通路抑制剂对Tax诱导的Bcl-3的影响及其在NF-κB激活中作用的研究

Study on the effect of pathway inhibit on Tax-induced Bcl-3 and its role on NF-κB activation

目的:通过研究不同通路的抑制剂在TaxP细胞(稳定表达Tax的Jurkat亚细胞系)中对Bcl-3(Human B-cellleukemia protein 3)表达的影响和shRNA Bcl-3对NF-κB转录激活作用的影响,探讨Tax阳性细胞中调控Bcl-3的通路及Bcl-3在NF-κB通路中的作用。方法:将TaxP细胞分别用NF-κB(Nuclear factor-κB)抑制剂、GSK(Glycogen synthase kinase)抑制剂和蛋白酶抑制剂处理后,Western blot检测Bcl-3蛋白的表达情况;将Bcl-3的shRNA质粒转入TaxP细胞中,RT-PCR检测Bcl-3mRNA的表达抑制情况;共转染Bcl-3的shRNA和pNF-κB-luc质粒至Jurkat和TaxP细胞中,检测抑制Bcl-3表达后对NF-κB转录活性的影响。结果:Bcl-3的表达不受GSK抑制剂的影响,但在蛋白酶抑制后有明显的升高;RT-PCR的结果表明,在转染Bcl-3 shRNA质粒后,Bcl-3的mRNA表达有明显的下降,荧光素酶活性结果显示Bcl-3在Jurkat和TaxP细胞中被抑制后NF-κB的转录活性明显下降(P<0.05)。结论:Tax诱导的Bcl-3表达不依赖于GSK通路,而NF-κB通路参与了Bcl-3的调控,说明Bcl-3在NF-κB的激活中发挥着促进作用。

Objective：To investigate the pathways involved in the regulation of Bcl-3 and the role of Bcl-3 in NF-κB pathway by means of the effects of inhibitors of different pathways on the Bcl-3 expression in TaxP cells,a subline of Jurkat that stably expresses Tax protein,and the effect of shRNA Bcl-3 on the NF-κB activation.Methods：TaxP cells were treated with NF-κB（nuclear factor-κB）inhibitor and GSK（glycogen synthase kinase）inhibitor and protease inhibitor,and the expression of Bcl-3 was detected by Western blot;Bcl-3 shRNA plasmids were transfected into Jurkat cells by using transfection agent Tfx-50,the mRNA expression of Bcl-3 was detected by RT-PCR;Bcl-3 shRNA plasmid and pNF-κB-luc were co-transfected into Jurkat and TaxP cells,the luciferase activities were tested.Results：The Bcl-3 expression was not effected by GSK inhibitor but was increased by protease inhibitor.The mRNA expression of Bcl-3 was significantly down regulated after transfection of Bcl-3 shRNA in TaxP.NF-κB activation was significantly decreased（P0.05）after transfection of Bcl-3 shRNA in Jurkat and TaxP cells.Conclusion：Expression of Bcl-3 by Tax-induced is not depending on GSK pathway,however,NF-κB pathway involves in the regulation of Bcl-3.Our result show that Bcl-3 plays a positive role in NF-κB activation.