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目的:通过RNAi下调K562细胞X染色体连锁的凋亡抑制蛋白(XIAP)的表达,观察XIAP下调后细胞凋亡及对化疗药物敏感性的变化情况。方法:K562细胞分为3组,实验组和阴性对照组分别用XIAP siRNA及阴性对照siR-NA转染,空白对照组不做任何转染处理。用实时荧光定量PCR和蛋白质印迹法检测转染后各组细胞XIAP mRNA及蛋白的表达,CCK-8法检测各组细胞对阿糖胞苷和依托泊苷敏感性的变化,流式细胞仪和Hochest染色法检测各组细胞加入不同化疗药物后细胞的凋亡情况。结果:转染后48h,实验组XIAP mRNA及蛋白表达量较阴性对照组分别下降约70.0%和60.0%,P<0.05;实验组细胞对依托泊苷和阿糖胞苷的敏感性分别提高2.77和2.28倍,P<0.05;转染siR-NA 48h后,实验组与阴性对照组、空白对照组间凋亡率差异无统计学意义,P>0.05;转染siRNA 6h后加入依托泊苷孵育48h,实验组凋亡率(41.3±1.6)%较阴性对照组(34.3±1.9)%和空白对照组(30.6±1.7)%明显增加;加入阿糖胞苷孵育48h,实验组凋亡率(47.4±1.1)%较阴性对照组(37.0±1.8)%和空白对照组(35.5±1.7)%明显增加,P<0.05。结论:XIAP siRNA能特异性下调K562细胞的XIAP mRNA和蛋白质水平的表达,提高K562细胞对依托泊苷和阿糖胞苷的敏感性,同时伴化疗药物诱导的细胞凋亡增强。
OBJECTIVE: To investigate the effect of down-regulation of XIAP on the expression of XAP-linked inhibitor of apoptosis (XIAP) in RNA interference (RNAi) cells. Methods: K562 cells were divided into three groups. The experimental group and the negative control group were transfected with XIAP siRNA and negative control siR-NA, respectively. The blank control group was not transfected. The expression of XIAP mRNA and protein in each group was detected by real-time fluorescence quantitative PCR and Western blotting. The sensitivity of cytarabine and etoposide to each group was detected by CCK-8 method. Flow cytometry and Hochest staining was used to detect the apoptosis of cells in each group after adding different chemotherapy drugs. Results: At 48h after transfection, the expression of XIAP mRNA and protein in the experimental group decreased by about 70.0% and 60.0%, respectively, compared with that in the negative control group (P <0.05). The sensitivity of cells in the experimental group to etoposide and cytarabine increased by 2.77 And 2.28-fold, P <0.05; 48h after transfection with siR-NA, there was no significant difference in apoptotic rate between experimental group and negative control group and blank control group (P> 0.05) (41.3 ± 1.6)% in the experimental group was significantly higher than that in the negative control group (34.3 ± 1.9)% and the blank control group (30.6 ± 1.7)% respectively; the apoptosis rate in the experimental group 47.4 ± 1.1% compared with the negative control group (37.0 ± 1.8)% and the blank control group (35.5 ± 1.7)%, P <0.05. Conclusion: XIAP siRNA can specifically down-regulate the expression of XIAP mRNA and protein in K562 cells, increase the sensitivity of K562 cells to etoposide and cytarabine, and enhance the apoptosis induced by chemotherapy drugs.