长链非编码RNA FOXD2-AS1作为“分子脚手架”调控非小细胞肺癌恶性进程的机制研究

The molecular mechanism of tumorigenesis in non-small cell lung cancer (NSCLC) is hot and difficult in the present. Recent studies have shown that lncRNAs play an important role in the process of tumor formation. Through high-throughput gene sequencing, we found lncRNA FOXD2-AS1 was significantly upregulated in NSCLC tumor tissues. Cytological experiments further confirmed FOXD2-AS1 could regulate the malignant phenotype of NSCLC cells. More importantly, FOXD2-AS1 was induced by transcription factor E2F1, and through binding to heterogeneous nuclear ribonucleoprotein K (hnRNP K) and Y-box binding protein 1 (YBX1), thus regulated the expression of E2F1 and other genes. So, we hypothesised that in the development of NSCLC, lncRNA FOXD2-AS1 could be activated by E2F1 and through scaffolding hnRNP K and YBX1, thus upregulated the expression of E2F1. LncRNA FOXD2-AS1 and E2F1 formed a positive feedback loop, which resulting in a vicious cycle. Simultaneously, FOXD2-AS1 bound to hnRNP K and YBX1 thus regulated the expression of many tumor promoting or suppressor genes. Our study reported that lncRNA FOXD2-AS1 had an important role in the pathogenesis of NSCLC. We will verify our hypothesis by using ChIP、RIP and RNA-pull down et.al, and reveal the precise molecular mechanisms in our future work. Our study could contribute to enrich the research of molecular mechanism of NSCLC development, and would provide new ideas for the diagnosis and treatment of NSCLC.

针对肿瘤发病的分子机制学探索一直是肺癌研究的热点和难点，近年来研究表明lncRNA与肿瘤发病密切相关。课题组通过基因测序发现，FOXD2-AS1在肿瘤组织中显著上调，细胞学实验证实其可以调控NSCLC细胞恶性表型，同时，FOXD2-AS1高表达受E2F1转录诱导，并可绑定hnRNP K和YBX1转而调控E2F1及其它基因表达。由此提出科学假设：在NSCLC中， FOXD2-AS1可被E2F1 激活转录，并通过“分子脚手架”机制同时绑定hnRNP K和YBX1激活E2F1表达，FOXD2-AS1 与E2F1成正反馈调控环路，导致恶性循环，同时调控一系列下游基因表达，从而促进NSCLC恶性进展。本研究提出FOXD2-AS1在肺癌发病中具有重要作用，并通过ChIP、RIP 和RNA-pull down等实验首次揭示其作为“分子脚手架”导致肺癌恶性进展的分子学机制，为NSCLC的诊治提供新的思路

针对肿瘤发病的分子机制学探索一直是肺癌研究的热点和难点，近年来研究表明lncRNA与肿瘤发病密切相关。首先，我们通过对课题组收取的肺癌组织样本检测数据，结合TCGA肺癌数据，分析发现FOXD2-AS1在癌组织中表达显著上调，并且高表达的FOXD2-AS1提示肺癌不良预后。我们还采用干扰和过表达技术从正反两方面在细胞和活体动物模型上验证了FOXD2-AS1在肺癌发生发展中的功能。我们通过细胞核质分离实验发现FOXD2-AS1 主要分布在细胞核内，提示其可能主要通过转录水平调控基因表达，之后通过RNA pull down（下拉实验）联合银染-质谱实验鉴定出FOXD2-AS1能够特异性的绑定hnRNP K 蛋白（heterogeneousnuclear ribonucleoprotein K）和Y-box 结合蛋白1(Y-box binding protein 1，YBX1），且实验证实了这两个蛋白之间也存在相互作用，因此提示FOXD2-AS1可以作为“分子脚手架”同时绑定hnRNP K和YBX1蛋白，从而调控下游的基因表达。综上所述，我们的研究发现，位于上游的E2F1蛋白可以通过调控FOXD2-AS1转录，激活FOXD2-AS1的表达, FOXD2-AS1可同时通过绑定hnRNP K和YBX1蛋白，发挥转录调控作用，调控包括E2F1和Cyclin D1在内的一系列基因表达，FOXD2-AS1同E2F1之间会形成了一个正反馈调控环路，从而促进非小细胞肺癌恶性进展。FOXD2-AS1是我们通过对肿瘤组织转录组测序，并结合TCGA大数据分析、筛选获得的肺癌发生发展相关的重要功能性分子，本研究的成功实施，进一步丰富了lncRNA调控肺癌发生的分子机制，为肺癌的诊疗提供潜在新靶标和新思路。