RYBP基因沉默对HL-60细胞对化疗药物敏感性的影响

Effect of RYBP Gene Silencing on Sensitivity of HL-60 Cells to Chemotherapeutic Drugs

目的:用RNA干扰技术探讨白血病HL-60细胞株RYBP基因沉默后对化疗药物敏感性的影响。方法:构建针对RYBP基因的shRNA干扰质粒并建立稳定沉默RYBP基因的白血病HL-60细胞株,通过实时荧光定量PCR和Western blot检测确认RYBP基因在mRNA和蛋白水平的沉默效果。然后用DNA ladder及Annexin V标记的流式细胞术检测各组HL-60细胞凋亡的情况,用CCK-8法检测各组HL-60细胞对化疗药物阿糖胞苷和柔红霉素的敏感性。结果:成功构建的RYBP shRNA慢病毒载体能够有效沉默HL-60中RYBP基因的表达。在没有加入化疗药物时RYBP shRNA组凋亡率低于空载体对照组(NC组)(P<0.05);用阿糖胞苷处理时RYBP shRNA组凋亡率和抑制率均低于NC组(P<0.05);用柔红霉素处理时RYBP shRNA组凋亡率虽然低于NC组,但是抑制率差异不明显。结论:HL-60细胞RYBP基因沉默能抑制细胞的凋亡,明显降低对阿糖胞苷的敏感性,但是对柔红霉素敏感性的改变不明显。

Objective:To investigate the effect of RYBP gene on sensitivity of HL-60 cells to chemotherapy drugs by using RNA interference.Methods:Plasmid expressing RYBP specific shRNA was constructed and then was used to establish the RYBP knockdown stable HL-60 cell line.Q-PCR and Western blot were used to confirm the efficacy of RYBP gene silencing at mRNA and protein level respectively;then the DNA ladder and Annexin V labeled flow cytometry were used to detect cell apoptosis;CCK-8 was used detect the sensitivity of HL-60 cells to the chemotherapeutic drug cytarabine or daunorubicin.Results:The lentiviral-RYBP-shRNA vector was succesfully and effectively inhibit the expression of RYBP at mRNA and protein in HL-60 cells.It was found that without chemotherapy drug treatment the apoptosis rate of RYBP shRNA group was lower than that of the empty vector control group(NC group).When treated with cytarabine,the apoptosis rate and inhibitive rate of RYBP shRNA group were lower than those of NC group.Besides,when treated with daunorubicin,the apoptosis rate of RYBP shRNA group was lower than that of NC group,while the inhibitive rate had no significant difference.Conclusions:RYBP gene silencing can inhibitive the apoptosis of HL-60 cells and significantly reduce the sensitivity to cytarabine,but this gene silencing can't affect the sensitivity to daunorubicin.