EBV下调miR-34c对促进鼻咽癌增殖转移的作用及机制研究

A lot of research shows that Epstein-Barr virus (EBV) infection and abnormal expression of microRNAs are important to study the pathogenesis of nasopharyngeal carcinoma (NPC). Our previous studies have found the low expression of miR-34c in NPC, which was involved in tumor proliferation and metastasis. And the protein EBNA1 encoded by EBV promoted the NPC metastasis. But the relationship between miR-34c downregulation and EBV is not yet clear. To figure this out, we found miR-34c was downregulated in EBNA1 positive NPC cells, which was associated with methylation levels. Therefore, we speculated that EBNA1 rescued the miR-34c expression by inducing miR-34c promotor hypermethylation, and regulated the target gene expression to influence the tumor progression. In this project, we will explore the association between miR-34c methylation and EBV infection. Next, we will analyze the molecular mechanisms of the miR-34c promotor hypermethylation induced by EBNA1, and reveal the key role of EBNA1 downregulating miR-34c in NPC proliferation and metastasis. In our study, we want to take further step to elucidate the mechanism of EBV in NPC, which will provide better understanding for developing molecular targeting treatment of NPC.

大量研究表明：EB病毒感染和miRNA的异常表达在鼻咽癌的发生发展中扮演重要作用。课题组前期研究发现miR-34c在鼻咽癌中表达下调，与肿瘤的增殖转移密切相关；同时也发现EB病毒的EBNA1蛋白促进鼻咽癌细胞的转移。然而，EB病毒与miR-34c下调之间的关系及机制不明。为明确二者的关系，课题组在过表达EBNA1的鼻咽癌细胞中发现miR-34c表达量降低，并与甲基化水平相关。据此，我们提出科学假说：EBNA1通过诱导miR-34c启动子甲基化下调其表达，进而调控相关靶基因参与鼻咽癌的增殖转移。本课题拟探讨miR-34c甲基化与EB病毒的关系，应用染色质免疫共沉淀等技术明确EBNA1致miR-34c甲基化的机制，通过动物实验证明EBNA1致miR-34c甲基化在促进鼻咽癌的增殖与转移中的关键作用。通过该研究，将进一步揭示EB病毒在鼻咽癌中的作用机制，为研究其分子靶向治疗提供更坚实的理论依据。

研究背景：.EBV作为鼻咽癌特异性的致病因素，探讨它如何促进肿瘤的发生发展对于突破临床治疗瓶颈具有重要意义。而m6A修饰是一类广泛存在于真核生物mRNA上的转录后修饰，可以导致促癌基因和抑癌基因的异常表达，并驱动了肿瘤的发生和恶性进展。已有文献表明m6A修饰与鼻咽癌密切相关，但尚无EBV与m6A修饰在鼻咽癌中的研究报道。因而我们尝试利用m6A修饰来解释EBV的致病机理，更深入地探讨EBV导致肿瘤形成的分子机制。.实验方法：.我们采用qRT-PCR、western blot技术筛选EBNA1阳性细胞株中m6A修饰相关蛋白的差异表达。瞬时转染及慢病毒包装转染构建敲低METTL14等m6A修饰相关蛋白表达细胞株。再通过细胞功能实验检测处理后细胞增殖迁移能力。利用斑点免疫印记检测各类细胞的m6A修饰水平。结合免疫组化实验验证METTL14在鼻咽癌组织中表达水平。在体内动物实验，我们通过皮下成瘤验证METTL14对肿瘤形成的影响。.实验结果：.在过表达EBNA1鼻咽癌细胞株中检测发现miR-34C表达水平下调，且细胞增殖及迁徙能力增加。EBNA1阳性细胞m6A修饰水平升高。通过筛选，我们得到差异表达的m6A修饰蛋白METTL14。并发现下调METTL14表达可抑制鼻咽癌细胞增殖能力，并可逆转miR-34C下调情况。更进一步的实验表明METTL14与BCL2表达呈正相关，下调METTL14可降低BCL2表达水平，而过表达miR-34C同样可导致BCL2表达下调。.结论：.综上所述，m6A修饰在EBV导致鼻咽癌形成的过程中具有不可忽视的作用，EBNA1可能通过METTL14/miR34C轴调节BCL2表达促进肿瘤增殖。本研究为探讨EBV致癌机制，临床制定分子靶向治疗提供更坚实的理论基础。