运用siRNA抑制K562细胞中端粒酶逆转录酶基因表达的研究

Using siRNA to inhibit hTERT gene expression in K562 cell line

目的 建立利用小分子干扰RNA(siRNA)技术抑制白血病细胞系K562细胞中端粒酶活性的方法,为肿瘤的基因治疗提供理论依据。方法 设计端粒酶逆转录酶(hTERT)基因特异性siRNA,用体外转录方法合成hTERT基因的siRNA并转染K562细胞,培养48小时后,收集细胞,应用实时荧光定量RT PCR和westernblot方法检测转染细胞中hTERT基因mRNA水平和蛋白表达量的变化,并运用TRAPELISA方法检测细胞内端粒酶活性的变化。结果 转染siRNA后,与对照组相比,实验组hTERTmRNA水平和蛋白表达量明显降低,抑制率分别为75%和60%,同时TRAPELISA方法检测发现实验组端粒酶活性仅为对照组活性的45%。结论 hTERTsiRNA能特异性的抑制hTERT基因的表达,降低端粒酶活性,因此运用siRNA来抑制hTERT的表达可达到降低端粒酶活性的效果。

Objective To establish a method to inhibit telomerase activity in leukemia cell line K562 by small interfering RNA(siRNA).Methods The hTERT gene specific siRNA was designed according to its sequence,and was made by in vitro transcription.The hTERT siRNA was transfected into K562 cell,and the cells was cultured for 48 hours before harvesting.hTERT mRNA and protein levels were monitored by fluorescence real time reverse transcription-polymerase chain reaction (RT-PCR)and western blot,and the telomerase activity was measured by TRAP-ELISA.Results Compared with control group,there was a significant decrease in the hTERT mRNA and protein levels in experimental group,and the inhibitory efficiency could reach 75% and 60%, respectively.The telomerase activity in experimental group was only 45% when compared to control group.Conclusion Our results suggested that the hTERT siRNA can specifically inhibit the expression of hTERT gene in K562 cells and can decrease telomerase activity.Our results also indicated that hTERT is a regulatory factor of telomerase.