摘要
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AML-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 siRNA72h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72h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Kasumi-1细胞在G1期，进而抑制细胞增殖。
Objective By inhibiting AML1-ETO fusion gene expression in Kasumi-1 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 Kasumi-1 ceils by eleetroporation, the non-specific siRNAs transfected cells were taken as control. EGFP plasmid 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 transfeetion [ （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 proportion in G1 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 Kasumi-1 cell, and then caused the cell cycle blocked in G1 stage and eventually inhibited the cell proliferation.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2008年第9期607-610,共4页
Chinese Journal of Hematology
