外泌体（exosomes）介导的细胞间信息传递在肺癌细胞顺铂耐药的作用及机制研究

Lung cancer is the leading cause of cancer-related mortality in the word. Although several target drugs such as erlotinib and cetuximab could increase the overall survival of NSCLC(non-small-cell lung cancer) patients, platinum-doublet chemotherapy remains the most important treatment for patients with advanced NSCLC. Cisplatin (DDP) is still one of the most important chemotherapy drugs in the treatment of lung cancer. As the development of individualized therapy, researchers are trying to find out biomarkers which could predict the efficiency of DDP. However, all phase III clinical trials got negative results..Exosomes are small extracellular membrane vesicles, which could be secreted by many kinds of cells. Exosomes are reported to contain DNA, miRNA, mRNA and protein and participated in cell-to-cell communication. Our preliminary study found that when DDP was added to A549 cells, exosomes secretion was strengthened and addition of the secreted exosomes to other A549 cells increased the resistance of these A549 cells to DDP. This resistance was probably mediated by miRNAs and mRNAs exchange by exosomes via cell-to-cell communication..To the best of our knowledge, there was no systematical research concerning the involvement of exosomes on lung cancer cells’ resistance to DDP. Based on our preliminary results, we would establish DDP resistant A549 cells and confirm the effect of exosomes in DDP resistance. We would investigate how does these exosomes regulate signal pathway of lung cancer cells and explore the possible mechanism of lung cancer cells’ resistance to DDP.

顺铂是肺癌治疗中最重要的药物之一，人们一直致力于寻找能预测肺癌患者顺铂耐药的分子标志物，但最终均以失败告终。外泌体是直径为30-100 nm的膜性囊泡，可由多种细胞分泌，其内包裹DNA、miRNAs、mRNAs、蛋白质等物质并参与细胞间交流。我们在前期研究中率先发现与顺铂接触后的肺癌细胞A549分泌的exosomes可使其他既往未接触过顺铂的A549细胞对顺铂相对耐药，且这种耐药很可能受到exosomes中携带的特定mRNA和miRNA调控。本项目拟在前期研究的基础上，建立顺铂耐药肺癌细胞系，在耐药细胞中进一步确认exosomes对肺癌细胞顺铂耐药的影响，研究exosomes对肺癌细胞重点信号通路的调控，系统研究和评价exosomes在肺癌细胞顺铂耐药中发挥的作用，明确肺癌细胞对顺铂耐药的可能分子机制，为预测肺癌患者顺铂治疗疗效和克服肺癌患者顺铂耐药的研究提供必要的前期理论基础和实验依据。

肺癌是全世界最常见也是死亡率最高的恶性肿瘤。化疗仍然是肺癌患者的重要治疗手段，而顺铂是肺癌化疗方案中最基础、最重要的药物之一。但肺癌患者顺铂耐药的机制研究及分子标志物研究仍未得到一个肯定的结论。本研究主要探讨耐顺铂的肺腺癌细胞分泌的外泌体是否参与肺癌细胞对顺铂的耐药并对其传递机制进行深入研究。本研究首先建立顺铂耐药肺癌细胞系，在耐药细胞中进一步确认exosomes对肺癌细胞顺铂耐药的影响，研究exosomes对肺癌细胞重点信号通路的调控，系统研究和评价exosomes在肺癌细胞顺铂耐药中发挥的作用。.通过以上研究，我们得到了以下结果：（1）A549/DDP（顺铂耐药肺腺癌细胞）细胞分泌的外泌体可以向A549细胞传递顺铂耐药，具体表现为：A549细胞经过与A549/DDP细胞分泌的外泌体（R-exo）共培养后，导致A549细胞对顺铂的IC50增加、凋亡率下降，并且能够将更多的细胞阻滞在G1期；（2）芯片结果表明A549细胞和它分泌的外泌体之间以及A549/DDP细胞和它的外泌体之间的microRNA表达谱具有正相关性。miR-197-5p、miR-4443、miR-642a-3p、miR-27b-3p以及miR-100-5p是细胞及外泌体中差异倍数前5的miRNA，实时荧光定量PCR验证差异表达的miRNA，其与芯片结果一致；(3)microRNA-100-5p是耐药细胞和亲本细胞及外泌体中差异表达最为明显的miRNA。该miRNA可以通过靶向mTOR影响肺腺癌细胞对顺铂的敏感性，并且可通过外泌体来传递信息；(4)体内实验证明外泌体中的microRNA-100-5p可以改变裸鼠肺腺癌移植瘤对顺铂的敏感性。 .本研究中，我们证实了miR-100-5p的降低导致了肺癌细胞A549对顺铂的耐药，并进一步通过体内和体外实验证实肺癌耐药细胞来源的外泌体通过传递miR-100-5p并靶向mTOR从而给受体细胞传递耐药性。据我们所知，这是首次关于外泌体中的miR-100-5p影响肺癌细胞对顺铂敏感性的系统研究。相关文章已经发表。