT-mTOR信号通路和NF-κB通路调控WWOX的表达； SETD2基因在鼻咽组织和鼻咽癌细胞株的表达下降及其与EBV-LMP1基因表达相关；WWOX通过G2阻滞和caspase 3通路抑制鼻咽癌细胞的增殖，诱导细胞凋亡；人基因表达谱芯片分析WWOX过表达前后基因mRNA表达水平变化，经过筛选得到有显著差异上调基因数772个，下调基因数340个，经IPA分析软件分析，推测目的基因WWOX在人鼻咽癌细胞上调控Role of BRCA1 in DNA Damage Response、Estrogen-mediated S-phase Entry和Protein Kinase A Signaling等通路，并提示该基因在proliferation of cells、Viral Infection和organismal death等疾病和功能中发挥作用；根据基因表达谱芯片数据，按照WWOX过表达后表达量下调最明显的方向进行排序，HCS细胞增殖筛选了56个基因，最终选择ERMAP基因并进行细胞生物学功能实验，发现shRNA慢病毒沉默ERMAP基因后可抑制鼻咽癌细胞的增殖，克隆形成及诱导细胞凋亡；通过生物信息学分析及qRT-PCR验证，获得了NUAK1、CDCA2共两个与鼻咽癌与正常黏膜组织表达有差异的基因，为筛选诊断生物标志物、研发新治疗靶标提供新思路。