E2F6调控LINC00152促进非小细胞肺癌吉非替尼耐药的机制研究

Advanced NSCLC patients are beset by EGFR-TKI secondary drug resistance.Our findings revealed that LINC00152 was highly expressed in gefitinib-resistance cells and tissues.Bioinformatics showed that E2F6 could bind to the site of LINC00152 promoter region. Subcellular fractionation and RIP assay verified that LINC00152 directly interacted with HuR. High throughput sequencing results illustrated that PSG1 transcription was regulated by LINC00152.Gene ontology analysis confirmed that LINC00152 was participated in apoptotic pathway.Consequently, we hypothesized that E2F6 induced LINC00152 transcription, LINC00152 could bind with HuR to hold PSG1 mRNA stability and increase PSG1 levels,thus decreasing cell apoptosis and leading to gefitinib resistance in NSCLC. This study will testify the hypothesis through luciferase reporter, ChIP and pulldown assays, and provide novel proof and targets for clinical treatment of EGFR-TKI secondary drug resistance.

EGFR-TKI继发性耐药是困扰晚期非小细胞肺癌（NSCLC）患者治疗的一大难题。我们研究发现LINC00152在NSCLC吉非替尼耐药细胞及组织中高表达。生物信息学分析显示E2F6与LINC00152启动子区域具有结合位点。核质分离及RIP实验证实LINC00152可绑定RNA结合蛋白人抗原R（HuR）。高通量测序表明LINC00152调控PSG1的转录。GO富集分析发现LINC00152与细胞凋亡相关。据此我们提出以下科学假说：E2F6促进LINC00152转录，LINC00152通过绑定HuR维持PSG1mRNA稳定性，提高PSG1mRNA表达水平，减少耐药细胞凋亡，从而增强NSCLC吉非替尼耐药性。本课题组将从细胞、动物及临床组织三个水平，拟采用荧光素酶报告、ChIP、pulldown等实验证实以上假说，为临床逆转TKI继发性耐药提供新的治疗依据及靶标。

EGFR-TKI耐药对很多非小细胞肺癌（NSCLC）患者的治疗及预后造成了不良影响。在本项研究中，我们通过筛查GEO数据库发现CASC9在吉非替尼耐药细胞及组织中高表达。利用细胞功能试验及体内试验证实抑制CASC9后可以增强耐药细胞对吉非替尼的敏感性，过表达CASC9可以促进吉非替尼耐药的发生。接着利用核质分离试验及RIP试验发现CASC9可与PRC2的核心亚基EZH2相结合。高通量测序表明在抑制CASC9表达后，DUSP1表达显著升高。ChIP试验就一步表明CASC9可与DUSP1的启动子区域结合，从而调控DUSP1的转录水平。而过表达DUSP1可增强耐药细胞对吉非替尼的敏感性。基于以上研究结果，我们可得出最终结论：CASC9通过募集EZH2，抑制下游DUSP1转录，促进耐药细胞增殖，减少耐药细胞凋亡，从而增强NSCLC吉非替尼耐药性。本项研究为临床逆转EGFR-TKI耐药提供了新的靶点和研究思路。