目的：探讨慢病毒载体介导短发夹RNA （shRNA，siRNA前体）靶向沉默HOXA10基因对U937细胞增殖、凋亡和形态的影响。方法：设计并构建4条针对HOXA10基因的shRNA质粒表达载体，并构建HOXA10基因的过表达质粒，将4条干扰质粒分别和过表达质粒共转染293T细胞，用Western blot 检测出敲减效果最好的1条质粒并包装成慢病毒（lenti-shHOXA10）；将U937细胞分为干扰组（lenti-shHOXA10）、阴性对照组（lenti-NC）和未处理组，通过流式细胞仪测定慢病毒对U937细胞的感染效率并用real-time PCR、Western blot方法测定对HOXA10基因的沉默作用；瑞氏染色观察3组细胞形态上的变化；MTT法检测细胞增殖抑制率；流式细胞术检测3组细胞凋亡率。结果：成功构建了有效沉默HOXA10基因的慢病毒-shRNA载体。干扰组HOXA10 mRNA的沉默效率为(92.3±1.3)%，蛋白表达水平下降91.1％，干扰组细胞抑制率为（43.9±0.7）%，与阴性对照组、未处理组相比差异有统计学意义（P<0.05）；瑞氏染色显示干扰组细胞核质比减小、核分裂相少见；干扰组细胞凋亡率为（27.1±1.4）%，显著高于阴性对照组的（19.4±1.9）%和未处理组的（5.5±1.3）%（P<0.05）。结论：慢病毒载体介导的shRNA可稳定地降低HOXA10基因的表达水平，有效抑制U937细胞增殖和促进其凋亡，HOXA10基因有望成为白血病基因治疗的新靶点。
Abstract
OBJECTIVE: To investigate the effects of lentivirus-mediated RNA interference targeting HOXA10 gene on the proliferation, apoptosis and morphology of leukemic cell line U937. METHODS: Four different shRNA plasmids were designed and built to interfere with HOXA10 gene. The four interference plasmids were transfected into 293T cells with the HOXA10 over expression plasmid and then the RNAi efficiency of the four interference plasmids was determined by Western blot. The best one was chosen to transfect 293T cells with lentiviral helping plasmids to produce packaged lentivirus (lenti-shHOXA10). U937 cells were divided into interference group (lenti-shHOXA10), negative control group and untreated group. After infection with the packaged lentivirus, infection efficiency of lentivirus for U937 was detected by flow cytometry, and the expression of HOXA10 gene mRNA and protein was detected by real-time PCR and Western blot. Cell survival was determined by MTT assay. Apoptosis rate was detected by flow cytometry. RESULTS: Lentiviral-shRNA vector of HOXA10 gene was successfully constructed. Compared with the negative control and untreated groups, mRNA level of HOXA10 decreased by (92.3±1.3)%, protein levels decreased by 91.1%, and the inhibition rate of U937 cells ［(43.9±0.7)%］ increased in the interference group (P<0.05). Wright′s staining showed that the ratio of karyon to cytoplasm was reduced and mitotic phase was rare in the interference group. Apoptosis rate in the interference group ［(27.1±1.4)%］ was significantly higher than in the negative ［(19.4±1.9)%］and untreated groups ［(5.5±1.3)%］ (P<0.05). CONCLUSIONS: Lentivirus mediated RNAi can reduce the expression level of HOXA10, effectively inhibit proliferation and promote apoptosis of U937 cells. HOXA10 gene is expected to become a new target for the treatment of leukemia at gene level.
关键词
HOXA10基因 /
白血病 /
慢病毒 /
RNA干扰 /
U937细胞
Key words
HOXA10 gene /
Leukemia /
Lentivirus /
RNA Interference /
U937 cell
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