Zhao Bao-Xia, Liu Si-Qi, Dong Si-Cong, Jing Ru-Nan, Meng Xiu-Xiang
Department of Clinical Hematology, Laboratorial Medicine College of Dalian Medical University, Dalian 116044, Liaoning Province, China.
Department of Laboratorial Medicine, Municipal Hospital Affiliated to Xuzhou Medical College，Xuzhou 221005, Jiangsu Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2020 Jun;28(3):758-766. doi: 10.19746/j.cnki.issn.1009-2137.2020.03.008.
To investigate the effect of Bmi-1 gene silencing on drug resistance of leukemia cell K562/ADR and to explore its possible mechanism.
After two sequences of Bmi-1-siRNA were transfected into drug-resistant 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.
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.
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.
探讨Bmi-1基因沉默对白血病细胞K562/ADR耐药性的影响，并探讨其可能机制。
将两条序列的Bmi-1-siRNA转染至耐药K562/ADR细胞后，检测Bmi-1基因的mRNA和蛋白表达。Bmi-1基因沉默后，检测P-gp和MDR1的表达，并通过流式细胞术检测阿霉素在K562/ADR细胞中的蓄积情况，以确定Bmi-1基因沉默对K562/ADR细胞耐药性的影响。Bmi-1基因沉默后分析NF-κB的蛋白表达。然后用NF-κB抑制剂PDTC处理K562/ADR细胞后，分析P-gp的蛋白表达及其功能变化，以确定NF-κB对白血病细胞耐药性的影响。检测Bmi-1基因沉默后PTEN、AKT和p-AKT的蛋白表达，确定Bmi-1基因沉默对耐药细胞中PTEN/PI3K/AKT信号通路的影响。用PI3K/AKT通路抑制剂LY294002处理K562/ADR细胞后，分析NF-κB和P-gp的蛋白表达，以确定AKT对NF-κB和P-gp表达的调控作用。用PTEN抑制剂BPV处理Bmi-1-siRNA转染细胞后，检测AKT、p-AKT、NF-κB和P-gp的蛋白表达。上述mRNA表达通过RT-PCR检测，蛋白表达通过Western blot检测。
Bmi-1基因沉默后，K562/ADR细胞中Bmi-1基因的mRNA和蛋白表达均降低，阿霉素蓄积增加。Bmi-1-siRNA转染细胞中MDR1/P-gp的表达低于K562/ADR细胞（P＜0.05）。Bmi-1基因沉默后，NF-κB的活性降低。使用NF-κB抑制剂（PDTC）可抑制NF-κB的活性和P-gp的表达，并降低K562/ADR细胞中P-gp的功能。Bmi-1基因沉默后，PTEN的蛋白表达增加，而p-AKT的蛋白表达降低（P＜0.05）。使用PI3K/AKT抑制剂LY294002可显著抑制p-AKT、P-gp的蛋白表达和NF-κB的活性（P＜0.05）。用PTEN抑制剂BPV处理Bmi-1-siRNA转染细胞后，NF-κB的活性和P-gp的蛋白表达恢复。
Bmi-1在K562/ADR细胞多药耐药相关的多药耐药中起关键作用，其可能通过激活PTEN/AKT通路调控NF-κB介导耐药。
