HMGB1基因沉默对阿霉素诱导K562/A02细胞凋亡增敏作用的研究

目的 探讨高迁移率族蛋白1(HMGB1)基因沉默对白血病细胞耐药逆转的作用.方法 将HMGB1基因特异性干扰RNA(HMGB1 siRNA)导入K562/A02细胞中,通过Western blot和RTPCR方法检测HMGB1基因在转染前后的表达;WST8法检测阿霉素(ADM)对转染前后K562/A02细胞的半数抑制浓度(IC50);流式细胞术检测凋亡细胞百分率;Western blot检测线粒体促凋亡蛋白Smac/DIABLO的释放;采用caspase活性定量检测试剂盒分析caspase-3的活性.结果 ①与未经处理的K562/A02细胞组相比,转染HMGB1 siRNA的K562/A02细胞组HMGB1 mRNA和蛋白水平分别下降86%和71%;②HMGB1基因沉默使K562/A02细胞对ADM的药物敏感性增强,其IC50值从转染前的(4.83±0.08)μg/ml降低到(1.33±0.10)μg/ml,并在ADM浓度为1μg/ml和5μg/ml时,细胞凋亡百分率分别增加27%、32%;③HMGB1基因沉默可促进ADM所致Smac/DIABLO从线粒体向胞浆释放,并增加caspase-3的活性.结论 HMGB1基因沉默能明显增加K562/A02细胞对ADM的敏感性,逆转K562/A02细胞对ADM耐药.

Enhancive effect of HMGB1 gene silence on adriamycin-induced apoptosis in K562/A02 drug resistance leukemia cells

Objective To investigate the effect of high mobility group box1 (HMGB1 )gene silence on adriamycin(ADM)-induced apoptosis in K562/A02 drug resistance leukemia cells.Methods K562/A02 cells were transient transfected with HMGB1-small interference RNA(siRNA)vector,and the levels of HMGB1 gene differential expression pre-and post-transfection were measured by RT-PCR and Western blotting.50% inhibition concentration(IC50)of ADM on K562/A02 was determined by WST-8 assay.Cell apoptosis was assessed by flow cytometry.The release of Smac/DIABLO from the mitochondria to the cytoplasm was assayed by Western blotting.Activity of Caspase-3 was assayed with a Caspase Colorimetric Assay Kit.Results ①The HMGB1 expression at mRNA and protein levels in HMGB1 siRNA transfected K562/A02 cells were decreased by 86%and 7 1%respectively compared with control.②Suppression of HMGB1 by siRNA in K562/A02 cells resulted in a reversal of the resistance to ADM,and decreased IC50 from(4.83±0.08)μg/ml to(1.33±0.10)μg/ml.1 μg/ml and 5 μg/ml of ADM treatment increased cell apoptotic rate by 27%and 32%respectively.③HMGB1 suppression in K562/A02 cells significantly promoted ADM-induced Smac/DIABLO release from the mitoehondria to the cytoplasm,and increased the activities of Caspase-3.Conclusion HMGB1 gene silence can enhance sensitivity of K562/A02 cells to ADM and reverse cell resistant to ADM.