摘要
目的:观察慢病毒介导的短发夹RNA(short hairpin RNA,shRNA)沉默同源盒A9(homeobox A9,HOXA9)基因对U937细胞增殖和凋亡的影响。方法:前期从4条针对HOXA9基因的RNA干扰(RNA interference,RNAi)慢病毒载体中筛选出了1条敲减效率最高的包装成慢病毒HOXA9/GV118RNAi-LV#2。实验分为对照组(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5d后,FCM检测U937细胞感染效率,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法和绘制细胞生长曲线检测细胞增殖抑制情况;FCM检测细胞凋亡及细胞周期;RT-PCR法检测细胞c-myc mRNA及p27mRNA表达情况。结果:慢病毒感染效率>90%,KD组细胞中HOXA9mRNA的沉默效率>55%,HOXA9蛋白的表达量明显减少,其中KD组相对表达水平达(0.223±0.024),显著低于NC组(0.884±0.009)及CON组(0.891±0.013),差异有统计学意义,F=1 566.202,P<0.001。MTT结果显示,KD组细胞的IR值为(40.469±1.756)%,明显高于NC组及CON组(F=1 311.928,P<0.001),细胞生长曲线表明该组细胞生长速度减慢;FCM结果显示,KD组、NC组及CON组凋亡率分别为(26.762±2.245)%、(6.819±0.547)%和(6.113±0.349)%,KD组与NC组(q=25.501,P<0.001)及CON组(q=26.418,P<0.001)比较差异均有统计学意义,提示该载体显著增加细胞凋亡率;其细胞周期G0/G1期细胞比例较CON组及NC组明显增加,提示该载体使细胞周期阻滞于G0/G1期,F=27.769,P=0.001;RT-PCR结果表明,U937细胞KD组较CON组及NC组c-myc mRNA表达水平下降,p27mRNA表达水平上升。结论:靶向HOXA9基因shRNA慢病毒载体能稳定沉默HOXA9表达,可显著抑制U937细胞增殖并促进其凋亡,同时可下调c-myc表达,上调p27表达,使细胞周期阻滞于G0/G1期。
OBJECTIVE：To investigate the effects on the proliferation and apoptosis of U937 cells by lentivirus-medi- ated shRNA targeting homeobox A9 gene. METHODS： The plasmid expressing shRNA targeting HOXA9 with the high- est level of knockdown was determined from four different plasmids and it was used to construct lentiviral vector named HOXA9/GVllSRNAi-LV# 2 in earlier stage. 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. Five days after U937 cells were infected with HOXA9/GVllSRNAi-LV ~ 2, the infection rate was measured by flow cytometry, the silence efficiency was examined by real-time fluorogenic quantitative PCR, and the expression of HOXA9 protein was detected by Western blotting. The effect of cell proliferation inhibition was assessed by MTT and cell growth curve. The apoptosis rate and cell cycle were measured by flow cytometry, c-myc mRNA and p27 mRNA expression were determined by RT-PCR. RESULTS： Lentivirus infection efficiency was above 90 %. The silence efficiency of lentivirus-mediated shRNA targeting HOXA9 in U937 cells was more than or equal to 55% in KD group,and the expression level of HOXA9 protein was decreased obviously, in which the relative expression level of KD group （0. 223±0. 024） was significantly lower than that of NC group（0. 884±0. 009） and CON group（0. 891± 0. 013）（F=1 566. 202,P〈0. 001）. IR value（40. 469± 1. 756）% of KD group was obviously higher than that of the NC group and CON group（F=l 311. 928,P%0. 001） by MTT in which group the cell growth was slowed by cell growth curve. The apoptosis rate of the KD group,NC group and CON group were （26. 762±2. 245）%, （6. 819±0. 547）% and （6. 113± 0. 349）~ respectively and compared to NC group and CON group, KD group was increased significantly which suggest that lentiviral-shRNA vector increased the rate of apoptosis. The cell cycle Go/G1 phase of KD group was increased signif- icantly compared to CON group（q=26. 418,P〈0. 001） and NC group（q=25. 501,P〈0. 001） which suggests that lenti- viral-shRNA vector make cell cycle arrested in Go/Gl phase（F= 27. 769, P= 0. 001 ）. Lentiviral shRNA vector could re- duce e-myc expression, but raise p27 mRNA expression compared to CON group and NC group of U937 cells by RT-PCR. CONCLUSIONS： Lentiviral-shRNA vector targeting HOXA9 gene can steadily silence HOXA9 gene expression,and can effectively inhibit the proliferation and induce apoptosis of U937 cells. At the same time it can reduce c-myc mRNA ex- pression, but raise p27 mRNA expression, which make cell cycle arrested in G0/G1 phase.
出处
《中华肿瘤防治杂志》
CAS
北大核心
2014年第3期187-192,共6页
Chinese Journal of Cancer Prevention and Treatment
基金
山东省科学技术发展计划(2010GSF10264)
山东省自然科学基金(Y2007C018)
