长链非编码RNA AFAP1-AS1促进鼻咽癌侵袭转移的机制研究

In our previous projects, we have found that a long non-coding RNA, termed AFAP1-AS1, is overexpressed in nasopharyngeal carcinoma and that AFAP1-AS1 levels predict poor outcomes; however, the biological function of AFAP1-AS1 and its mechanism are largely unknown. We have further elucidated that knock-down of AFAP1-AS1 could inhibit invasion and migration of nasopharyngeal carcinoma cells by regulating the assembly of microtubules and microfilaments, and the reorganization of the cytoskeleton, both in vivo and in vitro. Using a proteomics strategy, we have also shown that several key molecules of the Rho/Rac signaling pathway were significantly dysregulated in cancer cells by knock-down of AFAP1-AS1. In light of these findings, clinical data analysis of a large cohort of cancer patients, the results of our CHIP-sequencing and RNA pull-down experiments, and bioinformatics prediction, we hypothesize that the well-known oncogene MYC and the SWI/SNF chromatin remodeling complex regulate the transcription of the AFAP1-AS1 gene. Acting as a component of the SWI/SNF complex, transcribed AFAP1-AS1 RNA affects the organization of the SWI/SNF complex, modulates SWI/SNF genomic binding, and antagonizes the SWI/SNF-mediated regulation of multiple genes of the Rho/Rac signaling pathway; in turn, the Rho/Rac signaling pathway regulates the assembly of microtubules and microfilaments, and the reorganization of the cytoskeleton, thus promoting invasion and migration of cancer cells. In this study, we aim to elucidate in detail the role and mechanism of AFAP1-AS1 in promoting nasopharyngeal carcinoma progression through regulating MYC-SWI/SNF-Rho/Rac gene signaling transduction.

我们发现长链非编码RNA AFAP1-AS1在鼻咽癌中表达上调且与不良预后相关，但其功能和机制还鲜有研究；体内外实验证实敲低AFAP1-AS1可影响细胞骨架重构，抑制侵袭和迁移；利用蛋白组学技术发现Rho/Rac通路多个分子表达改变；结合临床资料分析、CHIP-Seq、RNA pull-down及生物信息学预测，本项目提出了“MYC及SWI/SNF染色质重构复合物共同调控AFAP1-AS1的转录，转录出的AFAP1-AS1 RNA又作为SWI/SNF复合物的组分之一，影响SWI/SNF复合物的组装和活性，进而调节Rho/Rac通路多个关键分子的表达，Rho/Rac通路则通过调控微管微丝组装和细胞骨架重构，促进鼻咽癌细胞的侵袭和转移”这一假说。本项目将初步明晰AFAP1-AS1在MYC-SWI/SNF-Rho/Rac信号通路中的表达调控规律，并阐释它在鼻咽癌发生发展中的作用和机制。

我们前期发现长链非编码RNA AFAF1-AS1在包括鼻咽癌在内的多种恶性肿瘤中均表达显著上调，且与肿瘤转移及不良预后密切相关。但AFAP1-AS1促进鼻咽癌转移的分子机制，以及它为何会在肿瘤中表达上调却不清楚。.在本项目资助下，我们发现AFAP1-AS1通过竞争性内源RNA （ceRNA）形式，通过miR-423-5p调控Rho/Rac通路RAB11B、LASP1的表达，AFAP1-AS1还可以通过miR-423-5p调控AP1家族成员FOSL2的表达，并通过FOSL2启动LASP1的转录，最终通过Rho/Rac通路促进鼻咽癌细胞侵袭和迁移能力。此外，我们还通过生物信息学分析锁定了AFAP1-AS1潜在启动子区域，通过ChIP等实验证实AFAP1-AS1启动子区域存在c-Myc的结合位点，并进一步证实c-Myc可以调控AFAP1-AS1的表达，这可能是AFAP1-AS1在多种恶性肿瘤中表达升高的原因之一。.本研究从ceRNA角度解析了AFAP1-AS1在鼻咽癌发生发展中的作用机理，并首次克隆和鉴定了AFAP1-AS1的启动子区域，为理解AFAP1-AS1在多种肿瘤中表达上调的机制提供了线索。上述发现表明AFAP1-AS1及其相关上下游调控分子有可能成为鼻咽癌诊断治疗的潜在靶点和标记。