EBV-miR-BART11协同调控PD-L1/PD-1促进鼻咽癌免疫逃逸的机制研究

Immune escape is a hallmark of human malignant tumor. Nasopharyngeal carcinoma (NPC) is charactered with the presence of a substantial lymphocytes infiltration around tumor lesions. Highly expressed PD-L1 on surface of NPC cells and PD-1 on tumor-infiltrating T cells indicate that PD-L1/PD-1 might play an important role in the immune escape of NPC, but the underlying mechanisms need further investigation. Epstein-Barr virus (EBV) infection is a clear etiology of the NPC pathogenesis. We have confirmed that EBV infection was positively correlated with the expression of PD-L1 and PD-1. According to our previous research results and literature analysis, the scientific hypothesis and specific aims of this proposed project are as follows: EBV encoded miRNA, named BART11, targets and inhibits the expression of forkhead box P1 (FOXP1), and increases PD-L1 expression through regulating the FOXP1 downtream signaling pathway in NPC cells; EBV-miR-BART11 may also be transfered from NPC cells to adjacent tumor-infiltrating T cells through the exosomes, and then upregulate PD-1 expression in T cells; Coordinately up-regulation of PD-L1 on NPC cells and PD-1 on T cells results in immunosuppressive reaction and facilitates NPC cells escaping immune surveillance, ultimately promoting the occurrence and development of NPC. This study will reveal the molecular mechanism of immune escape from the perspective of the interaction between the oncogenic virus and host genes, and will also provide an important clue for the clinical application of anti PD-L1/PD-1 immunotherapy strategy in NPC.

免疫逃逸是恶性肿瘤的基本特征之一；鼻咽癌组织中常伴大量淋巴细胞浸润，癌细胞表面高表达PD-L1，癌旁浸润的T细胞表面高表达PD-1，提示PD-L1/PD-1在鼻咽癌免疫逃逸中发挥了重要作用，但具体机制有待深入探讨。EB病毒(EBV)感染是鼻咽癌发病明确的病因，我们证实EBV感染与PD-L1和PD-1表达呈正相关，根据我们前期研究结果结合文献分析，本项目的科学假说如下：鼻咽癌细胞中EBV通过其编码的miRNA BART11靶向抑制FOXP1的表达，并通过FOXP1下游信号通路上调PD-L1；同时BART11经外泌体从鼻咽癌转运到癌旁浸润的T细胞中，上调PD-1的表达；最终通过协同调控PD-L1/PD-1使鼻咽癌细胞逃避免疫监视，促进鼻咽癌的发生发展。本项目的完成将从致瘤病毒与宿主基因交互作用的角度明晰鼻咽癌免疫逃逸的分子机制，并为抗PD-L1/PD-1免疫治疗策略用于鼻咽癌临床提供重要参考。

EB病毒感染与鼻咽癌、胃癌及多种淋巴瘤的发生、发展密切相关，但EBV促进PD-L1表达诱导免疫逃逸的具体机制仍需阐明。在本项目中对EBV编码的miRNA对PD-L1在肿瘤免疫逃逸中发挥的作用进行了深入研究。.我们首先对GEO数据、TCGA数据分析，发现PD-L1在鼻咽癌组织中的表达明显高于正常鼻咽上皮，且与EBV感染呈正相关。qRT-PCR、原位杂交和免疫组化检测鼻咽癌和非肿瘤鼻咽上皮组织临床样本中PD-L1的表达，发现PD-L1在鼻咽癌组织中显著高表达，与EBV感染具有显著相关性。.利用qRT-PCR和Western blotting的方法分别在EBV阴性或阳性的肿瘤细胞中检测PD-L1的mRNA和蛋白水平，发现PD-L1在EBV阳性的细胞或感染了完整EBV基因组的Akata-EBV病毒的细胞中均高表达，而在EBV阴性的原代正常鼻咽上皮细胞NP69和EBV阴性细胞中表达很低，EBV对PD-L1的调节不是通过B95-8的EBV miRNA BART区部分缺失片段。过表达BART17-3p、BART11-3p、BART11-5p均能显著促进PD-L1蛋白表达上调，而EBV阳性细胞中敲低BART17-3p、BART11-3p、BART11-5p能下调PD-L1的表达。.生物信息学分析并实验验证BART11通过靶向抑制FOXP1， BART17-3p通过靶向抑制PBRM1, 而FOXP1与PBRM1共同结合于PD-L1的超级增强子区抑制PD-L1表达，BART11和BART17-3p上调PD-L1表达，促进肿瘤免疫逃逸。BART11通过靶向FOXP1的mRNA 3’-UTR抑制FOXP1表达， BART17-3p通过靶向PBRM1的mRNA 3’-UTR抑制PBRM1表达，促进PD-L1表达。FOXP1与PBRM1能共同结合于PD-L1基因的超级增强子区；抑制FOXP1或PBRM1，能促进组蛋白修饰，增强PD-L1基因的转录活性。FOXP1通过与PBRM1所在的PBAF染色质重塑复合物结合，共同作用于PD-L1增强子，竞争性抑制BAF染色质复合物对PD-L1增强子的激活作用；抑制FOXP1或PBRM1，结合于PD-L1增强子的BAF染色质复合物特异性亚基DPF2增多，从而增强PD-L1基因的转录。最终诱导肿瘤周围T细胞的凋亡，抑制T细胞杀伤活性，导致肿瘤免疫逃逸。