EFEMP1促进宫颈癌微血管生成的分子机制研究

. Our previous study has proved that EFEMP1 promotes the angiogenesis of cervical carcinoma through VEGF up-regulation. It is demonstrated that EFEMP1 binds to and activates EGFR. We also found the up-regulation of p-44/42 MAPK, VEGF and Jagged1 in the cervical cancer cells treated with EFEMP1. Jagged1 can activate the Notch signal transduction of endothelial cells to promote the tube formation of endothelial cells.These data reveals that EFEMP1 may be a key angiogenic regulator modulating simultaneously the angiogenesis of cervical carcinoma cells and endothelial cells by binding to EGFR to activate the MAPK signal transduction which up-regulates the target gene expression of VEGF and Jagged1 of cervical cancer. In this study, the methods of co-immunoprecipitation for demonstrating the interaction of EFEMP1 with EGFR, western blotting for detecting the expression level of phosphorylated MAPK,VEGF and Jagged1, and three-dimensional cell culture for assessment of endothelium tube formation are used to investigate the molecular mechanism by which EFEMP1 promotes the angiogenesis of cervical carcinoma , with the combined application of the methods of gene transfection, RNAi, cervical cancer nude mice model and assessment of microvessel denstiy of tumor tissues.

我们前期研究证实EFEMP1促进宫颈癌血管增生且其机制可能与上调VEGF水平有关。目前有研究表明，EFEMP1能结合并活化EGFR，而我们预实验结果显示EFEMP1作用下宫颈癌细胞p-44/42MAPK、VEGF和Jagged1表达升高。Jagged1能激活内皮Notch信号促进血管生成，故推测EFEMP1可能通过EGFR激活下游MAPK进而上调靶基因VEGF和Jagged1表达，从而同时调节肿瘤细胞血管诱生和内皮血管形成能力。本研究通过免疫共沉淀证实宫颈癌细胞中EFEMP1与EGFR的结合，western blotting 检测磷酸化MAPK、VEGF和Jagged1的表达，构建三维细胞培养体系观察内皮细胞管腔形成能力，并结合基因转染、RNAi、裸鼠肿瘤模型和肿瘤组织微血管密度检测等方法研究EFEMP1通过EGFR-MAPK-VEGF/Jagged1信号途径促进宫颈癌血管生成的分子机制。

前期研究已证实EFEMP1能促进宫颈癌微血管生成，本课题在此基础上进一步研究EFEMP1促进宫颈癌微血管生成的分子机制。实验结果显示，EFEMP1能与EGFR相互作用，且作用后宫颈癌细胞中p-EGFR（P< 0.001, P=0.004）和p-MAPK(P<0.001; P=0.001) 都分别显著高于未处理组和EGFR抑制剂与EEMP1联合处理组。EFEMP1处理组癌细胞上清液作用下的内皮细胞的活力（P=0.001；P=0.004）、迁移能力（P=0.002；P=0.008）和管腔形成能力（P=.001；P=0.009）都分别显著高于未处理组和联合处理组上清液作用下的内皮细胞。处理组宫颈癌细胞的p-MAPK（P=0.028；P=0.042），VEGF （P=0.013；P=0.008）和Jagged1(P=0.026; P=0.024)的表达水平都分别显著高于未处理组和MAPK抑制剂与EFEMP1联合处理组。此两方面实验结果说明，EFEMP1活化EGFR-MAPK信号，上调VEGF和Jagged1的表达而促进宫颈癌细胞血管诱生能力。LV-JAG1-RNAi成功地转染宫颈癌细胞，再用EFEMP1处理，取其上清液作用于内皮细胞，并与EFEMP1作用下正常宫颈癌细胞上清液处理的内皮细胞和该上清液与Notch抑制剂联合作用下的内皮细胞作比较，发现宫颈癌细胞Jagged1RNAi (P=0.006) 和内皮Notch信号抑制(P=0.008) 都能显著减弱内皮管腔形成能力,而直接抑制宫颈癌细胞Notch信号对内皮的管腔形成无明显影响。这说明Jagged1促进内皮管腔形成是是通过活化内皮Notch信号而不是癌细胞本身的Notch 信号。组织学水平实验显示，在人宫颈癌组织中，EFEMP1，p-MAPK和Jagged1的表达三者呈两两正相关，且p-MAPK和Jagged1与宫颈癌微血管密度正相关。动物实验结果显示，Jagged1干扰组肿瘤的平均体积（P=0.001）、重量（P=0.002）和瘤体微血管密度（P=0.014）都显著低于对照组。本课题证实，EFEMP1与EGFR相互作用激活MAPK-VEGF/Jagged1信号通路促进宫颈癌微血管生成，为以EFEMP1为靶点的抗肿瘤血管生成研究提供可靠的理论基础和实验依据。