从ZEB1-SCRIB双向途径探讨化坚解毒活血法干预大肠癌转移的分子机制

Metastasis is the main cause of death in patients with colorectal carcinoma. We have previously detected through in vivo and in vitro experiments that the "Scleroma Disintegrating, Detoxicating with Blood Circulation Stimulating" method performs as a suppressor of invasion and metastasis through inhibiting ERK-MAPK pathway. Based on this finding, we speculated that the method may bidirectionally regulate the ZEB1-SCRIB loop (ERK-ZEB1-SCRIB(Lgl and Dlg) way or SCRIB(Scrib)-ERK-ZEB1 way), which is necessary for both the two core events in metastasis, that is, epithelial-mesenchymal transition (EMT) and losing of apical-basal polarity. Normal epithelial phenotype, intercellular junction and asymmetric cell division will be regained in response to the balanced relationship of ZEB1 and SCRIB complex. Considering that the body is lack of vital qi while the tumor is the accumulation of evil factors like toxic heat, phlegm and blood stasis is the core pathomechanism of metastasis, this project plans to examine the effect of pharmacological intervention on highly metastatic human colorectal cancer Lovo cell line by the typical evil eliminating drug XiHuangWan. To clarify the molecular mechanism and identify the effectors of Traditional Chinese Medicine, we intend to observe the expression level of ZEB1, SCRIB (Scrib, Lgl2, Dlg), EMT phenotype and the polarity marker apical junctional complex (AJC), furthermore to analyze the transcriptional regulation of ZEB1 and the phosphorylation of Lgl2, by means of ChIP-chip, immunoprecipitation combined with Western blot, Real Time PCR, etc. The achievement of this study is supposed to provide insight and evidence for research of antimetastasis with Chinese Medicine therapy.

转移是大肠癌的重要死因，在前期体内体外实验证实化坚解毒活血法可通过抑制ERK-MAPK通路而抗大肠癌转移的基础上，课题组提出进一步假说，认为该法抗转移机制可能是通过调控ERK-ZEB1-SCRIB(Lgl、Dlg)或SCRIB(Scrib)-ERK-ZEB1双向途径起到恢复上皮表型、细胞间连接及不对称分裂的作用，从而逆转EMT和顶-底极性丧失这两大密切相关的转移关键事件。基于机体为正，癌瘤为邪；热毒痰结、血瘀阻络为转移核心邪实病机的认识，项目拟选取西黄丸干预高转移性大肠癌lovo细胞株，采用ChIP-chip、免疫沉淀结合Western blot、实时定量PCR等手段，对EMT转录因子ZEB1进行转录调控分析和靶基因筛选，并分析极性蛋白复合物SCRIB各成分含量及化学修饰情况，观察EMT表型和极性标志物等指标，以期从基因、蛋白、膜分子等多个层次探明该法的作用靶点，为中药抗转移研究提供思路。

在前期体内体外实验证实解毒活血法中药西黄丸可通过抑制ERK-MAPK通路的激活而抑制大肠癌转移，文献学习发现ERK在上皮间质转化(Epithelial-to-mesenchymal transition ，EMT) 和顶-底极性丧失这两大转移关键事件的关键分子ZEB1-SCRIB构成的环路中起沟通作用的基础上，本研究旨在探讨西黄丸抗转移的深层次机制，观察其对EMT表型和上皮细胞极性表型的影响以及对ZEB1-SCRIB环路的作用。同时，寻找转录抑制因子ZEB1的其他可能靶点，以进一步解释其在促进肿瘤进展中的作用，并研究西黄丸对哪些ZEB1-靶基因启动子的结合具有影响，以及对EMT过程中哪些其他的分子和通路具有影响。实验结果显示，西黄丸干预可以恢复大肠癌LoVo细胞株上皮表型分子E-cadherin的基因和蛋白表达，抑制间质表型分子N-cadherin、Fibronectin的基因表达，从而逆转EMT的发生；恢复细胞顶底极性标志物JAM-1、Occludin、Claudin-7的基因和蛋白表达，从而逆转顶底极性的丧失。对此效应的机理探讨发现抑制转录因子ZEB1的蛋白表达，提高极性蛋白Scrib和Lgl2的表达，恢复ZEB1-SCRIB环路的平衡，是西黄丸引起EMT表型分子和极性标志物变化，逆转EMT和极性丧失以抗转移的机制。此外，以ChIP-on-chip 法发现的115个ZEB1的疑似靶基因主要富集于包括引发凋亡和细胞运动等与肿瘤相关的生物学过程及G蛋白偶联受体通路，提示ZEB1的促进肿瘤进展作用可能与上述生物过程相关。西黄丸干预抑制了ZEB1和其疑似下游靶点IL19和MSX2的结合，从而恢复二者表达，与其抗肿瘤效应的发挥有关。 敲低ZEB1基因引起了三个与DNA损伤应答相关的疑似靶基因USP17、CHD1L和DUX4的mRNA水平的升高，进而引起了细胞的凋亡增加和G1/S期阻滞，增加了其对顺铂和依托泊苷的敏感度，该发现完善了ZEB1促进耐药的分子机制。以QIAGEN Human Epithelial to Mesenchymal Transition PCR芯片筛选受西黄丸影响的EMT相关基因发现西黄丸干预可下调PLEK2、PTP4A1、NOTCH1三个促转移基因的mRNA表达，完善了西黄丸抗转移的机制。