靶向HOXA9基因RNAi慢病毒载体增加人急性单核细胞白血病U937细胞的药物敏感性

Lentivirus-mediated shRNA targeting homeobox A9 gene enhances drug sensitivity of human acute monocytic leukemia U937 cells

目的:探讨慢病毒载体介导的短发夹RNA(short hairpin RNA,shRNA)沉默同源盒A9(homeobox A9,HOXA9)基因对人急性单核细胞白血病U937细胞药物敏感性的影响。方法:应用蛋白质印迹法从4条针对HOXA9基因的RNA干扰(RNA interference,RNAi)慢病毒载体中筛选出1条敲减效率最高的包装成慢病毒HOXA9/GV118RNAi-LV#2。实验分成未感染对照组(control,CON)、阴性对照组(negative control,NC)及敲减组(knock down,KD)。HOXA9/GV118RNAi-LV#2感染U937细胞后,应用实时荧光定量PCR法检测HOXA9 mRNA的沉默效率,蛋白质印迹法检测HOXA9蛋白的表达,MTT法和FCM检测病毒感染前后U937细胞对长春新碱(vincristine,VCR)和柔红霉素(daunorubicin,DNR)的敏感性及细胞凋亡率变化,RT-PCR法检测DNR作用后各组细胞中MDR-1mRNA的表达。结果:KD组细胞中HOXA9mRNA的沉默效率在55%以上,HOXA9蛋白的表达量明显减少。VCR及DNR作用KD组细胞的半数抑制浓度(half inhibitory concentration,IC50)值明显降低(P<0.01),药物敏感性明显增强;VCR和DNR作用后,KD组细胞的凋亡率明显增加(P<0.01);DNR作用后,KD组U937细胞中MDR-1mRNA的表达水平明显下调(P<0.01)。结论:靶向HOXA9基因的RNAi慢病毒可稳定沉默HOXA9基因的表达,在一定程度上提高U937细胞对化疗药物的敏感性。

Objective： To investigate the effect of lentivirus-mediated shRNA （short hairpin RNA） silencing HOXA9 （homeobox A9） gene on drug sensitivity of human acute monocytic leukemia U937 cells. Methods： RNAi （RNA interference） using a plasmid construct expressing shRNA targeting HOXA9 was detected by Western blotting. The plasmid expressing shRNA with highest level of knockdown was determined from four different plasmids and it was used to construct lentiviral vector HOXA9/ GV118RNAi-LV#2. In this experiment, three groups were designed： control （without lentivirus infection）, negative control （infected with negative lentivirus） and knockdown （infected with lentivirus containing shRNA targeting HOXA9） groups. After U937 cells were infected with HOXA9/GV118RNAi-LV#2, the silence efficiency was examined by real-time fluorogenic quantitative PCR, and the expression of HOXA9 protein was detected by Western blotting. The sensitivity of U937 cells to VCR （vincristine）/DNR （daunorubicin） after lentivirus infection was detected by MTT assay, and the apoptosis rate of U937 cells was detected by flow cytometry. The expression of MDR 1 （multidrug resistance protein 1） mRNA after DNR treatment in each group was determined by RT-PCR. Results： The silence efficiency of lentivirus- mediated shRNA targeting HOXA9 in U937 cells was more than or equal to 55% in knockdown group, and the expression level of HOXA9 protein was decreased obviously. The ICs0 （half-maximal inhibitoryconcentration） values of VCR and DNR for U937 cells infected with HOXA9/GV118RNAi-LV#2 were significantly reduced （P 〈 0.01）, and it revealed that the sensitivity to VCR/DNR was remarkably enhanced. The apoptosis rate of U937 cells in knockdown group was increased after VCR/DNR intervention （P 〈 0.01）. The expression level of MDR 1 mRNA in U937 cells in knockdown group was significantly reduced after DNR intervention （P 〈 0.01）. Conclusion： Lentivirus-mediated shRNA targeting HOXA9 can steadily silence the expression ofHOXA9 gene and enhance the drug sensitivity of U937 cells.