结肠腺癌细胞中eIF-4E调节NF-κB表达及对乙酰肝素酶转录表达和活性的影响  

Role of Eukaryotic Initiation Factor-4E (eIF-4E) in Regulation of Expression of NF-κB and Its Subsequent Influence on Transcription and Activity of Heparanase in Human Colon Adenocarcinoma Cell Line

作　　者：杨玉捷[1] 张亚历[1] 王继德[1] 赖卓胜[1] 王群英[1] 崔海宏[1] 

机构地区：[1]第一军医大学南方医院全军消化系疾病研究所,广东广州510515

出　　处：《癌症》2003年第10期1023-1029,共7页Chinese Journal of Cancer

基　　金：广东省自然科学基金(No.010643);国家骨干教师基金

摘　　要：背景与目的:近期研究显示,人类肿瘤细胞部分转录因子可能受真核细胞起始因子-4E(eukaryoticinitiationfactor4E,eIF-4E)在翻译水平上的调节,此关键转录因子表达水平的变化可从转录水平改变某些恶性相关基因产物的表达。本研究旨在探讨结肠腺癌LS-174T细胞中eIF-4E对转录因子NF-κB表达和活性的影响,并观察NF-κB活性水平对乙酰肝素酶基因转录的作用。方法:将与eIF-4EmRNA翻译起始区互补的反义寡核苷酸(antisenseoligodeoxynucleotide,ASODN)经脂质体包裹后转染人结肠癌细胞LS-174T,以阻抑eIF-4E表达。随之使用Westernblot和电泳迁移改变分析(electrophoreticmobilityshiftassays,EMSA)方法检测NF-κB蛋白表达量及活性水平。通过RT-PCR、Westernblot方法观察乙酰肝素酶转录水平和蛋白表达量的改变;乙酰肝素酶活力采用特异酶活性检测方法,以放射性标记的硫酸乙酰肝素做为底物,凝胶过滤层析分离酶降解产物来分析。结果:针对eIF-4E的20个残基的ASODN经脂质体转染LS-174T细胞后,eIF-4E基因表达在转录和翻译水平都受到显著阻滞。eIF-4E基因阻抑表达引起结肠癌LS-174T细胞NF-κB蛋白表达量及其活性的显著下降;此外,转录因子NF-κB的下调也导致乙酰肝素酶基因的转录表达下降。BACKGROUND &OBJECTIVE: Recent studies have revealed that some transcription factors may be translationally regulated by eukaryotic initiation factor 4E (eIF 4E) in human cancer. These modifications in the expression levels of the key transcription factors will alter the expression of some malignancy related gene products at the transcriptional levels. The current study was designed to investigate the effect of eIF 4E on the expression and activity of NF κB and observe how the activity level of NF κB contributes, as a secondary effect, to the transcription of heparanase in human colon adenocarcinoma LS 174T cells. METHODS: In order to repress the expression of eIF 4E, a 20 mer antisense oligodeoxynucleotide (ASODN) targeted against the translation start site of eIF 4E mRNA was transfected into human colorectal cancer cell line LS 174T via liposome reagent, followed by assessment of the activity and the protein expression of NF κB by an electrophoretic gel mobility shift assay (EMSA) and Western blot analysis respectively. The alterations of heparanase expression were examined by RT PCR and Western blot analysis, and heparanase activity in LS 174T cells was measured by specific enzymatic activity test using radiolabeled heparan sulfate as the substrate and gel filtration chromatography for the analysis of the degradation product. RESULTS: The 20 mer ASODN against eIF 4E specifically and significantly inhibited eIF 4E expression at both transcriptional and translational levels, and the repression of eIF 4E gene expression was correlated with decreased expression levels and activity of NF κB protein. Furthermore, this down regulation of the ubiquitous transcription factor NF κB led to reduced transcription of the heparanase gene, and the transfected cells also showed a considerable decrease in heparanase protein and activity. CONCLUSION: These results suggest that eIF 4E play an important role in translational regulation of NF κB expression in LS 174T cells.NF κB is an essential factor

关 键 词：结肠腺癌 EIF-4E NF-ΚB 乙酰肝素酶活性 核转录因子 

分 类 号：R735.3[医药卫生—肿瘤]