长链非编码RNA NEAT1通过诱导BIN1选择性剪接促进肺腺癌EGFR-TKI耐药机制研究

EGFR-TKI has become one of the most common and efficient option in treating lung adenocarcinoma. However, resistance severely restricted the treatment efficacy of EGFR-TKIs. The frequent mechanisms of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance include T790M mutation, c-MET amplification, PIK3CA mutation, pathological phenotype transition, MAPK pathway continuous activation, and epithelial mesenchymal transition, etc. Unfortunately, the mechanisms of about 30% cases of EGFR-TKI resistance remain elusive. Inactivation of pro-apoptotic protein is one of the hallmarkers of EGFR-TKI resistance. Due to its complexity, the mechanisms of pro-apoptotic protein anergy is still little understood. Long non-coding RNA (lncRNA) is a kind of non-encoding RNA transcripts > 200 nucleotides in length and recently identified as crucial regulators of gene expression in cancer. Our preliminary study revealed that the low expression of pro-apoptotic protein bridging integrator-1 (BIN1) is correlated with EGFR-TKI resistance. At the same time, we found that aberrant alternative splicing accounted for this low expression. Aiming at elucidating the related factors, we assayed the lncRNA expression profile with sequencing chips, then we used overlap analysis and found that lncRNA NEAT1 was correlated with BIN1 aberrant alternative splicing and EGFR-TKI resistance simultaneously. Taken above, we put forward a hypothesis that NEAT1 could promote lung adenocarcinoma EGFR-TKI resistance through inducing aberrant BIN1 alternative splicing. In this project, we plan to use molecular biological methods including RIP, splicing analysis, and TUNEL, etc. to explore the regulating mechanisms from clinical sample, cell experiment, and in vitro model aspects. Thereout, to systematically describe the molecular regulation network of NEAT1 in EGFR-TKI resistance and provide novel theoretical foundation to reverse EGFR-TKI resistance.

EGFR-TKI耐药是影响肺腺癌靶向治疗效果最主要原因，目前仍有部分机制不明。促凋亡信号分子失活是EGFR-TKI耐药重要标志，其调控因素复杂。lncRNA在包括选择性剪接调控在内的多种肿瘤相关事件中发挥关键作用。我们前期研究发现，促凋亡分子桥接整合因子1（BIN1）在肺腺癌中低表达并诱导EGFR-TKI耐药，同时异常选择性剪接是导致其低表达主要原因；通过基因测序和overlap分析发现lncRNA NEAT1在肺腺癌中同时与BIN1异常选择性剪接和EGFR-TKI耐药有关。由此提出假说：NEAT1通过诱导BIN1发生异常选择性剪接促进EGFR-TKI耐药。本项目将从临床样本、细胞实验、动物模型多层次，利用RIP、剪接模式分析和TUNEL等技术深入探讨其分子调控机制。系统性绘制NEAT1参与肺腺癌EGFR-TKI耐药过程的分子调控网络，为逆转EGFR-TKI耐药治疗提供新的理论依据。

治疗抵抗是肺腺癌EGFR-TKI治疗领域中亟待解决的关键临床问题。主研人基于既往研究和前期预实验结果，提出科学假说：肺腺癌EGFR-TKI耐药与BIN1介导的促凋亡信号关闭有关，选择性剪接是肺腺癌中BIN1蛋白低表达的主要机制，lncRNA NEAT1能够上调SRSF1的表达水平促进BIN1异常选择性剪接诱导肺腺癌EGFR-TKI耐药。项目按照研究计划执行，研究结果表明：1、利用基因芯片筛选与肺腺癌EGFR-TKI治疗抵抗密切相关的lncRNA和mRNA，利用生物信息学技术构建调控网络，2、明确NEAT1在EGFR-TKI耐药的肺腺癌细胞中表达显著上调，3、敲低NEAT1能够下调肺腺癌PC9细胞的增殖能力并诱导DNA损伤和细胞周期阻滞，4、SRSF1能够诱导BIN1发生异常选择性剪接而产生BIN1 +12A亚型进而抑制细胞凋亡，5、明确NEAT1在肺腺癌细胞中作为ceRNA上调AKR1C1进而促进EGFR-TKI治疗抵抗，敲低NEAT1可逆转EGFR-TKI治疗抵抗，6、NEAT1/AKR1C1能够作为生物标志物提示肺腺癌患者对EGFR-TKI治疗抵抗，7、AKR1C1介导的铁死亡逃逸是肺腺癌EGFR-TKI治疗抵抗的重要分子机制。在完成本项目原定研究目标的基础上，主研人还在本项目研究过程中对肺腺癌的另一种重要治疗手段，即免疫检查点抑制剂的治疗抵抗机制进行探索，发现肿瘤微环境中的FAP能够通过影响免疫细胞浸润促进肺腺癌对免疫检查点抑制剂治疗抵抗。本项目研究结果能够为深入理解肺腺癌治疗抵抗的分子机制提供新理论基础，为NEAT1/AKR1C1作为肺腺癌EGFR-TKI耐药的新治疗靶点和生物标志物提供分子循证学依据，对于制定新的肺腺癌精准治疗策略具有重要意义。