miR-143/145簇与DNMTs反馈通路调控EBV相关型胃癌抗病毒治疗的研究

Epstein-Barr virus-associated gastric cancers (EBVaGCs) is one of the four subtypes of gastric cancer classified by The Cancer Genome Atlas (TCGA) project, which is latently infected by Epstein-Barr virus (EBV), with the typical epigenetic changes of extreme CpG island methylator phenotype (CIMP). We previously found that miR-143/145 cluster was down-regulated with hypermethylation in its promoter region and various DNA methyltransferases (DNMTs) were direct target genes of miR-143/145 cluster miRNAs, so we proposed that the feedback between miR-143/145 cluster and DNMTs could exist in EBVaGCs. We further found that the EBV status of latent infection could be switched to lytic infection mediated by Rta via intervention of the above feedback in EBVaGCs. Once the lytic cycle started, the EBV protein kinase EBV-PK would be induced by Rta, causing the increasement of phosphorylated products of antiviral drugs such as Ganciclovir (GCV) and Acyclovir (ACV), which interfered with DNA synthesis and inhibited tumor growth. Moreover, miR-143/145 cluster could be restored via intervention of the above feedback, resulting in repression of their target oncogenes and enhancing the effects of the antiviral treatment. We will systematicly elucidate the role of the feedback between miR-143/145 cluster and DNMTs involved in the antiviral therapy for EBVaGCs, based on clinical samples detection and molecular mechanism exploration, using DNA methylation assay, miRNA quantitation and transfection, in vitro and in vivo drug sensitivity test, etc.

EB病毒相关型胃癌（EBVaGCs）是胃癌TCGA分型的重要亚型，DNA极度超甲基化构成其典型的表观遗传学改变，EB病毒在EBVaGCs中呈潜伏感染。课题组前期发现EBVaGCs中miR-143/145簇低表达，其启动子显著超甲基化，多种DNA甲基转移酶（DNMTs）均属其靶基因，由此，我们认为EBVaGCs中可能存在miR-143/145簇与DNMTs的反馈通路。深入研究发现，干预此反馈通路，可诱导EB病毒由潜伏进入Rta蛋白开启的裂解循环，使蛋白激酶EBV-PK上调，导致抗病毒药物磷酸化产物增加，干扰DNA合成，抑制癌细胞生长；而且干预此反馈通路，可恢复miR-143/145簇表达，抑制其多种原癌靶基因，发挥协同效应。课题组拟结合临床样本检测与细胞、分子机制研究，运用甲基化检测、miRNA定量及转染、体内外药敏检测等方法，阐明此反馈通路调控EBVaGCs抗病毒治疗的效能及作用机制。

本课题通过对免疫check-point蛋白PD-L1以及其上游调控蛋白IRF-1在胃癌中的表达情况进行了免疫组化分析，发现EB病毒相关胃癌以弥漫型居多，且与EB病毒非相关胃癌相比，IRF-1和PD-L1在EB病毒相关胃癌组织中表达均呈显著上调，提示EB病毒相关胃癌可能对免疫check-point抗体药物存在响应；同时，发现一系列可靶向抑制免疫check-point蛋白PD-L1的人类以及EB病毒来源的miRNAs，包括：hsa-miR-378g、hsa-miR-93-5p以及ebv-miR-BART2-5p、ebv-miR-BART7，提示此四种miRNAs在EB病毒相关胃癌中可能参与调控肿瘤免疫逃逸。并在上述四种miRNAs中，发现EB病毒来源的miRNA：ebv-miR-BART7可显著上调EB病毒裂解相关蛋白Rta的表达，说明上调ebv-miR-BART7表达，可刺激EB病毒相关胃癌中EB病毒由潜伏感染状态转变为裂解状态，提示此EB病毒来源的miRNA：ebv-miR-BART7，不仅可以通过抑制PD-L1表达，激活机体对EB病毒相关胃癌的免疫反应；亦可促进EB病毒裂解，从而提高EB病毒相关胃癌的自身的免疫原性，对EB病毒相关胃癌的免疫治疗具有重要的临床参考价值。同时，在当前大数据时代背景下，不仅建立了泛消化道肿瘤，包括胃腺癌、食管鳞癌、结直肠癌的循环（含血浆或血清）miRNAs特征表达谱并明确其诊断效能；而且在此基础上，完成了非消化系统癌种循环miRNA表达谱的情况，包括肺腺癌、肺鳞癌，以及鼻咽癌；对不同miRNA在泛癌发生、发展中的组织、器官特异性有了系统、全面的认知。此外，亦发现了miR-3622b-5p可靶向抑制抗凋亡蛋白BIRC5，部分逆转胃癌顺铂耐药；以及一组与胰腺癌预后密切相关的长链非编码RNA组合；进一步增加了非编码RNA调控肿瘤耐药、发生、发展机理的认识。本项目共发表相关论文12篇，其中SCI论文7 篇，完成了项目既定的研究目标，同时，为后续深入研究挖掘，打好了坚实的基础。