转录抑制剂DRB和α-Amanitin对A549细胞中Nrf2定位影响的研究

Effect of DRB/α-Amanitin on localization of Nrf2 in A549 cells

目的:利用细胞核亚细胞器结构的一些特异性蛋白质SC35、核孔复合体(nuclear porecomplex,NPC)、RNA聚合酶Ⅱ(PolⅡ)及Y12等标记亚细胞器,探讨转录抑制剂5,6-dichloro-1-β-D-ribofuranosylbenzimidazole(DRB)与α-Amanitin对人非小细胞肺癌A549细胞中Nrf2的表达与定位的影响。方法:①50 mg/L DRB和2.5 mg/Lα-Amanitin分别作用于A549细胞1 h和6 h后,Western blotting检测Nrf2及其相关蛋白的表达变化。②50 mg/L DRB和2.5 mg/Lα-Amanitin分别处理A549细胞1 h,利用免疫荧光细胞技术检测Nrf2、SC35、NPC、RNAPolⅡ、Y12形态结构的变化,并研究Nrf2定位的变化。结果:①与对照组相比,DRB和α-Amanitin作用1 h后,Nrf2及其调控的下游基因HO-1、AKR1C和NQO1的蛋白质表达变化不明显,但6 h作用后,这些蛋白质的表达明显降低。②激光共聚焦显微镜观察显示,细胞经DRB和α-Amanitin处理1 h后,SC35的荧光强度明显增强,PolⅡ的荧光强度减弱,而Nrf2的定位无明显改变;NPC、Y12的表达无明显变化,且与Nrf2无共定位现象。结论:DRB和α-Amanitin能抑制Nrf2及其HO-1、AKR1C和NQO1的表达,但是对Nrf2的定位没有显著影响。

Objective： To investigate the effects of transcriptional inhibitors 5,6-dichloro-1-b-D- ribofuranosylbenzimidazole （DRB） and α-Amanitin on the localization of Nrf2 in the nucleus. Methods： A549 cells were treated with DRB （50 mg/L） or α-Amanitin （2.5 mg/L） for 1 h and 6 h in serum-free medium,respectively. The expressions of Nrf2, HO-1, NQO1 and AKR1C were detected by Western blotting analysis. The localization of Nrf2 was determined by laser scanning confocal microscopy after cells were treated with either DRB or α-Amanitin for 1 h. Results： The expressions of Nrf2 and Nrf2-ARE gene batteries HO-1, AKR1C and NQO1 were decreased after 6 h treated with either DRB or α-Amanitin. The expression of SC35 was up-regulated but RNA Pol 11 was down-regulated; Y12 and NPC did not significantly change. The localization of Nrf2 in the cell nucleus did not change significantly. Conclusion： DRB and α-Amanitin can down-regulate the expression of Nrf2 and its targeting proteins HO-1 ,AKR1C and NQO1, but may have no effect on the localization of Nrf2.