乏氧诱导的外泌体在肿瘤侵袭转移、血管生成和辐射抵抗中的作用及其机制研究

It is well known that the solid tumor has hypoxia microenvironment. As an important carrior of messenger molecules in the tumor hypoxic microenvironment, exosomes have multiple biological functions and contribute to tumor progresses and developments. Our previous study observed a phenonmenon that the hypoxia treatment could promote exosome release from lung cancer cells, which was relative to hypoxia-radioresistance. In this project, we will use lung cancer cell lines of A549 and H446 with different radiosensitivity and human umbilical vein endothelial cells (HUVEC) to investigate the mechanisms of the effects of hypoxia-induced exosomes on cancer invasion, metastasis, angiogenesis and radioresistance by analysing the alterations of proteins and miRNAs in tumor cell exosomes after hypoxia treatment with proteomics, SILAC-LC-MS and Illumina Hiseq-2000 deep sequencing techniques, and by investigating the roles of gene up-regulation and down-regulations by siRNA and inhibitor in cell survival, apoptosis induction, micronucleus formation, tumor cell invasion and metastasis, angiogenesis, protein and mRNA expressions. It can be expected that some novel mechanisms of hypoxia-modulated radioresistance, tumor invasion and metastasis and angiogenesis will be achieved, which may provide theoretical fundation for developing new tumor targeted drugs and new strategy of tumor theray.

乏氧可以引起肿瘤微环境的改变，外泌体作为乏氧肿瘤微环境中的重要的信号因子携带者，具有多种生物学功能，参与肿瘤的多种重要生物过程。前期研究发现，乏氧会促进肺癌细胞外泌体的释放，增加了对射线的抵抗。本课题拟以具有不同辐射敏感性的肺癌细胞和血管内皮细胞为对象，通过SILAC-LC-MS和Illumina Hiseq 2000深度测序技术，结合基因上调、siRNA干扰和表达抑制，采用体外集落形成实验、微核试验、侵袭实验、血管形成、流式细胞术、Western blot、RT-PCR及动物实验等方法，在细胞水平和动物水平上深入探索乏氧微环境中肿瘤细胞外泌体及其所携带的差异的蛋白和miRNA在乏氧诱导的外泌体对肿瘤增殖、侵袭转移和血管生成的作用，明确其参与辐射抵抗促发肿瘤进展的作用机制，为寻找肺癌治疗新靶点、提高疗效提供理论依据。

乏氧可以引起肿瘤微环境的改变，外泌体作为乏氧肿瘤微环境中的重要的信号因子携带者，具有多种生物学功能，参与肿瘤的多种重要生物过程。前期研究发现，乏氧微环境会促进肺癌细胞外泌体的释放，同时可增加肿瘤细胞对射线的抵抗性。本课题选用具有不同辐射敏感性的肺癌细胞、血管内皮细胞、神经胶质瘤细胞和鼻咽癌细胞，通过TMT蛋白组学和基因测序技术获得乏氧诱导的细胞水平和外泌体水平的特异性蛋白和基因数据库。围绕乏氧外泌体携带的特异性蛋白，探索了乏氧标志性蛋白可浓缩于外泌体中，随细胞分泌进入肿瘤微环境，并进一步介导肿瘤细胞的辐射抵抗、细胞增殖、侵袭迁移和血管内皮细胞的生成。鉴于外泌体存在于机体的多种体液，获得相对容易，且具有生物稳定性，可在肿瘤微环境中较远距离运输信号因子及各类炎症因子，具有肿瘤早期诊断和治疗靶点的重要意义。同时，我们聚焦于对辐射抵抗机制的探索，围绕细胞自噬这一研究热点，在前期获得数据库中选择了信号转导与转录激活因子3 (STAT3)、组织蛋白酶D（CTSD）和膜连蛋白6（ANXA6），探索他们在乏氧肺癌细胞、神经胶质瘤细胞核鼻咽癌细胞中诱导自增加肿瘤细胞辐射抵抗性的分子机制，探索新的分子靶点，有望为寻找肿瘤治疗新靶点、提高肿瘤诊断和放疗疗效提供理论依据和新策略。