肝细胞癌中Hedgehog/Gli通路对EMT的调控及其在癌转移中的作用

项目名称： 肝细胞癌中Hedgehog/Gli通路对EMT的调控及其在癌转移中的作用

项目编号： No.81072052

项目类型： 面上项目

立项/批准年度： 2011

项目学科： 轻工业、手工业

项目作者： 刘青光

作者单位： 西安交通大学

项目金额： 10万元

中文摘要： 项目通过两个独立的临床研究分别发现GLI1与肝细胞癌（HCC）复发相关以及GLI1过表达与HCC的上皮细胞间质化（EMT）表型和转移相关。测定11种HCC细胞系和正常人原代培养肝脏细胞中的GLI1 mRNA含量后，选择Huh7细胞进行GLI1过表达实验，发现随着GLI1表达的增加，Huh7细胞的活性、增殖、迁移、侵袭以及细胞克隆形成能力均显著增强，同时诱导出明显的EMT表型；同时选择SNU398细胞进行GLI1沉默实验，得到了截然相反的结果。这提示过表达的GLI1蛋白促进了HCC的生长和转移，并其诱导了其EMT表型。在本项目的临床研究中，GLI1表达和EMT诱导因子SNAI1表达在mRNA水平上是正相关的。而针对Huh7和SNU398的体外实验也证实了GLI1上调了SNAI1的表达，进而通过染色质免疫共沉淀实验证实了GLI1蛋白与SNAI1启动子结合，直接上调其表达。而敲除SNAI1明显逆转GLI1诱导的EMT表型。这证明了GLI1通过直接上调SNAI1进而诱导HCC细胞EMT表型。同时，本项目也发现了GLI1可以被TGFβ#35825;导上调，并参与了其诱导HCC细胞EMT表型的分子机制。

中文关键词： GLI1；上皮细胞间质化；肝细胞癌；SNAI1；TGFβ

英文摘要： It has been shown the positive correlation between GLI1 and hepatocellular carcinoma (HCC) recurrence after surgery, and between GLI1 and the epithelial-to-mesenchymal transition (EMT) phenotype and metastasis respectively by two independent clinical investigation in this program. After detecting the GLI1 mRNA expression of 11 HCC cell lines and primary cultured normal hepatocytes, we selected Huh7 as GLI1-overexpressing model and SNU398 as GLI1-silencing model in vitro. Forced expression of GLI1 was verified to promote cell viability, proliferation, migration, invasion and colony formation of Huh7 cells. Particularly, GLI1 overexpression induced EMT phenotype in Huh7 cells obviously. Conversely, knockdown of GLI1 led to a decrease in all the above-mentioned cancer-associated phenotypes of SNU398 cells. In this present study, there was a significant positive relationship between the mRNA expression of GLI1 and SNAI1, a renowned EMT inducer, found in HCC tissues. SNAI1 expression was also verified to be increased by GLI1 overexpression in Huh7 cells and decreased by silencing GLI1 in SNU398 cells. In Huh7 cells, GLI1 protein was found to bind with the SNAI1 promoter and directly upregulate SNAI1 expression by chromatin immunoprecipitation assay (ChIP assay). In addition, silencing upregulation of SNAI1 induced by GLI1 in Huh7 cells repressed the EMT phenotype driven by forced expression of GLI1, which indicates that GLI1 induces EMT phenotype via upregulating SNAI1 directly in HCC. Finially, TGFβtreatment increased GLI1 expression apparently and induced EMT phenotype in Huh7 cells. Suppressing upregulation of GLI1 by induction of TGFβreversed TGFβdriven EMT phenotype. It indicates that GLI1 plays a critical role in the mechanism whereby TGFβleads to EMT in HCC. In summary, it is shown strongly in this investigation that GLI1 contributes to the growth, recurrence, metastasis and EMT of HCC and is involved in the mechanism by which TGFβinduces EMT phenotype. Additionally, TGFβis confirmed here to activate Hedgehog signaling in a non-canonical manner in HCC.

英文关键词： GLI1; EMT; HCC; SNAI1; TGFβ