EBV-miR-BART2-5p靶向m6A甲基转移酶METTL14调控EMT促进鼻咽癌侵袭转移的研究

In recent years, much attention has been paid to the biological functions of m6A mRNA modification at the RNA levels, however, the roles of m6A modification in the malignant progression of nasopharyngeal carcinoma (NPC) have not yet been reported. In our pilot study, we found that 1) the m6A modification may participate in NPC malignant progression and the m6A modification may be affected by EB virus；2) METTL14 expression in NPC tissues is significantly downregulated，and is related with the TNM stages and EMT of NPC；3) METTL14 overexpression inhibits EMT induction and migration of NPC cells；4) METTL14 is a target gene of EBV-miR-BART2-5p，and EBV-miR-BART2-5p overexpression promotes migration of NPC cells，suggesing that EBV-miR-BART2-5p regulates m6A modification in NPC cells by targeting METTL14，and thereby modulating EMT，invasion and metastasis in NPC. Aganist this background, this project intends to further carry out the following studies: 1) to reveal the levels of m6A modification in NPC cell lines and NPC tissues; 2) to explore the influences of EB virus on m6A modification levels; 3) to investigate the roles of EBV-miR-BART2-5p and its target gene METTL14 in NPC EMT, invasion and metastasis, and confirm whether the aforementioned functions of EBV-miR-BART2-5p can be mediated by its target gene METTL14; 4) to screen METTL14-mediated m6A modification in Gene-X by combined use of MeRIP-seq/m6A-seq or m6A-miCLIP-seq and RNA-seq technology, followed by functional verification; 5) to reveal the expression profile of EBV-miR-BART2-5p, METTL14 and Gene-X in NPC tissue samples, the expression correlation and clinical pathological significance, as well as the expression relationship between these genes and EMT-related genes. Expected achievement are anticipated to reveal the functions of m6A modification in NPC EMT, invasion and metastasis and underlying mechanisms, and provide valuable clues to discover EMT repressors.

目前m6A修饰的功能备受关注，其在鼻咽癌恶性进程中的作用尚未见报道。前期研究发现，EBV可能影响m6A修饰，该修饰进而参与鼻咽癌恶性进程；METTL14(M4)在鼻咽癌组织中表达下调，与TNM分期和EMT相关，M4过表达抑制鼻咽癌细胞发生EMT和迁移；M4是EBV-miR-BART2-5p(B2)的靶基因，B2过表达促进鼻咽癌细胞迁移；这提示B2靶向M4可能影响m6A修饰，进而调控EMT和侵袭转移。据此，拟开展以下研究：揭示鼻咽癌细胞和组织中m6A修饰水平；确证EBV对m6A修饰影响；探讨B2和M4在鼻咽癌EMT和侵袭转移中的作用，并确证B2上述功能可否由M4介导；miCLIP-seq和RNA-seq联用，筛选M4介导的m6A修饰的Gene-X及功能验证；揭示B2、M4及Gene-X等在鼻咽癌组织中表达谱、表达相关性和临床病理意义。预期成果有望揭示m6A修饰在鼻咽癌恶性进程中作用与机制。

目前m6A修饰的功能备受关注，其在鼻咽癌(NPC)恶性进程中的作用有待深入阐明。本研究发现，在NPC临床组织标本中，METTL3、ALKBH5、FTO和WTAP表达未见明显下调，而METTL14表达显著下调，且其表达水平与NPC分化程度、肿瘤T分期、颈部淋巴结转移(N)、远处转移(M)、临床分期、增殖指数Ki67以及NPC的EMT(上皮-间充质转化)存在显著关联，这预示METTL14可能与NPC发病密切相关。METTL14沉默促进NPC细胞迁移、侵袭和转移，其过表达结果则相反。METTL14过表达对NPC细胞体外增殖无明显影响，但METTL14过表达可增强NPC细胞对放疗的敏感性；METTL14沉默促进NPC细胞在NSG重度免疫缺陷小鼠皮下生长。METTL14是EBV-miR-BART2-5p的靶基因，EBV-miR-BART2-5p靶向下调其靶基因METTL14表达是NPC组织中METTL14表达下调的重要原因之一。EBV-miR-BART2-5p在体外能显著促进NPC细胞增殖、迁移和侵袭。EBV-miR-BART2-5p通过靶向下调METTL14表达促进了NPC细胞EMT及迁移和侵袭。虽然METTL14基因启动子区存在有经典的CpG岛，但NPC组织中METTL14表达下调或缺失可能不是由METTL14基因启动子区DNA甲基化造成的。RNA-seq和MeRIP-seq关联分析，我们从四象限图中的Hypermethylated-down的基因(如CD68、CD99和SNAI2)和Hypermethylated-up的基因(如PTGER4、RIOK3和USP25)中，初筛出44个与METTL14调控NPC进展可能密切相关的候选靶点基因，验证实验正在进行中。本项目的成果揭示了m6A修饰在NPC演进中作用与机制。课题执行期间，在Nature Communications(IF=17.694)、Autophagy (IF=13.391)和International Journal of Biological Sciences(IF=10.75)等上发表15篇SCI论文，最高影响因子为17.694，IF大于10的有3篇，另有2篇SCI论文在投。课题执行期间团队成员获2项国家自然科学基金项目资助，共培养博士4人和硕士2人。

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