EGFL7调控EGFR介导的Akt/MAPK通路促进垂体腺瘤侵袭及机制研究

Invasive pituitary adenomas are account for about a third of all primary adenomas. The clinical cure rate is very low and the recurrence rate was more than 30%. It is exactly the difficult and key issues in clinical treatment of pituitary adenomas and the underlying mechanism remains unclear. Our previous study have found that Epidermal growth factor-like domain-containing protein 7 (EGFL7) identified secreted protein, also known as vascular endothelial statin, up-regulated in invasive pituitary adenomas tissues detected by mRNA and protein microarray. Further studies showed that overexpression of EGFL7 could up-regulate Erk1/2 and Akt signal pathway and enhance the invasion of GH3 cells in vitro. The EGFL7 protein sequence consists of two centrally located EGF-like domains, and EGFR inhibitor blocked the invasion-inducing effect of EGFL7 in GH3 cells detected by Transwell assay. The aim of this study is to clarify the exact role and mechanisms of EGFL7 and Akt/MAPK signal pathway in the invasion of pituitary adenomas and to find invasive molecular markers, fortunately, which could provide guidance for clinical decision and open up a new direction for gene therapy. The effects of EGFL7 on cell growth, migration and invasive behavior were observed among GH3 cells, primary cultured pituitary adenoma cells and nude mice transplantation tumor model. To determine whether EGFL7 may have functions in the invasion of pituitary adenomas through Akt/MAPK pathway, we would analyze that the relation between difference expression of EGFR to reveal the regulation mechanisms of EGFL7 in invasive pituitary adenomas. Our study will testify the regulatory mechanism of EGFL7 on Akt/MAPK pathway through EGFR and provide new potential specific biomarkers of molecular diagnosis and molecular therapy of invasive pituitary adenomas.

侵袭性垂体瘤治愈率低，复发率高达30%，发生发展机制仍不明确。基因芯片筛查发现侵袭性垂体瘤中，与细胞运动迁移能力密切相关的胚胎血管发育调控关键基因EGFL7表达显著升高。EGFL7含EGF样结构域，可调控垂体瘤GH3细胞Akt和Erk1/2磷酸化，并增加细胞体外侵袭能力，EGFR抑制剂可阻断其促侵袭作用。本研究将基于临床信息分析EGFL7和Akt/MAPK通路在垂体瘤侵袭发生发展中的作用。利用GH3细胞系和原代细胞及裸鼠移植瘤模型，观察过表达外源性和沉默内源性EGFL7对垂体瘤上皮间叶转化和侵袭行为的影响；采用抑制剂实验探讨EGFL7是否利用EGFR传导信号实现对Akt/MAPK磷酸化的调控；通过信号通路靶点干预明确Akt/MAPK通路对垂体瘤侵袭的作用及EGFL7的调控机制。从组织、细胞及动物模型多层面系统研究EGFL7介导垂体瘤侵袭的分子机制，为其分子诊断和分子治疗提供潜在的作用靶点。

侵袭性垂体瘤治愈率低，复发率高达30%，发生发展机制仍不明确。侵袭性是影响手术效果的关键因素，并与患者预后高度相关。患者年龄、肿瘤体积和病理分类与垂体腺瘤侵袭性密切相关。在本研究中我们首次发现与细胞运动迁移能力密切相关的胚胎血管发育调控关键基因EGFL7在侵袭性垂体腺瘤中表达显著高于非侵袭性腺瘤，肿瘤体积、病理分类、侵袭性和肿瘤复发与EGFL7表达量密切相关。使用Kaplan-Meier法分析患者无复发进展期，long-rank test提示高表达EGFL7相对于低表达EGFL7的垂体腺瘤具有更差的无复发进展期，多因素Cox回归分析提示EGFL7为无复发进展期的独立预后预测因子。此外体外实验证明下调EGFL7表达抑制GH3细胞增殖能力和侵袭性，体内实验证明抑制EGFL7表达可以抑制GHPA生长。通过shRNA干扰和抑制剂实验验证了EGFL7参与调控垂体腺瘤增殖和侵袭性生物学行为是通过介导Notch信号通路的Notch2/DLL3和EGFR信号通路的EGFR/Akt/MAPK实现的。该成果表明EGFL7在侵袭性垂体腺瘤中特异性高表达，可以作为分子诊断标志物。EGFL7表达量与垂体腺瘤的侵袭性和复发密切相关，可以作为垂体腺瘤的侵袭和预后因子。该成果对于临床垂体腺瘤的分子诊断提供了潜在靶点，为垂体腺瘤的治疗提供了可能的新思路和新方法。