肺癌细胞通过微囊泡（MV)运载AEG-1调控微血管内皮细胞生物学行为的分子机制研究

The research on angiogenesis mechanism mediated by microenvironment is a promising branch of cancer itiology. Our prior research has showed that AEG-1(Astrocyte Elevated Gene-1) has the abilitity to regulate the biological behavior of MEC(Microvascular Endothelial Cell). Firstly, lung cancer cells secrete microvesicles with AEG-1. Then, microvesicles with AEG-1 can be engulfed by MEC, through which AEG-1 enters into MEC and plays a vital role in angiogenesis.This study combines microdissection technology with the lung cancer model, which displays diverse lesions including hyperplasia of bronchia mucosa, squamous metaplasia, atypical dysplasia, carcinoma in situ and infiltrating carcinoma, to explore how AEG-1 dynamicly regulate the gene expression profile of MEC and tumor angiogenesis. Simultaneously,we utilize cell co-culture and angiogenesis assay to observe the ultrastructure of MVs and their influence on the biological behavior of MECs. Combining plasmid transfection, RNA interference with GST-pull down technology, we explore the molecular mechanism about JAK/STAT pathway through which AEG-1 regulates the biological behavior of MECs and further mediates angiogenesis. Related assays were devised to search for the specific target protein with which AEG-1 interact, and angiogenesis factors being downstream of AEG-1/JAK/STAT in order to provide experimental basis for target therapy of lung cancer. Eventually, basing on tissue and blood samples,we analyze the correlationship between various genes and clinical pathology parameters utilizing SVM (Support Vector Machine）to provide scientific data for the early diagnosis, recurrence and prognosis of lung cancer.

微环境介导血管生成日益成为肿瘤病因学的研究热点。我们前期研究发现肺癌细胞可以分泌微囊泡(MV),且含有AEG-1的MV可以被微血管内皮细胞(MEC)吞饮,进而调控MECs的生物学行为。本课题拟利用具有从支气管粘膜增生、鳞状化生、不典型增生、原位癌到浸润癌的肺癌模型，结合显微切割等技术，检测AEG-1对MECs基因表达谱和血管生成的动态调控。采用细胞共培养和血管生成试验等实时观察MV的超微结构及对MEC生物学行为的影响；结合质粒转染、RNA干扰和GST-pull down技术，探究JAK/STAT通路在AEG-1调控MECs生物学行为进而介导血管生成的分子机制,明确AEG-1直接作用的靶蛋白,探明AEG-1/JAK/STAT下游直接调控的血管生成因子，为肺癌靶向治疗提供依据。结合临床组织和血液标本，利用SVM模型分析AEG-1等基因与临床病理参数的关系，为肺癌早期诊断、复发和预后判断奠定基础

大多数肺癌患者确诊时已属晚期，失去了根治性手术治疗的机会，放化疗效果也不理想，导致肺癌死亡率居高不下。阐明肺癌发病的分子机制，为肺癌临床治疗提供坚实的理论基础有着重要的意义。.转移和复发是恶性肿瘤导致患者死亡的主要原因，是肿瘤演进和预后变差关键步骤，故阐明转移过程及其分子机制显得尤为重要和迫切。肿瘤转移过程包括肿瘤细胞粘附性降低、从肿瘤原发灶脱离、水解基底膜、侵入血管或淋巴管、随着血液或淋巴回流到达新的的器官定植生长等一系列序贯而复杂的过程。涉及的机制包括上皮间质化(epithelial-to-mesenchymal transition, EMT)、肿瘤血管形成、炎症微环境的形成、自噬机制的紊乱和程序性细胞死亡(programmed cell death，PCD)障碍、免疫监视逃逸等。.AEG-1基因(Astrocyte elevated gene-1，AEG-1)是新发现的癌基因，异常高表达于乳腺癌、肺癌、膀胱癌、卵巢癌、胃癌及淋巴造血系统等恶性肿瘤。AEG-1通过影响多条信号通路在肿瘤细胞增殖、进展、血管生成和转移等过程中起着重要的作用。为了阐明AEG-1介导非小细胞肺癌转移的分子机制及其在不同组织学类型的肺癌的表达规律。本课题首先构建了慢病毒载体并感染A549和GLG-82肺癌细胞系，建立了AEG-1稳定表达肺癌细胞系，并利用miRNA芯片杂交构建miRNA差异表达谱。接着我们选择了表达变化显著的miR-203a进行后续研究对象，通过功能获得研究对转染了miR-203a mimic的人肺癌细胞进行检测，观察过表达外源性miR-203a对细胞迁移及侵袭能力的影响，同时通过功能缺失研究抑制内源性miR-203a的表达对肺癌细胞迁移及侵袭能力的影响；我们通过miRNA靶基因预测软件寻找miR-203a的靶基因，并通过荧光素酶报告实验进行验证，进而初步探讨miR-203a抑制肺癌迁移、侵袭的信号通路，明确miR-203a通过调控TLR4和LASP1的表达而发挥其影响肺癌侵袭转移的分子机理。同时确定了miR-203a是TLR4/AEG-1相互调控正反馈环的关键分子。本课题的研究成果将加深我们对肺癌转移分子机理的了解，为肺癌的转移治疗提供重要的分子靶点。上述研究结果将为抑制肺癌转移的靶向治疗提供新的理论基础。