长链非编码RNA CASC9通过与IRS1结合诱导肺腺癌奥希替尼耐药的机制研究

While osimertinib does improve the survival of the lung cancer patients, drug resistance is eventually inevitable. So far, researches on the mechanisms of osimertinib resistance mostly focus at the level of coding DNA, and the mechanisms of resistance in about 50% of the patients are not accounted for. After excluding those patients with osimertinib resistance of known mechanisms, we performed high-throughput sequencing on long non-coding RNA (lncRNA) in 17 patients of lung adenocarcinoma that are osimertinib-resistant due to unknown mechanisms, and discovered that the expression level of CASC9 is significantly alleviated in these patients. The result is verified on 6 cases in the validation group. To study the relationship between CASC9 and osimertinib resistance, we knocked CASC9 out in an osimertinib-resistant cell line with CRISPR/Cas9, and found that the cells regained susceptibility, indicating that high expression level of CASC9 could lead to osimertinib resistance. Further experiments we performed, including RNA pull-down, mass spectrometry and RNA immunoprecipitation, showed that CASC9 binds to the protein IRS1. We therefore hypothesize that such binding accounts for the osimertinib resistance. In this research, we plan to verify the interaction between the CASC9 and IRS1, and specify the downstream pathways that CASC9 and IRS1 influence that eventually affect osimertinib susceptibility.

奥希替尼改善肺癌患者生存的同时不可避免会耐药，目前关于奥希替尼耐药机制的研究主要集中在可编码蛋白的基因水平，约有50%的患者耐药机制不详。我们前期排除奥希替尼耐药机制已知的患者，在目前耐药机制不详的17例肺腺癌患者中行lncRNA高通量测序，发现CASC9在奥希替尼耐药患者中的表达水平明显增高，并在6例验证集患者中证实该结果。为验证CASC9与奥希替尼耐药的关系，我们在奥希替尼耐药细胞系中，通过CRISPR/Cas9敲除CASC9表达，发现细胞可恢复奥希替尼敏感性，提示CASC9高表达可导致奥希替尼耐药。进而我们通过RNA pull-down、质谱实验和RIP等实验发现CASC9与IRS1结合，由此我们提出CASC9与IRS1结合导致奥希替尼耐药。本课题拟在细胞、动物和临床标本上验证CASC9与IRS1间的相互作用，以及CASC9和IRS1通过影响哪些下游通路影响奥希替尼的敏感性。

既往关于奥希替尼耐药的研究大多集中在编码蛋白基因的继发性突变上，约40%的患者奥希替尼耐药机制尚不清楚。越来越多的研究表明lncRNA的调控与肿瘤耐药有关。本研究通过CRISPR/Cas9和质粒转染成功敲除PC9OR和HCC827OR中的CASC9。进一步发现，CASC9高表达可导致细胞凋亡减少，细胞增殖和迁移能力减弱，并在裸鼠实验中验证了这一结果。在之前的研究中，我们发现CASC9和IRS1的结合力最强。在本研究中，我们发现CASC9表达的增加可以诱导IRS1表达的增加，从而导致细胞凋亡减少，细胞增殖和迁移能力下降，并在裸鼠实验中验证了这一结果。在此基础上，本研究明确了CASC9和IRS1在奥希替尼耐药中的作用:确定高表达的CASC9可以与IRS1结合导致奥希替尼耐药，并在细胞系和裸鼠中进行了验证。本研究揭示了奥希替尼耐药的新机制，为奥希替尼耐药患者带来了新希望。