Effect of RNA Interference Combined with Vincristine on U937 Cells Growth in vitro and in vivo
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摘要: 目的 从体外和体内两个方面探讨慢病毒载体介导小发卡shRNA(short harpin RNA，shRNA)靶向沉默同源盒A10(homeoboxA10，HOXA10)基因联合长春新碱（Vincristine，VCR）对U937细胞增殖和凋亡的影响。方法 将U937细胞分为干扰（KD）组、阴性对照（NC）组、空白对照（BC）组，经Western blot方法测定对HOXA10基因的沉默作用；MTT法检测各组细胞的IC50；流式细胞术（Flow cytometry,FCM）检测各组细胞的凋亡率；通过流式细胞仪分选出干扰组和阴性对照组绿色荧光蛋白（Green Fluorescent Protein，GFP）阳性的细胞，建立裸鼠AML皮下移植瘤模型（细胞活力>90%），计算干扰组的抑瘤率。结果 流式细胞仪测得慢病毒对U937细胞的感染率>90%，干扰组HOXA10的蛋白水平为较BC组下降89.78%。下调HOXA10的表达水平可降低VCR对U937细胞的半数抑制浓度（half-inhibitory concentration，IC50），提高VCR诱导的U937细胞凋亡率，与对照组相比差异具有统计学意义(P<0.05)。体内实验：干扰组肿瘤组织增长受到抑制。结论 慢病毒载体介导的RNA干扰下调HOXA10的表达水平可在体外和体内增强U937细胞对VCR的敏感度。Abstract: Objective To explore the effects of lentivirus-mediated RNA interference targeting HOXA10 gene combined with VCR on the proliferation and apoptosis of U937 cell line in vitro and in vivo. Methods U937 cells were divided into interference (lenti-shHOXA10,KD)group, negative control (lenti-NC,NC) group and blank control (BC)group.The infection efficiency of lentivirus for U937 was detected by flow cytometry,and HOXA10 gene expression of U937 cells at protein level was detected by Western blot. IC50 was determined by MTT assay.The apoptosis rates of KD,NC and BC group were detected by flow cytometry.The NC and KD group's GFP-positive cells were sorted by flow cytometry and then U937 xenografts were established by subcutaneous injection of U937 cells (viability>90%) into the flanks of nude mice.Tumor inhibition rate of KD group were calculated after VCR injection for four times. Results The ratio of GFP positive cells was up to 90%,and protein levels of HOXA10 in KD group decreased by 89.78% compared with the control group.Down-regulation of HOXA10 could reduce the IC50 and improve the VCR-induced apoptosis rate of U937 cells.Compared to NC and BC group the differences were significant (P<0.05).In vivo tumor growth was inhibited in interference group mice. Conclusion Lentivirus vector-mediated RNA interference-targeted HOXA10 gene could enhance the sensitivity of U937 cells to VCR in vitro and in vivo.
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Key words:
- HOXA10 /
- U937 /
- Nude mice /
- RNA Interference /
- Vincristine
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