流体剪切力调控肝癌细胞上皮-间质转化和迁移的力学生物学机制探索

The epithelial to mesenchymal transition (EMT) is regarded as a significant mechanism for explaining invasion and metastasis of malignant epithelial tumor cells. Nowadays, most of the attentions are focused on the biochemical stimuli induced-EMT in tumor cells; however, there is not documented so far whether some biophysical factors such as shear stress could induce EMT and what is the intrinsic mechanism if it exists in tumor cells. Our previous investigation demonstrated that the epithelial markers such as E-cadherin could be down-regulated while the mesenchymal markers including N-cadherin, Vimentin and Snail were up-regulated in time-dependence manner in HepG2 cells when exposed to 0.14 Pa shear stress under steady laminar flow. Therefore, we hypothesize that fluid shear stress is an important biomechanical factor which could induce EMT in hepatocellular carcinoma cells (HCCs) and then lead to local invasion and distant metastasis, and that the phenotype transformation and the invasion/migration ability of HCCs are all regulated by the expression level of the crucial molecules in tumor cells. In this project, the multi-disciplinary methods and techniques will be used to establish a mechanical loading system, which could simulate mechanical environment in vivo. We will focus on studying the role of shear stress on EMT of HCCs, and analyzing the effect of invasion/migration ability of HepG2 cells with EMT. In addition, we will explore the potential biomechanical mechanism of shear stress regulated-EMT signal pathways. Specially, we will pay attention to the crucial mechanical transduction pathways such as "Integrin-ILK-GSK-3β(Akt)-ZEB1-Snail" signal axis. The finding of this project maybe help to reveal the mechanobiological mechanism of invasion and metastasis of HCCs with EMT, find potential drug targets, and provide new cues for prevention and cure of HCCs. Therefore, the research of this project has profound scientific significance and underlying application.

上皮-间质转化（EMT）是上皮来源的恶性肿瘤细胞侵袭和转移的重要机制。我们前期研究发现，流体剪切力可下调肝癌细胞的上皮标志蛋白E-cadherin的表达，同时上调间质标志蛋白N-cadherin、Vimentin等的表达，提示剪切力可能是诱导肝癌细胞发生EMT、继而引起其局部浸润和远处转移的重要生物力学因素。本项目将应用细胞生物力学、分子生物学等多学科的方法和技术，研究剪切力对肝癌细胞EMT的诱导作用；探讨肝癌细胞获得EMT特性对其侵袭和迁移的影响。在此基础上，分析剪切力调控肝癌细胞表型转化和细胞迁移的信号转导途径，重点考察"Integrin-ILK-GSK-3β(Akt)-ZEB1-Snail"等信号通路上关键分子的变化规律，从而解析剪切力调控肝癌细胞EMT和迁移的力学生物学机制。本项目研究将有助于发现新的抗癌药物靶标，为肝癌防治提供新线索。因此，具有深远的科学意义和潜在的应用前景。

本项目应用工程学、生物力学、细胞生物学和分子生物学等方法和技术，在体外构建了可提供不同流体剪切力的流室（flow chamber）实验系统，模拟诱导肝癌细胞发生上皮-间质转化（EMT）的力学环境，在此基础上研究了流体剪切力对肝癌细胞EMT的诱导作用，以及肝癌细胞获得EMT特性后其侵袭和迁移能力的变化。同时，探讨了流体剪切力调控肝癌细胞上皮-间质转化的力学生物学机制。最后，通过动物实验评价了流体剪切力诱导发生了EMT的肝癌细胞在体内成瘤、侵袭和迁移能力，以及在体内所处的力学微环境中，干扰Snail基因对肝癌细胞EMT的发生、发展以及侵袭、迁移能力的影响。研究结果表明：(1)在流体剪切力作用下，肝癌细胞上皮标志物E-cadherin时序性表达下调，而间充质标志物N-cadherin、β-catenin、Snail等时序性表达上调，表明流体剪切力可诱导肝癌细胞发生上皮-间质转化。(2)肝癌细胞发生EMT后，其侵袭和迁移能力显著增强。(3)流体剪切力主要通过“Integrins—ILK—PI3K”信号通路调控肝癌细胞EMT的发生，而Integrins是细胞膜上响应流体剪切力的重要力学感受器。(4)流体剪切力可诱导β-catenin进入肝癌细胞核，进而调控E-cadherin/N-cadherin的表达和分布；(5)发生EMT的肝癌细胞FAK磷酸化水平和Rho GTPases蛋白表达上调，增强了细胞的迁移能力。(6)撤销剪切力后，肝癌细胞发生反转的间质-上皮转化（MET）过程。综上所述，本研究揭示了流体剪切力诱导肝癌细胞上皮-间质转化的分子机理，从新的视角阐明了肝癌的发病机理，为肝癌防治提供了新线索。