肺癌细胞通过微囊泡（MV）运载CTHRC1调控EMT促进癌细胞侵袭转移的分子机制

The research on epithelial-mesenchymal transition (EMT) mediated by microenvironment is increasingly becoming a hotspot of tumor invasion and metastasis. Our prior research has showed that CTHRC1 (collagen triple helix repeat containing 1) function to regulate the biological behavior of lung cancer cells. Firstly, lung cancer cells secrete microvesicles containing CTHRC1, influencing the microenvironment balance. Then, microvesicles with high CTHRC1 expression can be engulfed by the adjacent cancer cells, through which CTHRC1 enters into cells and plays a vital role in tumor invasion and metastasis. Our present study combines microdissection and microvesicle purification technologies 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 CTHRC1 dynamically regulate the gene expression profile of Wnt/β-catenin and Wnt/PCP pathway and the EMT process. Simultaneously, we utilize cell co-culture, xCELLigence and electron microscopy assays to observe the ultrastructure of MVs and their influence on the biological behavior of cancer cells. Combining plasmid transfection, RNA interference with GST-pull down technology, we probe into the molecular mechanism about Wnt/β-catenin and Wnt/PCP pathway through which CTHRC1 regulates EMT and further mediates invasion and metastasis. Related assays were devised to search for the specific target protein with which CTHRC1 interact, and the EMT-related factors being downstream of CTHRC1/Wnt/β-catenin and CTHRC1/Wnt/PCP in order to provide experimental basis for elucidating the molecular mechanism of invasion and metastasis and target therapy of lung cancer. Eventually, basing on tissue, serum and circulation tumor cell samples, we analyze the correlationship between various genes and clinical pathology parameters utilizing SVM (Support Vector Machine）to provide scientific data for the early metastasis and prognosis of lung cancer.

微环境介导EMT日益成为肿瘤侵袭转移的研究热点。我们前期研究发现微囊泡(MVs)介导微环境的调控, 且高表达CTHRC1的MV可以与跨膜受体相互作用,进而调控癌细胞的生物学行为。本课题拟检测CTHRC1在肺癌发生过程中的动态变化及在微囊泡中的表达特征，分析其与Wnt/β-catenin和Wnt/PCP通路和EMT的动态关系。实时监测MV的超微结构及其对癌细胞侵袭转移的影响；探究Wnt/β-catenin和Wnt/PCP通路在CTHRC1调控EMT进而介导侵袭转移过程的分子机制,明确CTHRC1直接作用的靶蛋白,探明CTHRC1/Wnt/β-catenin和CTHRC1/Wnt/PCP下游直接调控的EMT相关因子，为阐明肺癌侵袭转移分子机制和靶向治疗提供理论依据。结合临床组织、血清和循环肿瘤细胞标本，利用SVM模型分析CTHRC1等基因与临床病理参数的关系，为监测早期转移和预后判断奠定基础。

细胞外囊泡（extracellular vesicle, EVs）是由细胞释放的各种具有膜结构的纳米级囊泡结构统称。其中外泌体（exosomes）、微囊泡（microvesicle, MVs）、膜微粒（microparticle, MP）均属于此范畴。其中外泌体直径30-150nm，目前认为这种微小囊泡中含有特异的蛋白、脂质和核酸，能作为信号分子传递给其他细胞，从而改变其他细胞的功能。申请人前期研究结果表明，CTHRC1在肺癌的发生发展中起到了非常重要的作用，可作为预测肺癌预后的独立因子。此外，在体内和体外试验中证明CTHRC1与肺癌的侵袭转移相关。本项目在前期工作基础上，进一步验证和发现CTHRC1是通过外泌体运载进而调控非小细胞肺癌（non-small cell lung cancer, NSCLC）细胞的侵袭迁移。通过超速离心法分离外泌体，电镜鉴定技术与粒径分析进一步确认外泌体形态和直径，分析确定CTHRC1通过外泌体运载促进NSCLC细胞的侵袭迁移。此研究为进一步阐明CTHRC1的促肿瘤发生发展机制、指导预后和寻找NSCLC治疗新靶点，提供理论依据。