罗勒多糖调控乏氧微环境下上皮间质转化（EMT）抑制肝癌侵袭转移的机制研究

Invasion and metastasis of tumor cells is a bottleneck which restricts the prognosis of Hepatocellular Carcinoma (HCC). Tumor hypoxic microenvironment is closely associated with the invasion and metastasis of tumor in which epithelial-mesenchymal transition (EMT) is the key link. Under the hypoxic condition, hypoxia-inducible factor-1alpha (HIF-1α) can regulate transcription factors of EMT and activate EMT process, which result in the reduction of expression of an epithelial marker --- E-cadherin, and the declination of the adhesive ability between cells, and lead to the invasion and metastasis of tumor. Basil polysaccharide, the blood-activating and stasis-dissolving drug, is an anti-metastatic drug which we found in our previous study. By down-regulating the expression of HIF-1α and up-regulating the expression of E-cadherin, basil polysaccharide can improve tumor hypoxia microenvironment and inhibit tumor metastasis. Therefore, we hypothesize that, by regulating the expression of HIF-1αin hypoxia micro-environment，basil polysaccharide could control the expression of EMT transcription factors, then regulate the expression of E-cadherin, and plays a role in prevention and treatment of HCC metastasis during the EMT-controlling process. Through the tumor hypoxic model and the tumor metastasis model, this research will observe the control of HIF-1αto EMT transcription factors --- Snail, SIP1, Twist, CAIX and ZEB1, and the change of the expression of E-cadherin in hypoxia micro-environment. The target of this project is revealing the functional mechanisms of basil polysaccharide against tumor metastasis by intervening in the process of EMT.

肿瘤细胞侵袭转移是制约肝癌预后的瓶颈。肿瘤乏氧与侵袭转移密切相关，上皮间质转化（EMT）是侵袭转移的关键环节。乏氧条件下，乏氧诱导因子HIF-1α能调控EMT相关转录因子，激活EMT过程，导致上皮细胞标志物E-cadherin表达降低，细胞黏附能力下降，引起肿瘤侵袭转移。活血化瘀组分中药罗勒多糖是课题组前期发现的抗肿瘤转移新药，能改善肿瘤乏氧状态，通过下调HIF-1α，上调E-cadherin的表达抑制肿瘤转移。因此我们提出假说：罗勒多糖可通过调节乏氧条件下HIF-1α的表达来调控EMT转录因子的表达，进而调节E-cadherin的表达，通过调控EMT过程起到抗肝癌转移的作用。本项目通过建立肿瘤乏氧和转移模型，观察乏氧条件下HIF-1α对EMT转录因子Snail、SIP1、Twist、CAIX、ZEB1的调控及E-cadherin的表达，揭示罗勒多糖干预EMT过程发挥抗肿瘤转移的作用机制。

肝癌是发病率和死亡率均较高的恶性肿瘤。侵袭转移是影响恶性肿瘤预后的最主要原因，上皮来源的肿瘤细胞通过上皮间质转化(EMT)获得侵袭转移能力，并从原发病灶中分离是肿瘤转移的关键步骤。在肿瘤缺氧微环境中，HIF-1α能诱导和调控EMT的过程。HIF-1α可激活EMT相关信号通路，上调EMT转录因子的表达，进而降低上皮细胞标志蛋白的表达，增加间质细胞标志蛋白的表达水平，促进通过EMT调控的肿瘤细胞的侵袭转移。罗勒多糖是活血化瘀中药罗勒的有效成分，前期研究显示罗勒多糖具有良好的抗肿瘤转移作用。.本项目选用高转移人肝癌细胞MHCC97H和低转移人肝癌细胞MHCC97L，采用缺氧诱导剂CoCl2诱导细胞缺氧，建立体外细胞缺氧模型。通过罗勒多糖干预，观察罗勒多糖对常氧和缺氧条件下两种不同转移潜能肝癌细胞的侵袭转移能力的影响。建立MHCC97H细胞裸鼠皮下瘤模型，观察罗勒多糖在体内对EMT过程的影响。在此基础上，选用walker-256细胞株，建立wistar大鼠移植性肝癌模型，并进行肝动脉结扎术（hemorrhoid artery ligatoion，HAL），造成大鼠移植性肝癌肿瘤组织缺氧，观察在体内缺氧条件下，罗勒多糖EMT过程的影响。通过罗勒多糖对上述体内和体外、常氧和缺氧模型，本研究旨在阐明罗勒多糖的抗肿瘤转移机制，探讨其抗肿瘤转移作用是否与EMT有关。.研究结果显示罗勒多糖能抑制MHCC97H/L细胞EMT的发生，这一作用与肿瘤缺氧微环境密切相关，并具有细胞差异性，对转移能力强的MHCC97H细胞表现出更显著的作用。对MHCC97H细胞，罗勒多糖能通过调节缺氧条件下HIF-1α的表达以及EMT相关的E-cadherin、β-catenin、N-cadherin、Vimentin、VMP1的表达，影响EMT的发生，抑制肿瘤细胞的侵袭和运动能力；而对MHCC97L细胞，罗勒多糖仅通过调节缺氧条件下HIF-1α的表达和EMT相关的E-cadherin的表达，抑制肿瘤细胞的运动能力，对侵袭能力无明显作用。罗勒多糖能从多个方面对肿瘤产生影响，从而起到抗肿瘤转移的作用。结合本实验和前期研究结果发现，罗勒多糖能改善肿瘤缺氧微环境、抑制肿瘤细胞EMT的发生、增强机体免疫力进而改善实验动物生存状态，其抗肿瘤转移是多靶点、多途径共同作用的结果。