c-Myc调控的长非编RNA FOXD3-AS1介导非小细胞肺癌EGFR-TKI耐药的作用机制研究

It is urgent to overcome EGFR-TKI resistance in NSCLC, and C-MET amplification is one of key factors contributing to EGFR-TKI resistance. LncRNAs play important roles in the development of cancer, but their roles in drug resistance have not been studied clearly. Our present study reveals that the FOXD3-AS1 expression was significantly upregulated in EGFR-TKI resistant EGFR mutant NSCLC cell lines. Knockdown of FOXD3-AS1 expression could enhance EGFR-TKI resistant cells' sensitivity to EGFR-TKI. Besides, c-Myc could induce the promoter region activity of FOXD3-AS1 in part. FOXD3-AS1 could bind to WDR5 which was identified by RNA-Protein pulldown/mass spectrometry analysis and RNA immunoprecipitation assay. After knockdown of WDR5 expression, the expression of C-MET was decreased significantly. So our hypothesis is that c-Myc induced lncRNA FOXD3-AS1 enhances NSCLC cells resistance to EGFR-TKI by binding to WDR5 to epigenetically increase the transcriptional activity of the promoter region of C-MET. The aim of this study is to demonstrate that FOXD3-AS1 regulates C-MET-mediated EGFR-TKI resistance and expands our understanding of drug resistance both in vitro and in vivo. Moreover, it will provide us a novel theoretical basis for clinical treatment.

EGFR-TKI耐药是临床急待解决的问题，C-MET扩增是导致其耐药的关键因素。许多研究表明lncRNAs在肿瘤发生发展过程中起重要作用，但其在肿瘤耐药中研究甚少且机制不明。前期结果显示：TKI耐药细胞中FOXD3-AS1表达显著升高，干扰FOXD3-AS1的表达后增加耐药细胞对TKI的敏感性；c-Myc可诱导FOXD3-AS1启动子区活性；RNA-Protein pulldown联合质谱分析、RIP实验发现FOXD3-AS1可与WDR5结合，细胞中封闭WDR5后C-MET表达下调。据此提出假设：c-Myc扩增促进FOXD3-AS1的转录，FOXD3-AS1通过绑定WDR5，介导C-MET启动子区H3K4三甲基化，激活其转录，从而增强TKI耐药细胞的耐药性。本课题拟在细胞、组织、动物模型三个层面，证实FOXD3-AS1调控C-MET介导的TKI耐药，完善耐药机制，为临床治疗提供理论依据。

表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKI）是治疗EGFR突变非小细胞肺癌（NSCLC）患者的一线药物，其有效率为50%-80%，但这些对EGFR-TKI治疗有反应的患者，最终大多都因产生耐药而导致治疗失败，耐药问题已经成为限制EGFR-TKI疗效进一步提高的瓶颈。许多研究表明lncRNAs在肿瘤耐药表型形成过程中起重要作用。课题组通过系统筛选，发现lncRNA FOXD3-AS1在肺癌耐药组织中明显上调，提示FOXD3-AS1可能与NSCLC患者的EGFR-TKI耐药相关。本课题从细胞、活体多个层面，全面评价FOXD3-AS1在肺癌TKI耐药过程及发生中发挥的功能角色。研究结果表明FOXD3-AS1可以在体内和体外调控NSCLC EGFR-TKI耐药，且在体内外调控肺癌发生。机制研究发现FOXD3-AS1上游可以被c-Myc调控，并且FOXD3-AS1下游可以通过募集绑定WDR5复合体，促进C-MET基因启动子区H3K4me3活性增高，增强C-MET的转录，从而诱导NSCLC EGFR-TKI耐药，促进肺癌的发生发展。我们将进一步完善lncRNA FOXD3-AS1调控C-MET介导EGFR-TKI的耐药机制，为临床治疗提供理论依据，以期为临床克服耐药提供新的靶点。