CAJ | 학술논문

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Western blotting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1α(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-BP1 (P＜0.05)的磷酸化,而且抑制肿瘤组织中HIF-1α (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.
목적 탐토ARHI단백대간세포암혈관생성적영향급기궤제.방법 이인간조직총RNA위모판통과RT-PCR획취전장ARHIcDNA.장해서렬극륭병구건pcDNA3.1-ARHI표체질립.장pcDNA3.1-ARHI표체질립전염인간세포암세포계Hep3B세포,통과G418사선은정표체ARHI적세포.응용MTT법검측과표체ARHI대세포증식활성적영향.분별용은전공재체화ARHI적Hep3B세포접충건립라서인간세포암이식류모형.매주측량류체적.5주후마취처사라서.면역조화법검측증식세포핵표지물Ki 67화혈관내피세포표지물CD31적표체.응용Western blotting법검측혈관생성상관적mTOR통로중mTOR하유파단백S6K1화4E-BP1적린산화정황,병검측종류조직혈관생성상관인자결양유도인자1α(HIF-1α)화혈관내피생장인자(VEGF)적표체.결과 ARHI과표체현저강저Hep3B세포적증식능력(P＜0.01).병차,해기인대라서인간암이식류유명현적생장억제작용,ARHI조류체현저감소,억류솔체76.4％ (P＜0.01).면역조화검측현시,ARHI현저억제세포증식핵항원단백Ki-67 (P＜0.01)화혈관내피세포표기물CD31(P＜0.05)적표체.Western blot법검측현시,ARHI현저억제S6K1 (P＜0.01)화4E-BP1 (P＜0.05)적린산화,이차억제종류조직중HIF-1α (P＜0.05)화VEGF단백(P＜0.01)적표체.결론 과표체ARHI억제간세포암생장화혈관생성,저가능여기억제mTOR/VEGF통로유관,제시과표체ARHI가능성위간세포암치료적신방법.
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 ARHI overexpression on tumor growth and angiogenesis were assessed.Because of the key role of mammalian target of rapamycin (mTOR)signaling in HCC progression,we also tested whether ARHI overexpression affected the mTOR pathway.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 microvessel count.Western blot analysis of the xenografts showed that ARHI overexpression substantially reduced the phosphorylation 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-inducible factor 1 alpha and vascular endothelial growth factor,were significantly downregulated.Conclusion These data highlighted an important role for ARHI in controlling HCC growth and angiogenesis,therefore offering a possible therapeutic strategy against this malignancy.