ZIC2在鼻咽癌中对EB病毒感染的调控作用及机制研究

EBV infection is correlated to the development of Nasopharyngeal Carcinoma (NPC) and exists in NPC in the latency phase. However, how EBV latent infection is established and maintained in NPC is still elusive. Through RNA-seq, we found that ZIC2 was highly expressed in NPC compared to in nasopharygeal non-tumor tissues. Immunohistochemical analysis showed that the higher expression of ZIC2 was related to the worse survival and might be a potential independent prognosis factor for NPC. In addition, we found that ZIC2 was highly expressed in EBV harboring NPC cell lines, such as C666-1, CNE2-EBV, and HNE1-EBV cells. Knocking-down of ZIC2 decreased the EBV DNA copies. These data suggested that ZIC2 is related to EBV infection. But how ZIC2 regulates EBV infection in NPC is still needed to further explore. Our preliminary data showed that silencing ZIC2 upregulates the expression of IFN pathway related genes. IP and Mass-Spec found that ZIC2 interacted with UPF1 and other Nonsense RNA Degradation (NMD) pathway. Knocking-down of ZIC2 downregulated the expression of EBER1. Therefore, our study is aimed to investigate the underlying direct and indirect mechnisms of how ZIC2 regulates EBV infection by RIP, ChIP-seq, and microarray et al, to eluminate how ZIC2 participate in the pathonogenesis of NPC, providing the insight for prognosis and targeted therapy for NPC.

EB 病毒感染与鼻咽癌的发生、发展密切相关，并以潜伏感染状态存在于鼻咽癌中，但潜伏感染建立和维持机制不清。我们通过RNA测序发现ZIC2在鼻咽癌组织表达显著高于鼻咽非癌组织，免疫组化提示ZIC2高表达与不良预后相关，为独立预后指标。ZIC2在C666-1，CNE2-EBV和HNE1-EBV细胞中高表达，敲低ZIC2使得EB病毒拷贝数下降，提示ZIC2与EB病毒感染有相关性。ZIC2如何在鼻咽癌中调控EB病毒感染？前期研究发现敲低ZIC2可上调IFN等免疫相关基因的表达；免疫共沉淀和质谱鉴定发现UPF1等无义RNA降解信号通路蛋白与ZIC2相互作用，敲低ZIC2降低EBER1基因的表达水平。因此，本项目拟通过RIP、ChIP-seq、表达谱芯片等方法深入探讨ZIC2如何通过直接和间接两种机制共同参与调控EBV感染，诠释ZIC2参与鼻咽癌发生发展的分子机制，为鼻咽癌预后判断和靶向治疗提供基础。

目前已有多种鼻咽癌的治疗方法，包括化疗、生物治疗、手术治疗和放疗。尽管在治疗方面取得了很大进展，但局部复发和远处转移仍是鼻咽癌患者预后不良的主要原因。因此，研究鼻咽癌的发生发展机制，寻找新的分子靶点对鼻咽癌的预后和治疗具有重要意义。我们的研究发现ZIC2在C666-1，CNE2-EBV和HNE1-EBV细胞中高表达，敲低ZIC2使得EB病毒拷贝数下降，提示ZIC2与EB病毒感染有相关性。我们的RNA-seq分析表明，在敲低ZIC2后，TNF-α通路显著下调，MCSF是ZIC2敲低后鼻咽癌细胞中差异表达最显著的细胞因子。有趣的是，通过ZIC2的ChIP-seq分析和RNA-seq分析，我们发现JUNB是ZIC2的下游靶基因，ZIC2可以在-123位点显著诱导JUNB的启动子活性。巨噬细胞与过表达ZIC2的鼻咽癌细胞共培养后，JUNB表达下调会抑制MCSF的分泌和TAM向M2型极化，表明ZIC2直接结合JUNB启动子以诱导JUNB表达，从而促进MCSF分泌和M2表型巨噬细胞极化。总之，我们的结果表明，ZIC2调节其下游靶基因JUNB以诱导TAM向M2表型极化。ZIC2和JUNB的高表达水平与鼻咽癌患者的不良预后呈正相关，这意味着ZIC2、JUNB和CD163可作为鼻咽癌的预后标志物，为癌症免疫疗法的提供新的治疗靶点。