EB病毒LMP1通过激活c-myc上调CD47的表达介导鼻咽癌免疫逃避及机制

CD47, which is highly expressed in the tumor and mediates immune evasion through the "don’t eat me" signal, has become a new target of immunotherapy. Our previous study found that CD47 was particularly expressed in EBV-positive nasopharyngeal carcinoma cells and was associated with poor prognosis. It has been reported that MYC can directly regulate the expression of CD47, and LMP1 and c-myc can synergistically regulation of tumorigenesis. Here, we hypothesize whether the proto-oncogene encoded by exogenous Epstein-Barr virus during infection can activate c-myc, which then transcriptionally regulate CD47 expression involved in immune escape? We will firstly verify the role of CD47 on the biological behavior of nasopharyngeal carcinoma cells and its participation in immune escape, and then explore the prospective mechanisms by which the EB virus encoding transforming protein LMP1 activates endogenous c-myc which up-regulates the expression of CD47 to mediate immune escape. Through this study, we will clarify the significance of CD47 expression in nasopharyngeal carcinoma and provide experimental evidence for the administration of anti-CD47 in nasopharyngeal carcinoma. It will also reveal the mechanism of LMP1 in immune escape by up-regulation CD47 expression through activating c-myc and enrich the EBV-related tumor immune escape theory.

目前，CD47因高表达于肿瘤并通过“别吃我”信号介导免疫逃避，已成为新的免疫治疗靶点。我们前期研究发现CD47尤高表达于EBV阳性鼻咽癌细胞并与不良预后相关。已有研究报道原癌基因MYC可直接调控CD47的表达，而LMP1与c-myc在肿瘤发生上存在协同调控作用。据此我们推测：在外源性EB病毒感染致瘤过程中，其编码的癌基因可能激活原癌基因c-myc，后者再直接调控CD47的表达参与免疫逃避。首先我们验证CD47对鼻咽癌细胞生物学行为的影响及参与免疫逃避的作用，再探讨EB病毒编码转化蛋白LMP1通过激活内源性癌基因c-myc上调CD47的表达参与免疫逃避的作用及具体机制。通过本课题的研究，我们将阐明CD47的表达在鼻咽癌中的意义，为anti-CD47治疗在鼻咽癌中应用提供实验依据；并揭示LMP1通过激活c-myc上调CD47的表达参与免疫逃避的作用及可能机制，丰富EB病毒相关肿瘤免疫逃避理论。

鼻咽癌高发于我国南方地区，复发转移仍是非转移性鼻咽癌治疗失败的主要原因，尚缺乏有效的预测因子。现研究普遍认为CD47高表达于恶性肿瘤组织中并与不良预后相关，其主要通过CD47/SIRPα信号轴介导肿瘤免疫逃避，是潜在治疗靶点。为了解鼻咽癌组织中CD47表达及其临床意义并探索CD47上游调控机制。我们检测了CD47、LMP1、CD68、miR-429在鼻咽癌组织及细胞中表达情况，并分析了CD47、LMP1、CD68表达与临床特征的关系；分析了miR-429对鼻咽癌细胞生物行为的影响，探讨了miR-429对CD47表达的影响；另分析和探索了EB病毒感染对鼻咽癌细胞CD47的表达影响及机制。结果表明CD47在鼻咽癌组织中的阳性表达率为56.1%；其阳性表达与淋巴结分期(N分期)、年龄、EBV-DNA定量水平及疾病复发均明显相关并预示更差的生存期；多因素分析提示CD47是独立的预后因子。相关性分析发现LMP1、CD68表达与临床特征无相关性，但与CD47表达呈正相关。miR-429差异性高表达于低转移性细胞系中且过表达miR-429可抑制鼻咽癌细胞系的增殖、迁移和侵袭。双荧光素酶报告基因结果提示miR-429不直接靶向于CD47。在人鼻咽癌细胞系中，EBV阳性的C666-1细胞系中CD47和LMP1的表达明显高于EBV阴性细胞系。CNE-2-EBV+和CNE-2/LMP1细胞中CD47蛋白和mRNA表达明显高于CNE-2细胞和CNE-2/Vector细胞中。应用LMP1-siRNA 敲低CNE-2-EBV+和CNE-2/LMP1细胞中的LMP1表达后CD47蛋白表达随LMP1表达而下调。CNE-2-EBV、CNE-2/LMP1细胞中c-myc、p-stat3、p-NF-κB蛋白表达上调明显而Bim蛋白下调明显，而stat3、NF-κB蛋白变化不显著，LMP1-siRNA 敲低CNE-2/LMP1细胞中LMP1表达后，c-myc、p-stat3、p-NF-κB蛋白表达随LMP1下降而下降。可见，CD47高表达于鼻咽癌中并预示不良预后且与巨噬细胞浸润水平、EB病毒感染相关，是鼻咽癌的潜在治疗靶点，为anti-CD47抗体在鼻咽癌中临床应用提供依据。EB病毒可通过LMP1介导的信号通路上调CD47的表达，丰富了鼻咽癌肿瘤免疫逃避的理论。