胞外囊泡释放的EBV非编码RNA通过模式受体和表观遗传机制激活内皮细胞促进肿瘤血管生成

EBV encoded RNAs (EBERs) consist of two non-caped RNAs without poly-A tail, which can not be tanslated. Despite their high abundance, the role of EBERs in carcinogenesis remain ambiguous. Our preliminary data indicate EBERs promote the transition from inflammation to malignance in nasopharyngeal carcinoma (NPC) via recognition by pattern recognition receptors (PRRs) namely TLR3 and RIG-I and the consequent proinflammatory signaling pathway. Of note, we confirmed EBERs were present in endothelial cells of NPC microenvironment, in spite of their function and underlying mechanisms remain to be elucidated. Based on these observations, in this project we will focus on the accurate role of EBERs in the process of angiogenesis related to NPC progression and the detail of underlying molecular mechanisms. To achieve this, we will first ascertain the involvement of extracellular vesicles (EVs) released EBERs in NPC angiogenesis via EBV positive NPC cell- human umbilical vein endothelial cells (HUVECs) co-culture assay, NPC cell derived Evs- HUVECs assay and tube formation experiment. Next, angiogenesis associated genes included PCR array will be applied to identify the candidate cytokines participating in EBERs promoted NPC angiogenesis. In consideration of their expresson in both cytoplasm and nucleus in HUVECs, we will first explore the involvement of TLR3/RIG-I in cytoplasmic EBERs related HUVECs angiogenesis based on the results of TLR3/RIG-I knockdown, ChIP and EMSA experiments, then we will identify the role of nucleus-located EBERs-PAX5 complexes in the epinegetic regulation of angiogenetic cytokines by the application of CHART and RAP assay. Finally, we will validate these observations with animal experiment and clinical samples. The establishment of Evs released EBERs in the progress of NPC angiogenesis and the unveiling of related signaling pathway will benefit our knowledge toward the function of EBERs and help reducing the healthy load brought by NPC progression.

EBV编码的非翻译RNA（EBERs）在EBV感染细胞中具有极高丰度，但其功能不明确。我们的前期研究显示EBERs通过模式识别受体TLR3/RIG-I识别促进鼻咽癌的炎癌转化，且肿瘤微环境的内皮细胞中也呈EBERs阳性，但功能不明。本项目将研究内皮细胞中EBERs的功能和其中的分子机制。我们首先通过共培养实验、胞外囊泡处理内皮细胞以及血管生成实验确定囊泡递送的EBERs促进鼻咽癌血管生成，并利用PCR array确定促进血管生成的细胞因子；其次通过干扰TLR3/RIG-I、ChIP确定EBERs在细胞质中通过TLR3/RIG-I/NF-kB通路激活细胞因子的转录；进一步通过CHART、RAP实验确定EBERs-PAX5在细胞核中通过表观遗传调控激活细胞因子的转录；最后我们将利用matrigel plug等实验确定胞外囊泡释放的EBERs在体内的促血管生成能力，并在临床样本上验证。

EB病毒非编码RNA（Epstein–Barr病毒编码RNA，EBERs）被认为在淋巴瘤和鼻咽癌（nasopharyngeal carcinoma，NPC）的进展中起关键作用（NPC）。然而，其在肿瘤细胞与间充质细胞之间相互作用方面的功能和精确机制尚未阐明。本项目研究发现，除了NPC细胞外，在EBV感染鼻咽癌组织的内皮细胞中也发现了EBER，这表明可能NPC来源的细胞外囊泡（EV）将EBER传递至内皮细胞。为了证明这一假设，我们首先确定了NPC培养上清液中分离出的胞外囊泡将EBERs转移至内皮细胞。然后，我们证明了胞外囊泡来源的EBERs可以通过促进VCAM-1表达来促进血管生成。最后，我们探讨了TLR3和RIG-I对EBER识别在NPC血管生成中的作用。我们的结果表明鼻咽癌细胞胞外囊泡来源的EBERs促进血管生成的功能和机制，阐明了EBV感染的NPC细胞与肿瘤微环境之间相互作用的机制基础。