GPC3调控肝细胞癌侵袭和转移的信号通路研究

Epithelial-mesenchymal transition(EMT) is an important mechanism for tumor invasion and metastasis.Our previous studies indicated that GPC3 was closely correlated with hepatcelluar carcinoma (HCC) cells undergoing EMT. Furhtermore, we firstly demostrated that Brachyury, a T-box transcriptional factor, aslo a target gene of Wnt pathway, was highly expressed in HCC and it was modulated by GPC3. It is well known that GPC3 can activate Wnt signal pathway through binding to Wnt3a. Based on these findings, we hypothesize that GPC3 induces HCC EMT and promotes tumor invasion through modulating Wnt/β-catenin/Brachyury pathway.To test this hypothesis, we decide to carry out the following research. First, we will analyze the association between GPC3, Brachyury and EMT through clincal samples study. Then we will observe the influence of GPC3 on the biological behaviors (including EMT, migration and invasion, and so on) of HCC cells and the change of the key molecules and downstream target genes of Wnt/β-catenin/Brachyury pathway. Thirdly, through in vivo animal model, we want to testify that GPC3 induces EMT through Wnt/β-catenin/Brachyury pathway and promotes progress of HCC. Finally, we will reveal that the expression of GPC3 is regulated by Brachyury through promoter luciferase reportor assay and chromatin immunoprecipitation (ChIP) assay. This project aims to elucidate the molecular mechanism that GPC3 modulates HCC invasion and metastasis, which may provide new evidence and therapeutic target for GPC3-based HCC therapy.

上皮-间质转化（EMT）是肿瘤侵袭和转移的重要机制。我们前期研究发现GPC3表达与肝癌细胞EMT密切相关,而且我们首次发现T-box转录因子Brachyury在肝癌中高表达，并受GPC3调控。鉴于GPC3能与Wnt3a结合，我们推测GPC3可能通过Wnt/β-catenin/Brachyury通路诱导EMT，促进肝癌细胞侵袭和转移。本项目拟首先通过临床标本分析GPC3、Brachyury和EMT的相关性；其次观察GPC3对肝癌细胞EMT及侵袭和转移等生物学行为的影响，并检测该过程中Wnt/β-catenin/Brachyury通路主要成员及下游靶基因表达水平的变化；然后采用动物模型证实GPC3通过激活该通路诱导EMT促进肝癌侵袭和转移；最后通过细胞实验证实GPC3表达上调受Brachyury调控。本项目旨在阐明GPC3调控肝癌侵袭和转移的分子机制，为以GPC3为基础的靶向治疗提供新靶点。

上皮-间质转化（EMT）是肿瘤侵袭和转移的重要机制。我们前期研究发现GPC3表达与肝癌细胞EMT密切相关,而且我们首次发现T-box转录因子Brachyury在肝癌中高表达，并受GPC3调控。鉴于GPC3能与Wnt3a结合，我们推测GPC3可能通过Wnt/β-catenin/Brachyury通路诱导EMT，促进肝癌细胞侵袭和转移。本项目拟首先通过临床标本分析GPC3、Brachyury和EMT的相关性；其次观察GPC3对肝癌细胞EMT及侵袭和转移等生物学行为的影响，并检测该过程中Wnt/β-catenin/Brachyury通路主要成员及下游靶基因表达水平的变化；然后采用动物模型证实GPC3通过激活该通路诱导EMT促进肝癌侵袭和转移；最后通过细胞实验证实GPC3表达上调受Brachyury调控。在肝癌细胞系中通过对GPC3敲除组和过表达组的研究发现，GPC3除了能通过激活AKT/Snail以及brachyury信号通路诱导EMT，同时也能激活NF-κB通路。现已完成了GPC3相关的动物实验，通过GPC3过表达细胞系SMMC-7721和SK-HEP-1在裸鼠中的皮下瘤模型和肝原位模型中发现GPC3可以促进瘤体形成和转移，也可以促进肿瘤远处转移的发生。机制上证明了GPC3通过激活Brachyury通路调控EMT。另外，本实验还进行以下探索性研究：通过基因芯片技术发现，GPC3与多种促进转移的干性指标相关，并通过通路富集分析揭示了GPC3能通过MAPK/CREB通路调节与侵袭相关的MMP2、MMP3以及细胞因子CCL3、CCL5，CCL3、CCL5能促进巨噬细胞分化成M2型并对M2型巨噬细胞有招募作用。本项目旨在阐明GPC3调控肝癌侵袭和转移的分子机制，为以GPC3为基础的靶向治疗提供新靶点。