肿瘤细胞来源外泌体（exosome）选择性转运miR-423促进肺癌上皮-间质转化及转移研究

Lung cancer is one of the most common and lethal cancers worldwide. Metastasis constitutes a critical step in the pathogenesis of lung cancer. Epithelial-mesenchymal transition (EMT), a process is regarded as a critical event during tumor metastasis. Exosomes are small vesicles (30-150nm) secreted by various types of cells containing DNA, RNA and protein, which are emerging as local and systemic cell-to-cell mediators of information through the horizontal transfer of signaling macromolecules in various pathophysiologic process including tumor development. miRNA is one of the main functional component of exosome. In our early study, we have analyzed difference of small RNA profiling of exosome derived lung cancer cells A549 and normal bronchial epithelial cells 16HBE using high-throughput sequencing. Several miRNAs were screening out and some of them were related to EMT and metastasis. miR-423 was enriched in exosome of A549, which was higher than A549 cells and also higher than exosome of 16HBE. In this research, we will explore the mechanism of exosome transferring cancer-secreted miR-423 to promote EMT and metastasis of lung cancer by exosome/cell co-culture and tail vein injection of exosome experiment. Finally, we will analyze the diagnostic value of exosomal miR-423 in diagnosis and prognosis of lung cancer by clinical serum samples.

肺癌发病率和死亡率居恶性肿瘤之首，转移是肺癌的恶性标志和特征。上皮-间质转变（EMT）是转移的重要机制之一。外泌体（exosome）是由细胞主动分泌的30-150nm微小囊泡，其携带了大量的蛋白和核酸分子可介导细胞间信息传递而发挥促癌作用。miRNA是exosome主要的核酸类型之一，前期预实验中，利用高通量测序技术对肺腺癌细胞A549和正常支气管上皮细胞16HBE来源的exosome内小RNA表达谱分析，筛选出与EMT和转移有关的多种差异表达的miRNA。其中A549 exosome中有miR-423富集，不仅比A549细胞水平升高，也比16HBE-exosome高。本课题将应用exosome/细胞共培养及exosome尾静脉注射动物模型技术，研究exosome选择性转运miR-423促进肺癌EMT及转移机制，并利用临床血清样本探讨其作为循环标志物应用于肺癌诊断及预后的可能性。

肺癌发病率和死亡率居恶性肿瘤之首，转移是肺癌的恶性标志和特征。外泌体（exosome）是由细胞主动分泌的30-150nm微小囊泡，其携带了大量的蛋白和核酸分子可介导细胞间信息传递而发挥促癌作用。研究表明上皮-间质转变（EMT） 是引起肺癌远处转移的关键步骤，一方面，exosome可能通过EMT途径参与肿瘤发生发展，另一方面，某些特殊来源的exosome 会促进受体细胞发生EMT。miRNA是exosome主要的核酸类型之一，前期预实验中，利用高通量测序技术对肺腺癌细胞A549和正常支气管上皮细胞16HBE来源的exosome内小RNA表达谱分析，筛选出与EMT和转移有关的多种差异表达的miRNA。本课题中继续利用TGF-β1诱导肺腺癌A549细胞构造EMT模型，分离出间质型细胞（M-cell）来源外泌体(M-exsome)、上皮型细胞（E-cell）来源外泌体（E-exosome）以及正常肺癌上皮细胞（Con-exosome）来源外泌体（Con-exosome）；细胞共培养实验证明M-exosome可以进入E型上皮细胞并促进上皮细胞发生EMT转变及转移能力增强；进一步分析M-exosome、E-exosome、Con-exosome、M-cell、E-cell及Con-cell六种水平小RNA表达谱发现，exosome miRNA表达谱伴随EMT的表型表型变化而变化，并筛选出包含miRNA-423多种差异基因，主要靶向调控细胞间紧密连接、粘附功能及 TGF-β等与肿瘤EMT和转移密切相关的信号通路；最后，临床癌诊断价值研究中，建立肺癌血清样本库，并完成肺癌晚期转移组（M-serum-exosome），早期未转移组（E-serum-exosome），非肺癌其他肺部疾病对照组以及健康对照组四种患者血清来源exosome的小RNA测序，联合细胞exosome差异基因筛选出多种候选标志物（如miRNA-1426, miRNA-192, miRNA-320c），在大样本验证后，构建的exosome多指标联合检测模型，对于肺癌EMT转移监测及预后评估将有重要应用价值。