阻滞eIF-4E对乙酰肝素酶mRNA核浆转运及其表达的影响

目的：确定结肠癌Ls-174T细胞中过量表达的真核细胞起始因子-4E(eukaryotic initiation factor-4E，eIF-4E)是否影响乙酰肝素酶(heparanase)mRNA的核浆转运，并改变乙酰肝素酶表达水平．方法：脂质体包裹与eIF-4E mRNA翻译起始点互补的asODN转染人大肠腺癌细胞Ls-174T．使用Western blot，RT-PCR方法分别检测eIF-4E被阻抑后其转录和翻译水平的改变．乙酰肝素酶mRNA在细胞内核浆分布水平采用Northern blol，检测，其蛋白表达采用Western blot检测．结果：asODN转染显抑制elF-4E基因和蛋白表达．伴随eIF-4E被阻抑，Northern blot结果显示更多乙酰肝素酶mRNA滞留在细胞核，且其蛋白表达量也下降．结论：阻抑eIF-4E可影响LS-174T细胞乙酰肝素酶mRNA核浆转运，并降低乙酰肝素酶蛋白表达．此反义策略可应用于实验性和临床抗人类大肠癌转移的基因治疗．

Effect of eIF-4E on nucleocytoplasmic transport and protein expression of heparanase mRNA

AIM: To determine whether the over expressed eukaryotic initiation factor 4E(eIF 4E) is involved in the nucleocytoplasmic transport of heparanase mRNA, and expression of heparanase in human colon adenocarcinoma cell line LS 174T. METHODS: A 20 mer antisense s oligodeoxynucleotide (asODN) targeted against the translation start site of eIF 4E mRNA were introduced into LS 174T cells by means of liposome mediated DNA transfection. eIF 4E protein and mRNA levels were determined by Western blot analysis and RT PCR respectively. Northern blotting was employed to assess the nucleocytoplasmic distribution of heparanase mRNA and the alterations of heparanase expression were confirmed by Western blot analysis. RESULTS : The 20 mer asODN against eIF 4E specifically and significantly inhibited eIF 4E protein expression. Following eIF 4E inhibition, a larger proportion of heparanase mRNA was confined to the nucleus, and, simultaneously, heparanase protein expression was signigicantly decreased. CONCLUSION : The inhibition of eIF 4E affects nucleocytoplasmic transport of heparanase mRNA in LS 174T cells and results in an apparent reduction in heparanase protein expression. This targeting strategy in antisense chemistry may potentially find practical applications in experimental or clinical anti metastatic gene therapy for human colorectal carcinoma.