LncRNA AFAP1-AS1结合TDP43调控RNA可变剪接促进鼻咽癌拟血管生成

我们前期发现一个新lncRNA AFAP1-AS1在鼻咽癌中高表达并促进癌细胞侵袭迁移，导致患者预后不佳；最近我们捕获到AFAP1-AS1可与RNA可变剪接的关键调控蛋白TDP43结合，通过二代和三代测序技术证实了AFAP1-AS1通过TDP43影响鼻咽癌细胞中RNA可变剪接，并初步锁定AFAP1-AS1可能调控AXIN2基因的可变剪接促进鼻咽癌细胞的拟血管生成。本项目将深入解析AFAP1-AS1通过TPD43影响可变剪接复合物组装的结构基础，AFAP1-AS1/TDP43调控AXIN2基因可变剪接的原理，及AXIN2可变剪接产生的新蛋白变异体促进鼻咽癌细胞拟血管生成的具体机制。本项目将为AFAP1-AS1这个我们已研究多年的lncRNA开辟调控RNA可变剪接的研究新领域，并从拟血管生成的视角解析鼻咽癌转移的新机理，可变剪接产生的新蛋白及其下游通路也将为鼻咽癌的诊疗提供潜在分子标志和靶点。

We have previously reported that a novel long noncoding RNA (lncRNA), the actin-filament-associated protein 1 antisense RNA 1 (AFAP1-AS1), is upregulated in nasopharyngeal carcinoma (NPC). Our in vitro and in vivo experiments demonstrated that AFAP1-AS1 could promote NPC cell migration, invasion, and metastasis. The high expression of AFAP1-AS1 was significantly associated with a poor prognosis in NPC patients. Recently, through RNA pulldown experiments, followed by mass spectrometry analyses validated by RNA immunoprecipitation (RIP), we determined that AFAP1-AS1 could bind to the protein TDP43, an important RNA splicing regulator. Using second- and third-generation sequencing technology, we then performed transcriptome profiling of the NPC cells under conditions of AFAP1-AS1 overexpression and knockdown and verified that AFAP1-AS1 could interfere with RNA alternative splicing in the NPC cells. The results from further experiments implied that AFAP1-AS1 might have induced a vasculogenic mimicry in the NPC cells through regulating the alternative splicing of the AXIN2 gene. In this proposed project, we will thoroughly resolve the structural basis of the interaction between AFAP1-AS1 and TDP43, as well as their effects on the spliceosome assembly and will analyze the detailed mechanisms by which AFAP1-AS1 regulates the alternative splicing of downstream gene(s) such as AXIN2 and produces novel protein isoforms, ultimately promoting a vasculogenic mimicry in the NPC cells. Achieving the goals in this proposal will further reveal the novel biological function of AFAP1-AS1, an important oncogenic lncRNA that we have studied for several years, from the viewpoint of RNA alternative splicing. It will also elucidate the novel mechanisms of NPC metastasis from the perspective of vasculogenic mimicry. Finally, the novel protein isoform(s) produced by RNA alternative splicing may provide potential biomarker(s) and therapeutic tas) for NPC.