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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 lentiviral vector-mediated short hairpin RNA (shRNA) silencing homeobox A9 (HOXA9) gene on the drug sensitivity of human acute monocytic leukemia U937 cells. METHODS: Four knockdown RNAi (HOXA9 / GV118RNAi-LV # 2) plasmids were screened from four RNA interference (RNAi) lentiviral vectors targeting to HOXA9 by Western blotting. The experiment was divided into control (CON), negative control (NC) and knock-down (KD). After U937 cells were infected with HOXA9 / GV118RNAi-LV # 2, the silencing efficiency of HOXA9 mRNA was detected by real-time fluorescence quantitative PCR. The expression of HOXA9 protein was detected by Western blotting. The expression of HOXA9 mRNA was detected by MTT and FCM before and after vincristine , VCR) and daunorubicin (DNR). The expression of MDR-1 mRNA in each group was detected by RT-PCR. Results: The silencing efficiency of HOXA9 mRNA in KD cells was over 55%, and the expression of HOXA9 protein was significantly decreased. VCR and DNR increased the IC50 value of KD group (P <0.01), and increased the sensitivity of KD group. The apoptosis rate of KD group increased significantly after VCR and DNR treatment (P <0.01). After DNR treatment, the expression of MDR-1 mRNA in U937 cells of KD group was significantly down-regulated (P <0.01). CONCLUSION: RNAi lentivirus targeting HOXA9 gene can stably silence the expression of HOXA9 gene and to a certain extent increase the sensitivity of U937 cells to chemotherapeutic drugs.