Netrin-1通过增加隧道纳米管形成促进EMT肝癌细胞侵袭转移

Intercellular communication is critical for tumor metastasis，tunneling nanotubes（TNTs）are recently recognized as cellular conduits for long distance intercellular communication，possibly involved in cancer cell metastasis. However, the role and mechanism of TNTs in cancer pathobiology remain unclear. Hepatocellular carcinoma cells underwent epithelial mesenchymal transformation（EMT）express lost of polarity，disrupted cell-cell junction and decreased intercellular communication over short distances. We speculate that hepatocellular carcinoma cells underwent EMT might acquire enhanced ability of metastasis by inducing TNTs formation. Our previous researches demonstrated that TNTs existed both in HCC tissues in vivo，and cultured hepatocellular carcinoma cells in vitro. MHCC-97H cells with mesenchymal feature had significant more TNTs than epithelial Huh7 cells. Netrin-1 protein clearly promoted EMT of Huh7 cells，and induced TNTs formation. Base on the present theories and our previous results, we plan to investigate the role of TNTs in Netrin-1 promoted metastasis of hepatocellular carcinoma cells underwent EMT by multiple methods and models，including immunofluorescence, electronic speculum, scratch test, 3D cell culture model, and so on. We intend to verify the effect of Netrin-1 on the regulation of hepatocellular carcinoma metastasis by in vivo imaging technology in zebrafish xenograft model and indirect orthotopic liver transplantation tumor model in mice，and explore the role of TNTs in this process. Gene-chip and protein-chip assays are carried to clarify the potential regulatory mechanism of Netrin-1 on TNTs formation. In conclusion, this project which aims at clarifying the role and molecular mechanism of Netrin-1 induced TNTs formation in hepatocellular carcinoma cells underwent EMT will provide new clues and ideas to inhibit HCC metastasis.

细胞通讯是肿瘤转移的重要基础，隧道纳米管（TNTs）是新发现的长距离细胞通讯方式，研究提示其可能参与肿瘤转移，但具体作用和机制不清。肝癌细胞EMT后极性消失、细胞间黏附减少或缺失，细胞短距离通讯减少。因此，我们推测EMT肝癌细胞可通过TNTs增强转移能力。本项目前期研究发现，肝癌组织及体外培养细胞中均存在TNTs结构，间质表型的MHCC-97H细胞较上皮表型Huh7细胞的TNTs明显增多，Netrin-1蛋白诱导Huh7细胞EMT，促进TNTs形成。本项目在前期研究Netrin-1调控肝癌细胞EMT基础上，拟借助划痕实验和3D细胞模型，运用免疫荧光、电镜等方法证实Netrin-1通过增加TNTs形成促进EMT肝癌细胞侵袭转移，斑马鱼和裸鼠模型观察Netrin-1对肝癌细胞转移的影响，探索TNTs在其中的作用，基因和蛋白芯片阐明Netrin-1调控TNTs的机制，为抑制肝癌转移提供新的思路。

细胞通讯是肿瘤转移的重要基础，隧道纳米管（TNTs）是新发现的长距离细胞通讯方式，研究提示其可能参与肿瘤转移，但具体作用和机制不清。肝癌细胞EMT后极性消失、细胞间黏附减少或缺失，细胞短距离通讯减少。因此，我们推测EMT肝癌细胞可通过TNTs增强转移能力。显微镜下观察人肝癌手术标本组织中存在较多典型的TNTs结构，而癌旁组织中表达较少或无。TNTs 培养基培养肝癌细胞株，显微镜下观察到所有肝癌细胞株中均存在TNTs结构。TNTs培养基培养的肝癌细胞缺氧培养后， E-cadherin减少， Vimentin增加，TNTs指数增加。过表达ZEB1后，肝癌细胞E-cadherin减少， Vimentin增加，TNTs指数增加；干扰ZEB1表达后，肝癌细胞E-cadherin增加， Vimentin减少，TNTs指数降低。肝癌细胞中过表达Netrin-1抑制E-cadherin表达，促进Vimentin表达，TNTs指数增加；干扰Netrin-1后，肝癌细胞E-cadherin增加， Vimentin减少，TNTs指数降低，而加入rhNetrin-1蛋白处理后，EMT表型恢复，TNTs指数增高。过表达Netrin-1后，Huh7细胞侵袭能力增强；加入Actin解聚剂抑制TNTs后，细胞侵袭能力减弱。基因芯片检测分析发现Netrin1调控多种信号通路的关键分子，其中Wnt信号通路可能是其主要调控靶点。过表达Netrin-1后，WNT相关蛋白表达增加，干扰Netrin-1表达后，WNT相关蛋白表达降低。过表达Netrin-1后， TNTs指数增加，加入WNT通路抑制剂后TNTs指数下降。激光共聚焦观察脂筏在常氧状态下定位于细胞膜上，呈粗大的颗粒状；而缺氧状态下，细胞形态发生变化，发生间质样改变，同时，细胞膜上脂筏定位明显减少。MCD处理Huh7细胞后，细胞膜上脂筏明显减少，而TNTs指数明显增加。裸鼠实验进一步发现，过表达Netrin-1的肝癌细胞瘤块中TNTs指数明显多于对照组瘤块。裸鼠皮下成瘤，6 周后观察肝脏后观察过表达Netrin1组裸鼠肝内转移灶数目明显多于对照组裸鼠。提示Netrin1促进肝癌细胞转移，部分可能是通过调控TNTs表达。该项目研究为抑制肝癌转移提供新的思路。