靶向沉默同源盒A10基因对白血病NB4细胞的影响

目的 探讨慢病毒载体介导的短发夹RNA(shRNA)靶向沉默同源盒A10(HOXA10)基因对白血病NB4细胞增殖、凋亡和耐药的影响.方法 将NB4细胞分为干扰(lenti-shHOXA10)组、阴性对照组和未处理组,用慢病毒感染NB4细胞5d后进行相关实验.运用流式细胞仪测定慢病毒对NB4细胞的感染效率.实时荧光聚合酶链反应(RT-PCR)、蛋白质印迹法(Western blot)测定慢病毒干扰载体对HOXA 10基因的沉默作用;MTF法检测细胞的增殖抑制情况.流式细胞术检测3组细胞凋亡率的变化,Western blot方法测定干扰HOXA10基因对多药耐药1(MDR-1)蛋白的影响.结果 慢病毒对NB4细胞的感染率达90％左右.干扰组HOXA10 mRNA和蛋白的表达水平均较对照组下降,干扰组细胞抑制率和凋亡率分别为(52.12±4.02)％、(30.0±2.7)％,与阴性对照组和未处理组相比差异均有统计学意义(P＜0.05).Western blot结果显示干扰HOXA 10基因的表达能降低MDR-1基因的表达水平,逆转白血病细胞耐药.结论 慢病毒载体靶向干扰HOXA10基因的表达,能有效抑制NB4细胞增殖和促进其凋亡,逆转白血病细胞耐药.HOXA10基因有望成为逆转白血病耐药的新靶点.

Influence of targeted silence HOXA10 gene on leukemia NB4 cells

Objective To explore the effects of lentivirus-mediated RNA interference targeting HOXA10 gene on the proliferation, apoptosis and drug resistance of leukemic cell line NB4.Methods NB4 cells were divided into three groups: interference group, negative control group and untreated group.The infection efficiency of lentivirus for NB4 cells was detected by flow cytometry, and the expression of HOXA10 gene of NB4 cells at mRNA and protein level was detected by real-time PCR and Western blot.Cell survival was determined by MTF assay, and apoptosis and necrosis rates were detected by flow cytometry.Western blot was used to detect the influence of down-regulation HOXA10 gene on the multi-drug resistance-1 (MDR-1) protein.Results The ratio of GFP positive cells was up to 90 ％.HOXA10 gene mRNA and protein levels were decreased in interference group compared with control group.The inhibition rate of interference group was (52.12±4.02) ％, the apoptosis rate of interference group was (30.0±2.7) ％, and their differences in the interference group and in control groups (negative control group and untreated group) were significant (P ＜ 0.05).Western blot results showed that interfering HOXA10 gene significantly reduced the resistance gene MDR-1 expression level and reverse the drug-resistant of leukemia cells.Conclusions Lentivirns-shHOXA10 can steadily reduce the expression level of HOXA10, inhibit the leukemic cells proliferation, promote apoptosis and reverse drug-resistant.HOXA10 gene is expected to become a new target for reversing leukemia drug resistance.