PEDF调节鼻咽癌细胞EMT抑制鼻咽癌转移的作用及机制

We have reported for the first time that pigment epithelium-derived factor(PEDF) inhibts angiogenesis and tumor growth of nasopharyngeal carcinoma (NPC). Recently, we have found that PEDF expression is low in NPC tissues. Furthermore, PEDF expression is lower in nasopharyngeal carcinoma cell line S18 with meseschymal phenotype and high metastasis, contrarily its expression is higher in S26 cell line with epithelial phenotype and low metastasis. Moreover, PEDF can up-regulate the expression of E-cadherin and down-regulate that of vimentin and suppress migration and lymph node metastasis of S18 cells, which strongly suggesting that PEDF may inhibit NPC metastasis via regulating epithelial-mesenchymal transition(EMT). The design of this project is as follows: (1)To define whether PEDF participates in the EMT formation of nasopharyngeal carcinoma cells by observing the changes of cell phenotype、EMT molecular markers and cell behaviors in S26 cells with knockdown of PEDF and S18 cells with overexpression of PEDF. (2) Both of PI3K/Akt/GSK3β and Wnt/β-catenin pathway can down-regulate E-cadherin by stabilizing Snail and then induce EMT in nasopharyngeal carcinoma cells; NFκB could reduce E-cadherin expression through activating Twist. This project is to define the selective regulation of PEDF on these signaling pathways and to clarify the molecular mechanism by which PEDF inhibits EMT and NPC metastasis, thus providing a new drug candidate for the treatment of NPC metastasis.

我们首次报道PEDF抑制鼻咽癌血管新生和肿瘤生长；最近又发现人鼻咽癌组织低表达PEDF，鼻咽癌高转移细胞株S18呈间皮表型并低表达PEDF、低转移株S26呈上皮表型且高表达PEDF，PEDF上调E-Cadherin、下调Vimentin同时抑制S18细胞迁移和淋巴结转移，提示PEDF可能通过调节EMT抑制鼻咽癌转移。本项目设计内容如下：（1）观察S26细胞敲低PEDF表达和S18细胞过表达PEDF细胞表型、EMT分子标记和细胞行为的变化，明确PEDF是否参与鼻咽癌细胞EMT的形成。（2）PI3K/Akt/GSK3β和Wnt/β-Catenin通路均可通过稳定Snail下调E-Cadherin诱发鼻咽癌细胞EMT；NFκB通过激活Twist下调E-Cadherin；本项目拟明确PEDF对上述通路的选择性调控。旨在阐明PEDF抑制EMT和鼻咽癌转移的分子机制，为治疗鼻咽癌转移提供新的候选药物。

远处转移是鼻咽癌治疗失败的主要原因， 而上皮-间充质转化是鼻咽癌侵袭和转移的重要过程。参与鼻咽癌上皮-间充质转化过程的癌基因已经有一些研究，但是其相关的抑癌基因仍未见报道。色素上皮衍生因子(PEDF)具有强大的抗肿瘤作用，我们的研究表明相比于正常鼻咽上皮组织100%(12/12)的PEDF阳性表达率，鼻咽癌组织只有7.3%(16/218)，而且PEDF表达呈阴性的鼻咽癌病人临床分期更高。进一步研究显示，在临床样本中，PEDF的表达水平与E-cadherin表达正相关（r=0.427， p<0.05），和Vimentin的表达负相关（r=-0.414， p<0.05）。为了验证PEDF与鼻咽癌EMT之间的关系，我们设计相关实验内容并取得以下重要成果：(1) 在mRNA和蛋白质水平上验证了，鼻咽癌高转移株（S18和5-8F）和低转移株（CNE-2和SUNE-1）中PEDF的表达与细胞EMT 标志物存在密切关联。（2）运用慢病毒感染技术建立敲低PEDF的稳定细胞株，能够诱导低转移细胞株（CNE-2和SUNE-1）出现纺锤体样形态改变，伴随迁移、侵袭能力的增强，细胞极性丧失，克隆形成能力的增加及EMT 标记物的变化，以及体内脾种植瘤发生肝转移能力的增加。（3）慢病毒构建稳定过表达PEDF后能够恢复高转移细胞株（S18和5-8F）的上皮特性，抑制其迁移、侵袭能力和克隆形成能力，以及体内发生转移的能力。（4）机制方面，LRP6/GSK3β/β-catenin信号通路而不是AKT/GSK3β信号通路参与PEDF对EMT的调节。（5）鼻咽癌组织中PEDF表达的下调可能与miR-320c高表达有密切关系。总结以上结果，我们得出以下结论：首次发现PEDF在调节鼻咽癌EMT和转移中发挥抑癌基因的作用；被miR-320c直接调控的PEDF通过Wnt/β-catenin信号通路调节鼻咽癌的EMT和体内转移。因此，PEDF不仅可以成为一个辅助鼻咽癌诊断的分子标记物，而且有希望作为鼻咽癌预后和治疗的靶标。