Amotl2促进肝细胞肝癌血管生成拟态及EMT形成

Amotl2 promotes vasculogenic mimicry and epithelial-mesenchymal transition in hepatocellular carcinoma

目的:研究Amotl2对肝癌细胞迁移侵袭能力的影响及其在诱导肝癌上皮间充质转化(EMT)及血管生成中的作用。方法:将Amotl2过表达质粒和干扰质粒分别转染至肝癌细胞系HepG2和Bel7402中,Western blot检测转染前、后HepG2和Bel7402中Amotl2、EMT相关蛋白(E-cadherin、Vimentin)表达变化情况;划痕、侵袭实验检测Amotl2对肝癌细胞迁移和侵袭能力的影响;三维培养检测Amotl2对HCC细胞形成血管样结构的影响。结果:促进Amotl2表达后HepG2表现出EMT样改变,E-cadherin表达下降、Vimentin表达上升、细胞迁移侵袭和三维成管的能力增强。抑制Amotl2表达后Bel7402由间质样表型转变为上皮样表型,E-cadherin表达上升、Vimentin表达下降、细胞迁移侵袭和三维成管的能力减弱。结论:Amotl2可能通过诱导EMT促进原发性肝癌的迁移侵袭能力和血管生成拟态的形成。

Objective： To examine the expression of Amotl2 in hepatocellular carcinoma（HCC） cells and its effect on the migration and invasion. To evaluate the functions of Amotl2in inducing epithelial-mesenchymal transition（EMT）. Methods： A cDNA sequence containing Amotl2 over-expresslon plasmid was inserted into HepG2 cells to induce exogenous expression of Amotl2 protein while the Amotl2 shRNA sequence plasmid was inserted into Be17402 to interfere the quantity of Amotl2 protein. The expression of Amotl2, EMT- related protein （E-cadherin, Vimentin） in HepG2 cells and Be17402 cells were analyzed by Western blot before and after transfection; In wound healing assays, cell motility was assessed by measuring the movement of cells into a scarped and the invasion assay was used to determine the function of invasive potential;Matrigel 3D culture was utilized as a well-established in vitro model for investigating vasculogenic mimicry（~M） formation. Results： After transfection, the HepG2 cells showed significant changes from epithelial phenotype to interstitial phenotype. Accordingly, up-regulation group presented an E-cadherin expression down-regulated and Vimentin expression up-regulated in HepG2-Amotl2 cells and its motility and invasiveness were enhanced. The transfected Be17402 cells were converted from interstitial phenotype to epithelial phenotype, which increased E-cadherin expression and decreased Vimentin expression and its motility and invasiveness were inhibited. Conclusion： Amotl2 may affect hepatocellular carcinoma migration, invasion and VM formation by regulating the epithelial-mesenchymal transition process.