非细胞固态基质类型、硬度参与调节肝癌侵袭转移基础研究

Many previous studies on HCC invasion and metastasis mainly focused on biological properties changes of tumor cells including adhesion, migration, cytoskeleton etc, or interaction between tumor cells and ambient mesenchymal cells by dint of the secreted soluble factors. But, only few literatures were found on non-cell solid-state matrix, as a tissue barrier and a 3D cell framework support, involving in regulation of HCC invasion and metastasis, more particularly, little was known on type and stiffness of non-cell solid-state matrix regulating this pathological process. From the view of physical and chemical characteristics of non-cell solid matrix such as type, synergic regulation and stiffness, this project is mainly planned to elucidate the molecular mechanism of tumor cells attaching and invading into matrix under 2D and 3D growth mode in early stage of metastasis, and obtain several lines of evidences about type and stiffness of matrix regulating HCC invasion and metastasis. Furthermore, determine the most important matrix protein and synergic mechanism of different matrix proteins during the early metastasis, as well as stiffness changes modulating HCC invasion and metastasis. Undoubtedly,these anticipated results will provide the theoretical and experimental basis for intervention target screening of HCC cells and matrix interaction.

以往肝癌侵袭转移的研究多聚焦于肿瘤细胞粘附、运动、细胞骨架等自身病理特征改变，以及肿瘤细胞与周边间质细胞间分泌因子介导的信号传导协同作用，却长期忽视作为肿瘤组织侵袭屏障、肿瘤细胞三维结构和变形运动力学支撑结构的胞外固态基质成分的调控作用，而从非细胞固态基质类型、协同调控及基质硬度等角度对肝癌细胞恶性生物学特征的调控研究更是相对缺乏，本项目拟以非细胞固态基质理化特征的基础变化作为始动因素，通过三维和两维生长方式下肝癌细胞与基质蛋白相互作用的差异，阐述不同基质蛋白类型，基质力学硬度对转移源头阶段肿瘤细胞粘附、侵入基质病理过程的分子调控，获取不同固态基质类型、基质力学硬度改变影响肝癌细胞恶性生物学特征的系列证据，确定显著影响肝癌侵袭转移基质蛋白类型或多种基质蛋白协同调控的作用模式，以及基质硬度改变参与肝癌细胞侵袭转移的可能分子调控机制，为肝癌细胞-基质间特异转移干预靶点的寻找提供理论和实验依据。

临床数据显示，约80%以上的肝癌患者具有重度肝纤维化和肝硬化背景，具有肝硬化背景的肝癌患者中位生存期低，高肝脏硬度促进肝癌进展，目前肝脏硬度已成为临床肝癌发生、进展及预后的强预测指标。尽管肝脏硬度影响肝癌进展的临床数据已引起临床肿瘤学者的较多关注，但由于缺少硬度研究相关实验平台，基质硬度调控肝癌侵袭转移的分子机制研究依然相对不足，本课题利用建立的两种具有创新意义的基质硬度研究平台，即不同肝基质硬度背景大鼠肝癌模型和体外不同基底硬度细胞培养体系，对基质硬度参与肝癌侵袭转移的相关分子机制进行解析。课题结果显示基质硬度增加可通过激活Integrinβ1-PI3K/AKT通路而上调肝癌细胞VEGF表达；可通过激活Integrinβ1-GSK3β/ β-cantenin途径上调肝癌细胞OPN表达。另外，基质硬度增加可通过激活ITGB1/Akt/mTOR/SOX2信号通路增强肝癌细胞的干性特征。基质硬度增加还可独立诱导肝癌细胞EMT的发生，三条途径包括S100A11膜转位-NADPH氧化酶复合物-ROS，Raf1-eIF4E磷酸化，TGFβ1自分泌共同参与了硬度诱导肝癌EMT的调节。本研究解决了基质硬度体外机制研究缺乏理想硬度实验平台问题，获得了基质硬度参与肝癌细胞侵袭转移调控系列证据及其相关的调控机制，发现了发现基质硬度物理信号作为始动因素参与肝癌侵袭转移的一些新机制，为后续肿瘤硬度微环境干预研究的开展奠定了良好的基础。