RPS15a介导内质网应激反应在肺癌EGFR-TKI获得性耐药中的作用及机制研究

The incidence and mortality rates for non-small cell lung cancer (NSCLC) are the highest in the world. The tyrosine-kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR) have been approved for the treatment of NSCLC. However, the acquired resistance severely limits its application. In the previous study, gefitinib-resistant NSCLC HCC827 cell line harboring EGFR mutation was established. Analysis of gene expression profile between gefitinib-resistant HCC827 cell line and parent cells was performed. Among the differentially expressed genes, we found that RPS15a might be responsible for acquired EGFR-TKI resistance. Further study indicated that RPS15a was involved in endoplasmic reticulum (ER) stress induced apoptosis in lung cancer cells exposed to gefitinib. In this study, to investigate the role of RPS15a as a predictor of EGFR-TKI sensitivity, the expression of RPS15a protein and those ER stress associated proteins will be analyzed in NSCLC specimens. Down or up regulation of RPS15a expression will be performed using letivirus mediated gene function loss or enhancement. Moreover, identification of interacting proteins of RPS15a will also be done. As a result, the down-streaming signaling pathway will be established using microarray-assisted pathway analysis. Our study will explore a subtle mechanism of RPS15a modulating acquired EGFR-TKI resistance from the level of tissue, cell and protein in NSCLC, which could provide scientific basis for target therapy and reversal of drug resistance in NSCLC in the future.

尽管EGFR-TKI明显提高了肺癌的临床治疗效果，但其获得性耐药制约了临床应用价值，目前获得性耐药机制尚未完全阐明。申请人在前期研究中，通过基因芯片分析吉非替尼获得性耐药的EGFR敏感突变肺癌细胞株HCC827的基因表达谱，发现RPS15a高表达与肺癌细胞EGFR-TKI获得性耐药相关。后续研究表明：RPS15a介导内质网应激反应影响了吉非替尼对肺癌细胞的诱导凋亡作用。本项目拟在此基础上，通过在肺癌标本中检测RPS15a及内质网应激反应标记蛋白的表达，明确其与临床耐药的关系；利用慢病毒介导的基因功能缺失和增强，下调或上调RPS15a的表达，观察细胞株耐药能力的变化；采用蛋白相互作用的方法鉴定RPS15a结合的分子，通过蛋白信号通路分析建立起RPS15a调控的下游信号通路。由此从组织、细胞和分子水平阐明RPS15a在肺癌EGFR-TKI获得性耐药中的调控机制，为今后肺癌靶向治疗提供理论依据。

近年来，尽管EGFR-TKI明显提高了肺癌的临床治疗效果，但其获得性耐药制约了临床应用价值，目前获得性耐药机制尚未完全阐明。本研究前期研究中，通过基因芯片分析吉非替尼获得性耐药的EGFR敏感突变肺癌细胞株的基因表达谱，发现RPS15a高表达与肺癌细胞EGFR-TKI获得性耐药相关。因此，本研究利用RPS15a过表达和shRNA慢病毒载体感染肺癌PC-9细胞及其继发耐药细胞株PC-9R，建立稳定细胞株，验证RPS15a表达对于肺癌吉非替尼治疗敏感性的影响，检测内质网应激反应蛋白，初步探讨RPS15a作用于吉非替尼耐药的功能与机制。结果显示，在RPS15a过表达和沉默慢病毒感染肺癌敏感细胞株PC-9和耐药性细胞株PC-9R中，干扰RPS15a后细胞增殖能力变化、细胞周期分布变化、细胞凋亡情况变化均不明显。而加入吉非替尼后检测细胞敏感性变化，进一步研究表明在PC-9R中敲除RPS15a后，PC-9R细胞对吉非替尼的敏感性增强。而在PC-9细胞中过表达RPS15a后，PC-9细胞对吉非替尼的敏感性减弱，呈现一定的抗药性。在本研究中，我们使用特异性的sh-RNA下调A549细胞中RPS15a的表达，结果表明RPS15a表达降低能显著抑制肺癌细胞增殖和存活。并且随着RPS15a表达的降低，A549细胞明显积累在G0 / G1期，提示RPS15a的抑制可能通过细胞周期阻滞，诱导细胞增殖减少。免疫组织化学分析进一步显示RPS15a在手术切除的肺癌组织中存在过表达。综上所述，我们将RPS15a鉴定为肺癌发生的新型潜在致癌基因。本研究可能为肺癌的基因治疗提供初步的实验依据。项目资助发表SCI论文7篇，待发表论文2篇，协助培养硕士研究生2名，博士研究生1名。

内容获取失败，请点击重试