Netrin-1对肝癌细胞EMT的调控及其侵袭表型逆转的实验研究

项目名称： Netrin-1对肝癌细胞EMT的调控及其侵袭表型逆转的实验研究

项目编号： No.30873038

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 轻工业、手工业

项目作者： 田德安

作者单位： 华中科技大学

项目金额： 30万元

中文摘要： 在肿瘤的转移过程中，癌细胞主要通过上皮间质转化（EMT）获得迁移、侵袭能力，但具体的机制仍不清楚。Netrin-1是层黏连蛋白相关的分泌蛋白，参与调控神经轴突生长和肿瘤细胞的侵袭转移。本项目旨在研究netrin-1及其下游信号对肝癌细胞EMT的调节作用。建立肝癌细胞物理缺氧模型，通过细胞形态、标志蛋白及功能改变鉴定EMT细胞。检测缺氧培养肝癌细胞Netrin-1表达及其下游信号通路。 siRNA干扰Netrin-1的表达以及转染Netrin-1质粒对对肝癌细胞EMT的作用。我们发现缺氧条件下肝癌细胞形态改变，间质细胞标志蛋白表达增加，细胞骨架重排，侵袭能力增强，Netrin-1的分泌显著增加，下游信号分子激活。在缺氧条件下siRNA干扰 Netrin-1的表达后可以抑制缺氧诱导的肝癌细胞EMT。转染Netrin-1质粒后的hepG2细胞形态改变，标志蛋白和侵袭力显著增强。缺氧可以促进肝癌细胞EMT，Netrin-1及其下游信号通路在缺氧诱导EMT过程中发挥重要调节作用。进一步研究探讨抑制Netrin-1逆转EMT细胞恶性表型的可能性，为抑制肝癌早期转移提供干预靶点。

中文关键词： 肝癌；上皮间质转化；缺氧；Netrin-1；转移

英文摘要： In the process of tumor metastasis, cancer cells obtain migration and invasion ability mainly through the epithelial- mesenchymal transition (EMT), but the exact mechanism remains unclear. Netrin-1 is a laminin-related secreted protein, which regulate axonal growth and cell invasion and metastasis. The aim of this project is to study the role of netrin-1 in the regulation of EMT and downstream signaling pathways in HCC cells. We established the physical hypoxia model of hepatocellular carcinoma cells. The cell morphology, marker proteins and functional changes of HCC cells were measured to indentify EMT. The level of Netrin-1 secreted from hypoxic HCC cells was measured by ELISA. The expression of Netrin-1 was inhibited by siRNA and changes of EMT of HCC were studied. Furthermore, the downstream signling pathways were also tested.Netrin-1 expressing plasmid was transfected into HCC cells hepG2. The expression was detected by western blot. Netrin-1 induced EMT was studied. Under hypoxic conditions morphological changes and cytoskeletal rearrangements of HCC cells was found. The expression of mesenchymal marker proteins and the invasion of HCC cells were significantly increased. Netrin-1 secreted from hypoxic HCC cells was significantly increased. Downstream signaling pathways were activated. Under hypoxic conditions, inhibiting Netrin-1 by siRNA significantly inhibited hypoxia-induced EMT in HCC cells. In hepG2 cells with Netrin-1 plasmid stably transfected, cell morphology was changed, mesenchymal marker proteins and invasion were significantly enhanced. Hypoxia can promote EMT of HCC cells. Netrin-1 and its downstream signaling pathways play an important role in hypoxia-induced EMT.We studied the possibility of reversal of EMT malignant phenotype by inhibiting Netrin-1 The mechanism of early metastasis of HCC will be clarified and further study of metastasis in HCC will provides an important theoretical target.

英文关键词： Hepatocellular carcinoma;epithelial mesenchymal transition;Hypoxia;Netrin-1;Metastasis