小干扰RNA下调白血病细胞X连锁凋亡抑制蛋白的表达及其对化疗药物敏感性的影响

目的 探讨小干扰RNA(siRNA)下调白血病MOLT-4、HL-60、K562细胞的白血病细胞X连锁凋亡抑制蛋白(XIAP)的表达对化疗药物敏感性的影响.方法 采用实时荧光定量PCR和Western免疫印迹检测MOLT-4、HL-60、K562细胞及健康人外周血单个核细胞(PBMC)XIAP mRNA和XIAP蛋白表达水平.用NucleofectorTM核酸转染仪对高表达XIAP的K562细胞转染XIAP siRNA(实验组),同时以转染无同源性siRNA作阴性对照组,未转染任何siRNA的K562细胞作空白对照组,并检测3组细胞XIAP mRNA和XIAP蛋白表达水平.同时将实验组、阴性及空白对照组细胞暴露于不同浓度的依托泊苷(vp-16,0.01、0.1、1、10、100mg/L)及阿糖胞苷(Ara-c,0.1、1、10、100、1000、10 000mg/L),用CCK-8法检测细胞抑制率变化.结果 与健康人PBMC比较,MOLT-4、HL-60、K562细胞XIAPmRNA及蛋白表达均增高(均P＜0.05),其中K562细胞XIAPmRNA及蛋白表达水平最高,与MOLT-4、HL-60细胞比较差异有统计学意义(均P＜0.05).K562细胞转染XIAP siRNA48 h后,与阴性对照组、空白对照组比较,实验组细胞XIAP mRNA及蛋白表达水平均明显下降(mRNA:0.37±0.10比1.41±0.13比1.00±0.12,蛋白:0.37±0.03比0.99±0.08比0.98±0.07,均P＜0.05).在给予1 mg/L vp-16、10及100 mg/L的Ara-c后,实验组细胞抑制率与阴性对照组、空白对照组比较,差异有统计学意义(均P＜0.05);在给予其他浓度vp-16和Ara-c后,3组细胞抑制率差异均无统计学意义(均P＞0.05).结论 XIAP siRNA能特异性下调K562细胞XIAPmRNA和蛋白质水平的表达,增强K562细胞对化疗药物依托泊苷、阿糖胞苷的敏感性.

Effect of small interfering RNA-mediated down-regulation expression of X-linked inhibitor of apoptosis protein in leukemic cell-lines on sensitivity to chemotherapy

Objective To investigate the effect of small interfering RNA (siRNA)-mediated downregulated expression of X-linked inhibitor of apoptosis protein (XIAP) in leukemic cell-lines MOLT-4, HL-60 and K562 on the sensitivity to chemotherapy. Methods Real-time fluorescent quantitative PCR and Western blotting were used to detect the expression of XIAP mRNA and protein in leukemic cell-lines MOLT-4, HL-60,K562 and healthy peripheral blood mononuclear cells (PBMCs). The K562 cells with high expression of XIAP were transfected with XIAP siRNA (experimental group) or non-homologous siRNA (negative control group) by using NucleofectorTM nucleic acid transfection device, and those not transfected with any siRNA were assigned to blank control group. The expression levels of XIAP mRNA and protein in three groups were then detected.Meanwhile, the K562 cells of all groups were exposed to different concentrations of etoposide (vp-16: 0.01,0.1,1, 10, 100 mg/L)and Ara-C (0.1, 1, 10, 100, 1000, 10 000 mg/L)for determination of the cell growth inhibition rate with CC K-8 assay. Results The expression levels of XIAP mRNA and protein were elevated in MOLT-4,HL-60 and K562 cell lines compared with those in healthy PBMCs (all P＜0.05). Notably, K562 had the highest expression of XIAP mRNA and protein, which was statistically different from MOLT-4 and HL-60 (all P＜0.05 ).Compared with negative and blank control groups, the experimental group had a significant decline in the expressions of XIAP mRNA and protein after transfection with XIAP siRNA for 48 hours(mRNA: 0.37±0.10 vs 1.41 ±0.13 vs 1.00±0.12, protein: 0.37±0.03 vs 0.99±0.08 vs 0.98±0.07, all P＜0.05 ). The cell growth inhibition rate in the experimental group was remarkably different from those in negative and blank control groups when treated with 1 mg/L vp-16, 10 mg/L and 100 mg/L Ara-c (all P＜0.05 ), whereas no statistical difference was found among three groups when treated with other doses of vp-16 and Ara-c (all P＞0.05 ). Conclusion XIAP siRNA may specifically down-regulate the expressions of XIAP mRNA and protein in K562 cells and thereby enhance the sensitivity of K562 cells to vp-16 and Ara-C of chemotherapy.