Bmi-1介导的K562/ADR细胞多药耐药机制

Effect of Bmi-1 on Multidrug Resistance in K562/ADR Cells and Its Mechanisms

目的:观察Bmi-1基因沉默对白血病耐药细胞株K562/ADR耐药性的影响并初步探讨其机制。方法:将2种序列的Bmi-1小干扰RNA SiRNA转染到耐药细胞K562/ADR,检测Bmi-1基因m RNA和蛋白的表达以确定转染效果。检测Bmi-1基因沉默后耐药蛋白P-gp及其编码基因MDR1的表达情况,并采用流式细胞术检测细胞内阿霉素蓄积情况。检测Bmi-1基因沉默后NF-κB蛋白的表达变化;应用NF-κB抑制剂PDTC处理K562/ADR细胞抑制NF-κB活性后,检测P-gp蛋白的表达及其功能的变化。检测Bmi-1基因沉默后PTEN、AKT和p-AKT蛋白表达的变化。应用PI3K/AKT通路抑制剂LY294002处理K562/ADR细胞抑制p-AKT表达后,检测NF-κB和P-gp蛋白的表达。应用PTEN抑制剂BPV(phen)处理Bmi-1基因沉默后的细胞,检测AKT、p-AKT、NF-κB和P-gp蛋白的表达。以上mRNA表达用RT-PCR法检测,蛋白表达用Western blot法检测。结果:在mRNA水平和蛋白水平2种序列的小干扰RNA均使K562/ADR细胞Bmi-1基因表达降低。MDR1/P-gp在Bmi-1 siRNA干扰细胞中的表达明显低于在K562/ADR细胞的表达(P<0.05);干扰后细胞内阿霉素蓄积增多。Bmi-1基因沉默后,细胞NF-κB活性降低;NF-κB抑制剂抑制NF-κB活性后,K562/ADR细胞P-gp蛋白表达及药物泵出功能被抑制。Bmi-1基因沉默后PTEN蛋白表达增高,而p-AKT蛋白表达明显降低(P<0.05)。PI3K/AKT通路抑制剂LY294002抑制p-AKT表达后,NF-κB活性和P-gp蛋白表达明显下降(P<0.05)。PTEN抑制剂BPV(phen)处理Bmi-1基因沉默细胞后,NF-κB活性和P-gp蛋白表达得到重塑。结论:Bmi-1在MDR1/P-gp介导的K562/ADR细胞多药耐药中起关键作用,这种作用可能是通过激活PTEN/AKT途径调控NF-κB完成。

Objective:To investigate the effect of Bmi-1 gene silencing on drug resistance of leukemia cell K562/ADR and to explore its possible mechanism.Methods:After two sequences of Bmi-1-siRNA were transfected into drugresistant K562/ADR cells,the mRNA and protein expressions of Bmi-1 gene were detected.After Bmi-1 gene silencing the expression of P-gp and MDR1 were detected and the accumulation of doxorubicin in K562/ADR cells were detected by flow cytometry to determine the effect of Bmi-1 gene silencing on drug resistance of K562/ADR cells.The protein expression of NF-κB was analyzed after Bmi-1 gene silencing.Then after K562/ADR cells were treated with NF-κB inhibitor PDTC,the protein expression of P-gp and its functional changes were analyzed to determine the effect of NF-κB on drug resistance of leukemia cells.The protein expressions of PTEN,AKT and p-AKT after Bmi-1 gene silencing were detected and the effect of Bmi-1 gene silencing on PTEN/PI3K/AKT signaling pathway in drug-resistant cells was determined.After K562/ADR cells were treated with PI3K/AKT pathway inhibitor LY294002,the protein expressions of NF-κB and P-gp were analyzed to determine the regulation of AKT on the expression of NF-κB and P-gp.The protein expressions of AKT,p-AKT,NF-κB and P-gp were detected after the Bmi-1-siRNA transfected cells were treated by PTEN inhibitor BPV.Above-mentioned expression of mRNA was detected by RT-PCR,and the protein expression was detected by Western blot.Results:The expression of Bmi-1 gene in K562/ADR cells decreased at both mRNA and protein levels and the doxorubicin accumulation increased after Bmi-1 gene silencing.The expression of MDR1/P-gp in Bmi-1-siRNA transfected cells was lower than that in K562/ADR cells(P<0.05).After Bmi-1 gene silencing,the activity of NF-κB decreased.The activity of NF-κB and P-gp expression was inhibited and the function of P-gp in K562/ADR cells was reduced by using NF-κB inhibitor(PDTC).The protein expression of PTEN increased while the protein expression of p-AKT decreased after Bmi-1 gene silencing(P<0.05).The protein expressions of p-AKT,P-gp and the activity of NF-κB were inhibited significantly by using PI3K/AKT inhibitor LY294002(P<0.05).After the Bmi-1-siRNA transfected cells were treated by PTEN inhibitor BPV,the activity of NF-κB and the protein expressions of P-gp were restored.Conclusion:Bmi-1 plays a key role in MDR-mediated multidrug resistance in K562/ADR cells,which may be mediated by activating PTEN/AKT pathway to regulate NF-κB.