结肠腺癌细胞株eIF-4E对NF-κB表达的影响及诱导凋亡机理的探讨

Eukaryotic initiation factor-4E(eIF-4E) regulates expression and activity of NF-κB and its correlation with apoptosis in human colon adenocarcinoma cell line:LS-174T

目的 研究真核细胞起始因子 (EukaryoticInitiationFactor 4E ,eIF 4E)对NF κB表达及活性水平的影响 ,并对其阻滞后诱导肿瘤细胞凋亡的途径进行探讨。方法 应用脂质体包裹与eIF 4EmRNA翻译起始点互补的反义寡核苷酸 (asODN) ,处理人大肠腺癌细胞LS 174T。使用Westernblot和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。EMSA用来比较eIF 4E阻滞前后NF κB活性的改变。采用流式细胞仪检测LS 174T细胞凋亡。结果 eIF 4E被阻抑表达后 ,NF κB蛋白表达和活性水平也有显著下降。伴随eIF 4E表达和NF κB活性水平下降 ,LS 174T细胞的凋亡率显著升高。结论 本试验证明NF κB受eIF 4E的翻译调控 ,eIF 4E的抗凋亡作用部分可能是通过调节NF κB活性来实现的。此结论对于结肠癌试验性和肿瘤临床的基因治疗具有一定意义。

Objective To explore the effects of eIF-4E on the expression and activity of NF-κB and its correlation with the apoptosis in human colon adenocarcinoma cell line LS-174T. Methods A 20-mer asODN targeted against the translation start site of eIF-4E mRNA were introduced into LS-174T cells by lipid-mediated DNA-transfection. eIF-4E protein and mRNA levels were detected by Western blot analysis and RT-PCR respectively. Nuclear factor kappa B (NF-κB) DNA-binding activity was evaluated by electrophoretic gel mobility shift assay (EMSA). For the LS-174T cells treated with ODN,the development of apoptosis were examined mainly by flow cytometer. Results Western blot analysis revealed a dramatic decrease of eIF-4E expression and this was correlated with decreased NF-κB protein levels and its constitutive activity. In the meantime,the apoptosis-positive rate of LS-174T cells treated with asODN was significantly increased in compare with control cultures. Conclusion The study demonstrates that eIF-4E may be involved in translational regulation of NF-κB in colon adenocarcinoma cell line,and anti-apoptotic effect of eIF-4E may be paralleled with the level of active NF-κB. Partial loss-of NF-κB function may induce apoptosis in LS-174T cells. This targeting strategy in antisense chemistry may have practical application in experimental or clinical anti-cancer gene therapy for human colorectal carcinoma.