293-siDrosha稳定细胞系模型的建立及检测microRNA相关功能

基础医学与临床 ›› 2012, Vol. 32 ›› Issue (4): 381-385.

293-siDrosha稳定细胞系模型的建立及检测microRNA相关功能

郭蓉,刘力

北京协和医学院基础学院

收稿日期:2012-01-12 修回日期:2012-02-23 出版日期:2012-04-05 发布日期:2012-03-21
通讯作者: 刘力 E-mail:lliu@pumc.edu.cn
基金资助:
国家自然科学基金;2010年中国医学科学院基础医学研究所所院长基金

Establishment of 293-siDrosha stable cell line and detection of its potential use in the study of microRNA functions

Received:2012-01-12 Revised:2012-02-23 Online:2012-04-05 Published:2012-03-21

摘要/Abstract

摘要： 摘要：目的利用RNAi实验方法构建293-siDrosha稳定细胞系，并探讨该稳定细胞系在microRNAs功能研究中的应用。方法将质粒pSicoR－human Drosha1转入人胚肾293细胞，用G418筛选2-3周后，获得293-siDrosha#3稳定细胞系，并用RT-PCR、Western blotting、real-time PCR等方法检测293-siDrosha#3稳定细胞系中Drosha基因mRNA和蛋白质表达情况。并用poly(A) 实时定量 RT-PCR法检测野生293细胞，Mix细胞(未克隆的293-siDrosha细胞)，293-siDrosha#3细胞（克隆的293-siDrosha细胞）中内源hsa-miR-491-5p的表达情况。结果构建了293-siDrosha#3稳定细胞系，与对照组相比，293-siDrosha#3细胞中Drosha基因的mRNA和蛋白质表达水平均显著下降。检测内源性hsa-miR-491-5p在对照组和293-siDrosha#3细胞中表达情况，表明对照组细胞中表达的hsa-miR-491-5p是293-siDrosha#3细胞的近120倍。结论成功构建了293-siDrosha稳定细胞系，并且证明此稳定细胞系可以作为研究一些microRNAs相关功能的模式细胞系。

关键词: siDrosha, microRNAs, 293细胞

Abstract: Abstract：Objective To construct Drosha-1 deficient 293 stable cells (293-siDrosha) and to study microRNA processing by using the established 293 stable cells. Methods The pSicoR-human Drosha1 plasmid DNAs were transfected into 293 cells and selected for neomycin resistant clones with 1200g/ml G418 for 2-3 weeks. The mRNA and protein expressions of Drosha were detected by RT-PCR，real-time PCR and Western blotting. The expressions of hsa-miR-491-5p in 293 cells, uncloned 293-siDrosha cells (293-siDrosha mixed) and the cloned 293-siDrosha cells (293-siDrosha #3 cells) were detected with poly(A) real-time RT-PCR. Results Compared with the control cells, the mRNA and protein expressions of human Drosha1 in cloned 293-siDrosha cells were dramatically decreased. The expression levels of hsa-miR-491-5p in the cloned 293-siDrosha cells have about 120 fold reduction as compared with that of the control cells. Conclusion 293-siDrosha stable cells were successfully constructed. The cloned 293-siDrosha cells may be useful as a model system for the study of microRNA functions.

Key words: siDrosha, microRNAs, 293 cells

郭蓉刘力. 293-siDrosha稳定细胞系模型的建立及检测microRNA相关功能[J]. 基础医学与临床, 2012, 32(4): 381-385.