应用H1-U6双启动子RNAi载体筛选人泛素结合酶hUBE2W的RNAi有效靶点

Selecting Functional siRNA Target Sites of hUBE2W Based on H1-U6 Dual Promoter RNAi Plasmid

摘要:

hUBE2W是新近鉴定出的一种I型泛素结合酶, 可能在肿瘤发生和DNA损伤修复过程中起重要作用。RNA干涉是指通过形成局部双链RNA进而特异性地降解细胞内同源基因mRNA的机制, 目前已成为基因功能研究的有力工具。通过构建H1-U6双启动子RNAi载体, 可以在体内直接转录出针对hUbe2w基因的两段互补小RNA, 更接近于生理状态下小干涉RNA的作用。利用RT-PCR方法从293FT细胞总RNA中扩增出hUbe2w基因, 分别将其连接至质粒pGL3-Control、pCMV-myc和pDsRed-express-C1中, 以便在mRNA水平和蛋白水平检测RNA干涉效果。RNA干涉载体分别与上述报告载体共转染293FT细胞, 测定萤光素酶活性和hUBE2W蛋白表达水平, 结果显示靶点125和259能够在mRNA水平和蛋白质水平显著降低hUbe2w的表达。

Abstract:

hUBEW, a newly identified class I ubiquitin conjugating enzyme, probably plays an important role in tumorigenesis and DNA repair processes. RNA interference (RNAi) is a process in cells to degrade specific homologous mRNA by forming duplex RNA and has been developed into a powerful tool to study gene functions. In this study, the H1-U6 dual promoter RNAi plasmid was constructed and the target sequence for hUbe2w could be transcribed from both strands and form a double stranded RNA with two 5'Uridine overhangs, which closely resembles endogenous functional siRNA. The hUbe2w cDNA was amplified from reverse transcription of the 293FT total RNA by RT-PCR, and then cloned into the pGL3-Control, pCMV-myc and pDsRed-express-C1 plasmids respectively, which were selected as report vectors to detect the RNAi effects. The plasmids were co-transfected into HEK293FT cells, and then the luciferase activity and hUBE2W protein expression were measured respectively. The Resulted reduction of mRNA and protein level demonstrate that the targets of 125 and 259 could significantly inhibit the hUbe2w expression.