靶向沉默HOXA9基因对U937细胞体内外增殖的影响

Effects of RNA interference targeting HOXA9 on the proliferation of U937 cells in vitro and in vivo

背景与目的:多数急性髓系白血病(acute myelocytic leukemia,AML)患者高表达同源盒A9(homeoboxA9,HOXA9),而HOXA9高表达与患者预后差有关。本研究探讨慢病毒载体介导shRNA(short harpinRNA,siRNA前体)靶向沉默HOXA9基因对人急性单核白血病细胞株U937细胞在体内外增殖的影响。方法:将U937细胞分为干扰组、阴性对照组、空白对照组,转染效率>90%时,经蛋白质印迹法(Western blot)测定对HOXA9基因的沉默作用;FCM检测各组细胞周期的变化;绘制各组细胞生长曲线。将32只裸鼠腹腔接种U937细胞,随机分为4组,即阴性对照组、干扰组、阴性对照联合VCR组、干扰联合VCR组,每组8只,每只裸鼠腹腔注射环磷酰胺100 mg/kg,连续4 d。1周后向阴性对照联合VCR组和干扰联合VCR组裸鼠的腹腔注射VCR0.5 mg/kg,每周1次,连续4周。观察各组裸鼠的生存期。结果:流式细胞仪测得慢病毒对U937细胞的转染效率>90%,干扰组HOXA9的蛋白表达水平为46.56%±3.54%,较空白对照组和阴性对照组均下降(P<0.05)。干扰组细胞被阻滞于G0/G1期,其增殖明显受到抑制。慢病毒干扰HOXA9基因的表达联合VCR可显著延长裸鼠的生存期。结论:以慢病毒载体介导shRNA靶向沉默HOXA9基因表达,可明显抑制U937细胞在裸鼠体内外的增殖。

Background and purpose： In most acute myeloid leukemia（AML） patients, HOXA9 was highly expressed, the high expression of HOXA9 is related to the poor prognosis of patients. This study aimed to investigate the effects of lentivirus-mediated short hairpin RNA targeting HOXA9 gene on the proliferation of U937 cells in vitro and in vivo. Methods： U937 cells were divided into interference group, negative control group and blank control group. The infection efficiency of lentivirus for U937 was detected by flow cytometry, the expression of HOXA10 at protein level was detected by Western blot. Cell cycle was assayed by FCM. Cell growth curves were obtained by calculating the cell number. In vitro experiments were performed using nude mice injected intraperitoneally with the interference group U937 cell（or negative group U937 cell） and subsequently treated with VCR or PBS intraperitoneally. Results： The ratio of GFP positive cells was up to 90%; the levels of HOXA9 protein in interference group were decreased by 54.44% compared with the blank control group（P〈0.05）. Interference group cells were blocked in G0/G1 and their proliferation was significantly inhibited. In vivo： down-regulated expression of the HOXA9 gene combined with VCR could significantly prolong the survival time of nude mice. Conclusion： Lentiviral vector mediated shRNA can steadily reduce the expression level ofHOXA9 gene, and then inhibits the proliferation ofU937 cells in nude mice.