ARHI基因过表达对肝细胞癌血管生成的影响  

Overexpression of ARHI suppresses tumor angiogenesis in hepatocellular carcinoma

作　　者：赵晓海 蔡柳新[2] 卓建新 李谨峰 王成志 孔建兵 

机构地区：[1]浙江省乐清市第二人民医院普外科,325608 [2]浙江省台州医院肝胆外科

出　　处：《中华肝胆外科杂志》2012年第9期709-713,共5页Chinese Journal of Hepatobiliary Surgery

基　　金：浙江省温州市2011年第二期科技计划项目（Y20110106）资助

摘　　要：目的探讨ARHI蛋白对肝细胞癌血管生成的影响及其机制。方法以人肝组织总RNA为模板通过RT—PCR获取全长ARHIcDNA。将该序列克隆并构建pcDNA3．1-ARHI表达质粒。将pcDNA3．1-ARHI表达质粒转染人肝细胞癌细胞系Hep3B细胞，通过G418筛选稳定表达ARHI的细胞。应用MTT法检测过表达ARHI对细胞增殖活性的影响。分别用稳转空载体和ARHI的Hep3B细胞接种建立裸鼠人肝细胞癌移植瘤模型。每周测量瘤体积。5周后麻醉处死裸鼠。免疫组化法检测增殖细胞核标志物Ki-67和血管内皮细胞标志物CD31的表达。应用Westernblotting法检测血管生成相关的mTOR通路中mTOR下游靶蛋白S6K1和4E—BP1的磷酸化情况，并检测肿瘤组织血管生成相关因子缺氧诱导因子1n（HIF-1α）和血管内皮生长因子（VEGF）的表达。结果ARHI过表达显著降低Hep3B细胞的增殖能力（P〈0．01）。并且，该基因对裸鼠人肝癌移植瘤有明显的生长抑制作用，ARHI组瘤体显著减小，抑瘤率达76．4％（P〈0．01）。免疫组化检测显示，ARHI显著抑制细胞增殖核抗原蛋白Ki-67（P〈0．01）和血管内皮细胞标记物CD31（P〈0．05）的表达。Western blot法检测显示，ARHI显著抑制s6K1（P〈0．01）和4E-BPI（P〈0．05）的磷酸化，而且抑制肿瘤组织中HIF-1a（P〈0．05）和VEGF蛋白（P〈0．01）的表达。结论过表达ARHI抑制肝细胞癌生长和血管生成，这可能与其抑制mTOR／VEGF通路有关，提示过表达ARHI可能成为肝细胞癌治疗的新方法。Objective To investigate the effect of the Ras-related tumor suppressor gene aplasia Ras homolog member I （ARHI） on angiogenesis in hepatocellular carcinoma （HCC）. Methods We generated stable cell lines overexpressing ARHI in Hep3B cells, which lack endogenous ARHI. Cell proliferation was assessed by the MTT assay. The effects of ARH1 overexpression on tumor growth and angiogenesis were assessed. Because of the key role of mammalian target of rapamyein （roTOR） signaling in HCC progression, we also tested whether ARHI overexpression affected the roTOR path- way. Results Ectopic expression of ARHI significantly diminished cell proliferation in Hep3B cells （P〈0.01）. ARHI overexpression significantly retarded Hep3B xenograft growth by 76.4% in vivo, and caused a marked reduction in tumor angiogenesis assessed by CD31-stained rnierovessel count. Western blot analysis of the xenografts showed that ARHI overexpression substantially reduced the phospborylation of two mTOR substrates, S6K1 and 4E-BP1, indicative of an inactivation of the mTOR pathway. Accompanying with the mTOR inactivation, the angiogenic factors, hypoxia-induc- ible factor 1 alpha and vascular endothelial growth factor, were significantly downregulated. Conclu- sion These data highlighted an important role for ARHI in controlling HCC growth and angiogene- sis, therefore offering a possible therapeutic strategy against this malignancy.

关 键 词：ARHI基因 肝细胞癌 血管生成 

分 类 号：R735.7[医药卫生—肿瘤]