阻抑eIF-4E对大肠腺癌细胞乙酰肝素酶mRNA水平及其表达的影响

Effect of eIF-4E inhibition on heparanase mRNA and its expression in colon adenocarcinoma cell

目的确定阻抑大肠腺癌LS-174T细胞中过量表达的真核细胞起始因子-4E(eIF-4E)是否促进乙酰肝素酶mRNA的降解,并改变其翻译表达水平。方法应用脂质体包裹与eIF-4E mRNA翻译起始点互补的反义寡聚核苷酸,转染处理人大肠腺癌细胞LS-174T。Western blotting和RT-PCR方法分别检测eIF-4E被阻抑后转录和翻译水平的改变。采用Northern blotting定量检测乙酰肝素酶mRNA的细胞内水平,Western blotting检测其蛋白表达水平的改变。结果反义寡聚核苷酸经脂质体转染LS-174T细胞后,eIF-4E基因表达明显受到抑制,其蛋白表达产物也显著下降。伴随eIF-4E被阻抑表达,Northern blotting结果显示乙酰肝素酶mRNA水平下降,且其蛋白翻译表达量也降低。结论阻抑eIF-4E影响LS-174T细胞乙酰肝素酶mRNA的稳定表达,促使其降解,并降低乙酰肝素酶表达。

Objective To verify whether inhibition of the overexpressed eukaryotic initiation factor-4E (eIF-4E) in human colon adenocarcinoma cell line LS-174T may facilitate the degradation of heparanase mRNA and alter the translation and expression levels of heparanase protein. Methods A 20-mer antisense s-oligodeoxynucleotide (asODN) targeted against the translation start site of eIF-4E mRNA was introduced into LS-174T cells by means of lipid-mediated DNA-transfection, followed by Western blotting analysis and reverse transcription-PCR to determine eIF-4E protein and mRNA levels, respectively. Northern methods was applied to determine heparanase mRNA expression level, with the alterations of heparanase expression assessed by Western blotting analysis. Results The 20-mer asODN against eIF-4E specifically and significantly inhibited eIF-4E protein expression, and as a result, a significant reduction in heparanase mRNA level was observed by Northern blotting in conjunction with significantly decreased heparanase protein expression. Conclusion The inhibition of eIF-4E strongly reduces the stability of heparanase mRNA in colon adenocarcinoma cell line LS-174T and results in an apparent reduction in the expression of heparanase protein.