人工诱骗子的入核分布对核因子-κB活性的调控作用

Nuclear localization of oligonucleotides decoy effect on nuclear factor-κB activity

摘要:

探讨了由核定位信号 (NLS) 多肽介导的核因子-κB (NF-κB) 寡核苷酸诱骗子 (ODNs decoy) 进入HeLa细胞核的效率，以及对细胞核内NF-κB活性的调控作用。利用双功能交联剂 (Sulfo-SMCC) 共价交联末端氨基修饰的ODNs decoy和末端巯基修饰的NLS多肽，形成NLS多肽共价连接的ODNs decoy。依靠TransME 转染试剂的辅助转染NLS-ODNs decoy进入HeLa细胞，用荧光显微镜观察荧光标记的NLS-ODNs 在细胞内的分布。用MTT法检测HeLa细胞的活力，以凝胶迁移实验 (EMSA) 检测TNF-α诱导的HeLa细胞核抽提物中NF-κB的活性。结果表明，NLS多肽成功地连接到ODNs decoy上，NLS-ODNs可高效入核，入核率达到17.9％。转染NLS-ODNs进入HeLa细胞，对细胞活力无明显影响，而显著抑制核内NF-κB的活性。结果表明NLS多肽可提高ODNs decoy的入核效率，显著增强诱骗子对NF-κB活性的抑制效果。

Abstract:

To investigate the effect of the localization of oligonucleotides decoy (ODNs decoy) on the activation of nuclear factor-κB (NF-κB) in TNF-α induced HeLa cells. The mercapto group-modified nuclear localization signal (NLS) peptide was covalently conjugated to amino group-modified NF-κB ODNs decoy by Sulfo-SMCC cross-linker. The NLS-ODNs decoy was transfected into HeLa cells by TransME transfection reagent. The intracellular distribution of fluorescent labeled NLS-ODNs decoy was detected with a microscope. The cell viability was detected by MTT assay, and then the activity of NF-κB in cell nuclear extract was assayed by electrophoretic mobility shift assay (EMSA). The results showed that NLS peptide was successfully conjugated to ODNs decoy by Sulfo-SMCC cross-linker. The NLS-ODNs decoy effectively entered into nucleus with high rate of 17.9%. It was observed that the cell viability of HeLa cell was not significantly affected by the transfection of NLS-ODNs decoy, while NLS-ODNs decoy significantly inhibited the activation of NF-κB in TNF-α induced HeLa cells nuclear extracts. This experiment can provide a new covalent conjugation of NLS peptide to ODNs can effectively drive decoy into nucleus, and thus improve its inhibitory effects on the activation a transcription factor.