LncRNA GAS5逆转肺腺癌对EGFR-TKIs原发性耐药的功能和机制研究

Primary drug resistance restricts the application of gefitinib(EGFR-TKIs) in patients with advanced pulmonary adenocarcinoma. It was found in our previous study that the low expression of lncRNA GAS5 was associated with innate gefitinib resistance. Nonsense-mediated mRNA decay pathway is discovered as a RNA quality control system which eliminates aberrant transcripts with premature termination codons (PTC). Our previous study had found that the UPF1, as the key player of NMD, was upregulated in gefitinib-resistant lung adenocarcinoma. The GAS5 promoter sequence contains multiple PTCs that can be identified as substrates for NMD. Moreover, GAS5 can specified bind to UPF1. Furthermore, in our previous study, we also demonstrated that overexpression of GAS5 reversed gefitinib resistance by down-regulation of IGF-1R expression. Given the evidences, we hypothesized that up-regulation of UPF1 in NMD down-regulate GAS5 expression in gefitinib resistance lung adenocarcinoma cells by accelerating GAS5 degradation, as GAS5 can mediate IGF-1R gene promotors methylation by recruitment of DNA methyltransferase 1 (DNMT1) which lead to down-regulation of IGF-1R.The project will be conducted by BRIC kit, BSP, ELISA, RIP-Seq/RNA pulldown, rescue assay and tumor formation assay in vivo.We aimed to elucidate the molecular mechanisms of EGFR-TKIs resistance and explore the potential way to reverse the resistance of EGFR-TKIs in lung adenocarcinoma, which may provide new opportunities for solving the clinical problem of EGFR-TKIs resistance.

原发性耐药限制了吉非替尼(EGFR-TKIs代表药物)在肺腺癌患者中的应用。申请者发现LncRNA GAS5在吉非替尼耐药的肺腺癌组织和细胞系中表达下调，但机制不明。无义介导的RNA降解（NMD）是一种降解含有提前终止密码子(PTC)转录产物的RNA监测机制，是转录后mRNA质量控制重要环节。申请者还发现NMD关键蛋白UPF1在耐药肺腺癌组织中表达上调，且GAS5启动子序列中含有多个PTC及与UPF1结合位点；GAS5能够通过下调耐药相关蛋白IGF-1R逆转耐药，且在耐药细胞系中过表达GAS5后IGF-1R甲基化水平下调。我们推测，耐药肺腺癌细胞中UPF1表达上调促进GAS5降解增多，导致后者丰度下降；同时GAS5可招募DNA甲基转移酶1介导IGF-1R基因启动子区甲基化，导致IGF-1R表达下调逆转耐药。本项目旨在探究肺腺癌中吉非替尼耐药产生及逆转的分子机制，为解决耐药问题提供新的靶点。

表皮生长因子受体(EGFR)是一种具有酪氨酸激酶活性的跨膜糖蛋白。对EGFR敏感突变的肺腺癌患者，EGFR-TKIs已成为一线治疗方案。非EGFR敏感突变的患者，耐药限制了其应用。本研究对肺腺癌对EGFR-TKIs耐药性产生及逆转的机制进行探索。前期发现GAS5的低表达与肺腺癌细胞系A549对吉非替尼耐药相关，发现上调GAS5的表达可逆转肺腺癌细胞对吉非替尼的耐药，机制不明。. 课题探究是否EGFR-TKIs耐药性肺腺癌细胞中UPF1表达上调导致NMD效应增强引起GAS5降解增多，GAS5低表达；下调UPF1能否抑制NMD，上调GAS5表达，逆转耐药。GAS5是否通过结合DNMT1介导IGF-1R甲基化修饰，下调IGF-1R表达，逆转耐药。. 1、发现GAS5在耐药肺腺癌细胞A549降解速度快于非耐药肺腺癌细胞HCC827，检测NMD关键蛋白UPF1表达，耐药肺腺癌细胞A549中的UPF1表达高于非耐药细胞HCC827的表达水平；2、发现干扰UPF1后吉非替尼处理耐药细胞系A549的增殖、克隆及侵袭能力降低; 3、裸鼠体内实验，检测A549干扰UPF1表达后对吉非替尼耐药性的影响，成瘤实验表明在耐药细胞系A549中干扰UPF1表达，干扰UPF1后瘤体小于对照组; 4、RIP-Seq/RNA pulldown验证GAS5与UPF1相互结合作用; 5、回复UPF1表达，细胞和动物水平完成挽救实验，结果示回复UPF1表达后A549细胞的增殖和侵袭能力增强。6、检测对照组、GAS5组和空载体组的DNMT1酶表达水平。GAS5组中DNMT1蛋白表达水平高于其他两组。7、去甲基化处理后细胞IGF-1R表达水平增高，表明GAS5通过使IGF-1R基因DNA区甲基化抑制其表达。对IGF-1R的mRNA水平进行了检测，去甲基化处理后，IGF-1R的mRNA水平显著上升，证实GAS5通过使IGF-1R基因DNA区甲基化抑制其表达。RNA-Seq/RNA pulldown检测GAS5与DNMT1结合。ChIP-qPCR验证DNMT1与IGF-1R基因的启动子结合。体内裸鼠实验，检测A549-GAS5+细胞去甲基化后其耐药性改变；回复IGF-1R表达，证实该结论。. 本研究完善肺腺癌中EGFR-TKIs的耐药机制，寻找逆转耐药的分子机制，有一定的理论和现实意义。