核因子κB降低小鼠胚胎成纤维细胞中Lmp2基因转录

NF-κB decreases the level of Lmp2 genes in murine embryo fibroblasts

目的研究在小鼠胚胎成纤维细胞(MEF)中,核因子κB(NF-κB)对Lmp2基因转录的影响。方法利用NF-κB荧光素酶报告基因检测MEF和c-abl/arg缺失株(DKO)中内源性NF-κB活性;构建包含Lmp2启动子序列和NF-κB结合序列突变体的荧光素酶报告基因,通过双荧光素酶报告系统,研究NF-κB对Lmp2启动子转录活性的调控,并应用定量PCR、Western印迹比较Lmp2基因在MEF和DKO细胞中表达水平。结果 DKO细胞中内源性NF-κB活性上调;DKO细胞中Lmp2基因启动子的转录活性发生下调,并且NF-κB结合序列突变后,其转录活性升高;定量PCR和Western印迹结果表明,DKO细胞中Lmp2基因mRNA和蛋白表达水平比较MEF细胞均发生下调。结论 MEF中内源性NF-κB可负调控Lmp2基因转录,并降低其蛋白表达水平。

Objective To investigate the possibility of nuclear factor-κB（NF-κB） involved in the regulation of Lmp2 in murine embryo fibroblasts（MEF）.Methods The endogenous NF-κB activity is detected using a NF-κB luciferase reporter（pNFκB-Luc） in the MEFs and c-abl-/-/arg-/-MEFs（c-abl-/-/arg-/——double knockout,DKO）.The dual-luciferase reporter assay system was used to evaluate the effect of NF-κB on the transcription of Lmp2,and the expression level of Lmp2 was detected by quantitative PCR and Western blotting.Results The NF-κB activity was upregulated in c-abl/arg deficient fibroblasts.The promoter activity of Lmp2 was decreased in DKO cells,while increased when NF-κB box was mutated.Then,the mRNA and protein level of Lmp2 were downregulated in DKO cells.Conclusion NF-κB is a negative regulator of the Lmp2 genes in MEF.