RNA干扰对急性髓系白血病AML1-ETO融合基因表达的抑制作用

目的 应用RNA干扰技术抑制Kasumi-1细胞AML1-ETO融合基因的表达,研究随后出现的细胞增殖和细胞周期变化.方法 体外化学合成针对AML1-ETO融合基因的小干扰RNA(siRNA),并用电穿孔方法将AML1-ETO siRNA转染Kasumi-1细胞,以非特异性的siRNA转染细胞作阴性对照;电转带有增强型绿色荧光蛋白(EGFP)的载体,流式细胞术检测其绿色荧光以确定电转效率;荧光染料实时定量PCR及Western blot检测AML1-ETO siRNA的抑制效应;并应用CCK-8实验法检测细胞增殖率;采用碘化丙锭(PI)法测定细胞周期DNA含量.结果 电转增强EGFP的转染效率可达44.5%;电转AML1-ETO siRNA可以有效抑制AML1-ETO融合基因在mRNA和蛋白水平的表达;电转AML1-ETO siRNA 72 h后细胞增殖率(47.90±0.02)%]低于对照组(66.90±0.08)%](P<0.05);PI染色显示AML1-ETO siRNA转染细胞72 h后,G1期细胞比例为38.3%,对照组为31.6%,而处于G2/M期细胞分别为1.8%和2.4%.结论 化学合成的特异性siRNA能抑制AML1-ETO融合基因的表达,siRNA介导的AML1-ETo融合蛋白表达减少阻滞Kasnmi-1细胞在G1期,进而抑制细胞增殖.

Inhibitory effect of RNAi on AML1-ETO fusion gene expression in leukemia cells

Objective By inhibiting AML1-ETO fusion gene expression in Kasumi-I cells with RNAi, to investigate the changes in cell proliferation and cell cycle. Methods The small interference RNAs (siRNAs) specifically targeting the AML1-ETO fusion gene were synthesized in vitro and transfected into Ka-sumi-1 cells by electroporation, the non-specific siRNAs transfected cells were taken as control. EGFP plas-mid was transfected into Kasumi-1 cell and the transfection efficiency was detected by FCM. Inhibitory effect of siRNAs were detected by real-time RT-PCR and Western blots. Cell proliferation was measured by CCK-8 assay. DNA content was detected by PI assay. Results The transfection efficiency was 44.5%. The AML1-ETO specific siRNAs inhibited AML1-ETO expression at both mRNA and protein levels. The cell proliferation rate in siRNAs treated group was lower than that in control group 72 h after transfection (47.90±0.02)% vs (66.90±0.08)%, P<0.05]. The cell cycle was blocked at G1 phase 72 h after siRNAs treatment, the cell propertion in Gl phase being 38.3% and 31.6% in control group, while in G2/M phase being 1.8% and 2.4% respectively. Conclusions The synthesized siRNAs can inhibit AML1-ETO fusion gene expression. AML1-ETO specific siRNA induced the decline of AML1-ETO fusion protein in Kaanmi-1 cell, and then caused the cell cycle blocked in G1 stage and eventually inhibited the cell proliferation.