Elmo1诱导上皮-间质转化促进肝癌细胞侵袭转移的机制研究

Epithelial to mesenchymal transition (EMT) is an important mechanism for tumor invasion and metastasis. In our previous study, we found Elmo1 was over-expressed in hepatocellar carcinoma (HCC) tissues through two-dimentional electropheresis (2-DE) and mass-spectrometry. In addition, over-expression of Elmo1 promoted HCC invasion, accompanied with obvious EMT phenomenon (up-regulation of E-cadherin and down-regulation of Vimentin). In addition, this phenomenon could be reversed by PI3K pathway inhibitor. Based on these results, we propose that Elmo1 might induce EMT through activation of PI3K/AKT pathway, therefore promoting HCC invasion and metastasis. To test this hypothesis, we decide to carry out following research. First, we will analyze the association of Elmo1 with EMT through clinical sample examination. Then we will observe the influence of Elmo1 on the biological behaviors (including EMT, migration and invasion, and so on) of HCC cells and the change of PI3K/AKT pathway key molecules and downstream target genes. Thirdly, through in vivo animal model, we want to testify that Elmo1 induce EMT through PI3K/AKT pathway and promote advancement of HCC. Finally, through clinical samples and cellular experiment we will prove that the expression of Elmo1 is regulated by long non-coding RNA (lncRNA). This project aims to elucidate the molecular mechanism how Elmo1 induces EMT and promotes HCC invasion and metastasis, which may provide new therapeutic target for HCC.

上皮-间质转化（EMT）是肿瘤侵袭和转移的重要机制。我们前期采用双向电泳-质谱分析发现肝癌组织中Elmo1表达增加；Elmo1高表达能够增强肝癌细胞侵袭能力且伴有明显的EMT现象（E-cadherin下调，Vimentin上调），PI3K通路抑制剂可逆转该效应。我们推测Elmo1可能通过激活PI3K/AKT信号通路诱导EMT促进肝癌进展。本项目拟首先通过临床标本分析Elmo1和EMT的相关性；其次观察Elmo1对肝癌细胞EMT及侵袭和转移等生物学行为的影响，并进一步检测该过程中PI3K/AKT信号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Elmo1通过激活PI3K/AKT信号通路诱导EMT促进肝癌侵袭和转移；最后通过临床标本和细胞实验证实Elmo1表达上调受相关lncRNA的调控。本项目旨在阐明Elmo1诱导EMT促进肝癌侵袭和转移的分子机制，为治疗肝癌提供新的作用靶点。

我们前期研究表明Elmo1有可能通过上皮-间质转化（EMT）机制促进肝细胞癌（HCC）的侵袭、转移。我们首先构建了组织芯片对263对肝癌组织及癌旁组织进行免疫组化检测，发现Elmo1肝癌组织中表达高于癌旁组织，Elmo1高表达的肝癌患者其1、3、5年生存率分别为64.6%、 39.9% 和33.1%；低表达的肝癌患者1、3、5年生存率分别为77.1%、55.2%和49.2%（P=0.008）；而相应的无瘤生存期（DFS）分别为39.5%、25.5% 和50.5%、39%、34.1%（P=0.029）。继而我们构建了Elmo1过表达和下调表达的慢病毒载体并稳定转染了肝癌细胞株MHCC97L和MHCC97H。通过qRT-PCR、Western blotting、免疫荧光等方法，我们证实Elmo1上调表达后能够增加肝癌细胞MHCC97L的迁移、侵袭、克隆形成能力，伴有E-cadherin下调和N-cadherin上调；而下调Elmo1则能够降低MHCC97H细胞的迁移、侵袭、克隆形成能力，伴有E-cadherin的上调和N-cadherin的下调。进一步我们通过裸鼠皮下肝癌模型和肝脏原位肿瘤模型证实Elmo1能够增强肝癌细胞的增殖能力，促进肿瘤细胞的转移。通过基因芯片检测，我们发现Elmo1上调后的肝癌细胞PI3K/Akt信号通过活化明显，进一步通过Western Blotting、免疫荧光灯实验证实了Elmo1可以通过该通路激活Snail转录和细胞形态改变，诱发EMT。此外，我们还通过EMT芯片发现Elmo1很可能通过作用于Sox10起到上述作用，并通过细胞实验证实了上述假设。本项目证实了Elmo1可通过调控Sox10/PI3K/Akt信号通路诱导EMT，促进肝癌侵袭和转移，为治疗肝癌提供新的作用靶点。