NF-κB_3结合位点在人TNF-α基因转录调节作用中的研究

Study on the effcts of hTNF-α gene transcriptional regulation by NF-κB_3 binding sites

目的 :探讨TNF α启动子中 3个κB位点在调节基因转录中的作用及其与核蛋白作用的亲和力关系。方法 :用含不同调节区域的重组报告基因进行HL 6 0细胞的基因转染 ,检测LPS刺激前后及κB3 反义寡核苷酸封闭后的报告基因表达水平 ;提取LPS刺激 6h的HL 6 0的细胞核蛋白 ,用凝胶迁移率改变实验及竞争结合实验比较NF κB与TNF α的 3个NF κB位点的亲和力。结果 :尽管TNF α基因中 3个κB位点都参与该基因的诱导和非诱导性转录调节 ,但κB3 位点的作用更为重要 ;3个κB位点能同LPS刺激和非刺激的HL 6 0细胞核蛋白发生特异性结合 ,但LPS刺激的蛋白 DNA复合物电泳条带明显增粗 ,尤其κB3 位点出现 2条明显的特异性条带 ;3个κB位点与LPS诱导后HL 6 0核蛋白的亲和力强弱顺序依次为 :κB2 >κB1 >κB3。结论 :TNF α基因的 3个κB位点都能与NF κB结合 ,从而参与调节HL 6 0细胞TNF α组成性表达和LPS诱导性表达。尽管κB3 位点在对LPS刺激的反应性中发挥的作用最大 ,但这种效应与其同核蛋白亲和力的大小无关。

Objective:To explore the effect of 3 κB binding sites in TNF-α promoter region on transcriptional regulation of its gene,and compare the affinity of these sites with nuclear protein.Methods:Following the report gene containing different regulation region of TNF-α gene transfected into HL-60 cell,its expression level stimulated with/without LPS or blocked with κB 3 antisense was detected respectively;the nucleus extracts from LPS stimulated HL-60 cells were prepared.With the help of electrophoresis mobility shift assay(EMSA) and competitive binding assay,the affinity of three κB binding sites of TNF-α with NF-κB was compared.Results:It was shown that all the three κB binding sites in the TNF-α gene promoter region were involved in the induced and non-induced gene transcriptional regulation,but the effect of κB 3 site may be the most important;It was also shown that all the three κB kinding sites were capable of binding specificity to the nuclear protein of HL-60 cells stimulated or non-stimulated by LPS,however,the LPS stimulated protein-DNA complex band was apparently increased in width and in particular,two specific bands were clearly seen at the κB 3 binding site.The affinity between the LPS induced HL-60 nuclear protein and the three different κB sites were markedly different from one another,and they were in the order of κB 2>κB 1>κB 3.Conclusion:All the three κB binding sites in TNF-α gene were able to be bound by NF-κB and thus involved in the regulation of TNF-α constitutive and inductive expression by HL-60 cells.Although the κB 3 binding site plays the most important role in the three binding sites in response to LPS stimulation,the effects seem to be unrelated to the affinity between the binding site and the nuclear protein.