IRF7介导的TAM糖脂代谢重编程在骨肉瘤外泌体构建促肺转移微环境中的作用及淫羊藿干预的药效物质基础研究

Lung metastases are a frequent complication of osteosarcoma. Searching for the novel targets and mechanisms is benefit for treating and controlling lung metastasis of osteosarcoma. This study is focus on tumor microenvironment, which plays a critical role in facilitating tumor metastasis. Understanding the effect of exosome miRNA from high metastasis osteosarcoma cells on IRF7 expression in TAM, and how these changes contribute to glucose and lipid metabolic reprogramming of TAM and the M1/M2 phenotype of TAM may reveal novel therapeutic targets for lung metastasis of osteosarcoma. Based on these novel targets, the phenotypic, high-content microscopy screen was established to search effective compounds from the compounds library of Traditional Chinese Medicine Epimedium. To test the effect and mechanism of effective compounds on lung metastasis of osteosarcoma, the in vitro and in vivo lung metastasis models were established to evaluate the interaction between effective compounds and IRF7, as well as the regulation of effective compounds on glucose and lipid metabolic reprogramming of TAM and the M1/M2 phenotype of TAM. This study will provide theoretical and experimental basis for IRF7 as a novel therapeutic target for lung metastasis of osteosarcoma. Moreover, this study will provide evidence for the screening effective compounds on lung metastasis of osteosarcoma from Traditional Chinese Medicine Epimedium, which will benefit for the development of novel medicines with self-owned intellectual property.

肺转移是骨肉瘤患者预后不良的主要原因，探寻骨肉瘤肺转移过程中的新靶点、新机制，是治疗和预防肺转移性骨肉瘤的有效途径。本项目拟从肿瘤微环境的角度，阐明高转移性骨肉瘤的外泌体miRNA下调IRF7，诱导TAM糖脂代谢重编程促进其M2/M1表型比例升高，进而促进骨肉瘤细胞转移的作用机制。围绕骨肉瘤外泌体构建促肺转移微环境中的关键靶点，构建IRF7调控的TAM糖脂代谢和表型改变的抗骨肉瘤转移高内涵筛选模型，从淫羊藿中筛选抗骨肉瘤肺转移微环境的药效物质。利用体内外模型考察淫羊藿药效物质与IRF7蛋白的相互作用，对TAM糖脂代谢和表型的调控作用，以及对骨肉瘤肺转移的抑制作用，全面阐明淫羊藿药效物质抗骨肉瘤肺转移的作用及机制。本项目的实施为确证IRF7作为一个新的抗肺转移肿瘤微环境的靶点提供实验依据，为探究中药淫羊藿抗骨肉瘤肺转移微环境的药效物质提供科学支撑，为开发我国具有自主知识产权的新药奠定基础。

转移性骨肉瘤患者常见转移途径是血行转移，即骨肉瘤细胞侵入到血管中，随着血流转移到身体的远处器官如肺，形成转移瘤。其中，由肿瘤细胞自身通过上皮间质转化（EMT）驱动血管拟态（VM）形成的管状结构对肿瘤细胞生长、转移提供有利的微环境。外泌体可作为细胞间通讯的媒介。肿瘤外泌体诱导的肿瘤相关巨噬细胞（TAM）可显著性增加VM的形成。干扰素调节因子7（IRF7）是产生 I 型干扰素的重要调节因子。本项目组发现转移性骨肉瘤外泌体显著性降低TAM中IRF7的表达。敲低IRF7促进骨肉瘤外泌体诱导的TAM表型转变。miRNA簇是指在染色体上排列成簇的一组比单个miRNA具有更大调控作用的miRNA基因。本项目组发现转移性骨肉瘤外泌体携带miR-194-1簇靶向十四烷基化丙氨酸丰富蛋白激酶C底物（MARCKS）促进EMT驱动的VM形成。基于前述研究，本项目组利用四个miRNA数据库以及TAEGET数据库中的骨肉瘤临床数据构建miR-194-1簇特征基因集。然后将此特征基因集与Connectivity Map数据库中的小分子药物转录谱进行特征匹配，发现淫羊藿的主要成分淫羊藿苷(Icariin)的连接分数达到-97.72，提示淫羊藿中的活性产物可能具有miR-194-1簇抑制剂作用。结合文献报道和试验筛选，本项目组发现淫羊藿苷的体内活性代谢产物淫羊藿次苷II（Icariside II）是中药淫羊藿中抑制转移性骨肉瘤EMT化的药效物质，其通过结合PGK1对抗miR-194-1簇介导的EMT化作用。EMT、VM介导的免疫逃逸也是导致转移性骨肉瘤肺转移的原因之一。尽管免疫疗法不断扩大适应症，但在骨肉瘤中响应率极低。本项目组发现淫羊藿次苷II显著性升高转移性骨肉瘤中M1型TAM、CD8＋T细胞、记忆性T细胞、下调髓系抑制细胞，肿瘤抑制率为54.99%。本项目组还发现淫羊藿次苷II和穿心莲内酯可产生协同效果。二者在摩尔比为1:1时，协同指数（IC）为0.42。本项目组将两者共包封在谷胱甘肽响应的脂质体（Combo-NPs）中，达到在肿瘤细胞中按最佳协同比精准释药的目的。骨肉瘤原位小鼠模型表明，Combo-NPs促发抗肿瘤免疫效应，表现出高效的协同抗肿瘤作用，肿瘤抑制率为76.61%。此外，本项目组发现了一个新型血管拟态抑制剂，研究其与TLR7免疫激动剂的联合作用，并基于转录组学进行协同机制研究。