cyclin E基因siRNA抑制K562细胞生长的研究

南方医科大学学报 ›› 2005, Vol. 25 ›› Issue (04): 361-365.

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cyclin E基因siRNA抑制K562细胞生长的研究

宋海星¹, 马文丽¹, 宋艳斌¹, 张宝¹, 郑文岭²

1. 南方医科大学分子生物学研究所, 广东, 广州, 510515;
2. 广州军区广州总医院医学实验科, 广东, 广州, 510010

出版日期:2005-04-20 发布日期:2005-04-20
基金资助:
收稿日期:2004-11-23。
基金项目:National Natural Science Foundation of China(39880032);Key Science Research Project of Guangzhou(99-Z-022-01)
作者简介:SONG Hai-xing,Tel:020-61648210,E-mail:shx@fimmu.com
通讯作者:MA Wen-li,Tel:020-61648210

Growth inhibition of K562 cells by cyclin E gene-specific small inter-fering RNA

SONG Hai-xing¹, MA Wen-li¹, SONG Yan-bin¹, ZHANG Bao¹, ZHENG Wen-ling²

1. 南方医科大学分子生物学研究所, 广东, 广州, 510515;
2. 广州军区广州总医院医学实验科, 广东, 广州, 510010

Online:2005-04-20 Published:2005-04-20

摘要/Abstract

摘要： 目的应用RNAi技术,研究cyclinE基因小干扰RNA(siRNA)对K562细胞生长的抑制作用。方法针对cyclinEmRNA设计siRNA序列,经PCR方法体外扩增,得到含有U6启动子以及siRNA序列的PCR产物,以LipofectamineTM2000脂质体法转染K562细胞,分别设置为干扰组、阴性对照组以及空白对照组,转染48h后进行细胞计数、RT-PCR及通过PI染色进行流式细胞仪检测,观察其干扰效果。结果干扰组与阴性对照组和空白对照组相比,活细胞计数减少约80%,G₁期细胞百分比增高30%,细胞基本停止增殖。干扰组cyclinEmRNA表达明显减弱,相对阴性对照组及空白对照组,干扰组mRNA表达下降70%。阴性对照组与空白对照组无显著性差异。结论应用RNAi技术可以有效地干扰cyclinE基因的表达,并进一步抑制细胞的生长,但对于干扰的长期有效性尚无实验结论,需通过长效表达载体实验验证。

Abstract: Objective To study the inhibitory effect of small interference RNA(siRNA) of cyclin E gene on the growth of K562 cells. Methods siRNA targeting the 940 bp site of the cyclin E mRNA were designed and generated by PCR amplification. The PCR products containing U6 promoter and the siRNA were then transfected into K562 cells via LipofectamineTM2000. The cells transfected with non-functional siRNA served as the negative control group and those only treated with serum-free RPMI1640 as the blank control group. Cell counting, reverse transcriptase (RT)-PCR and flow cytometry were employed to evaluate the effect of RNA interference. Results Compared with the negative and blank control groups, the viable cell count in the interference group was decreased by approximately 80%, the ratio of G₁-phase cells increased by nearly 30%, and growth arrest was observed. Cyclin E mRNA expression in the cells of the interference group was significantly lowered by about 70% as compared with that of the negative and blank control groups, whereas the latter two groups had similar expression levels. Conclusion RNA interference induces obvious inhibition of cyclin E gene expression, which consequently affects the proliferation of K562 cells.

中图分类号:

Q789

宋海星¹, 马文丽¹, 宋艳斌¹, 张宝¹, 郑文岭². cyclin E基因siRNA抑制K562细胞生长的研究[J]. 南方医科大学学报, 2005, 25(04): 361-365.

SONG Hai-xing¹, MA Wen-li¹, SONG Yan-bin¹, ZHANG Bao¹, ZHENG Wen-ling². Growth inhibition of K562 cells by cyclin E gene-specific small inter-fering RNA[J]. Journal of Southern Medical University, 2005, 25(04): 361-365.

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cyclin E基因siRNA抑制K562细胞生长的研究

Growth inhibition of K562 cells by cyclin E gene-specific small inter-fering RNA

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