采用蛋白组学技术分析质粒介导siRNA的“Off-target”效应

Evaluation of "Off-target" Effects on Plasmid-based siRNA Delivery by Proteomic Analyses

siRNA的"脱靶效应"(off-target effects)是RNA干扰应用研究领域的热点问题.采用蛋白组学技术对质粒介导的siRNA稳定沉默原癌基因c-myc可能存在的"off-target"效应进行初步研究,为siRNA靶向特异性的系统评价奠定理论与实验基础.构建靶向c-myc的siRNA真核表达质粒p-Mat01-1及其错配质粒p-Mis09-1,空质粒pEGFP-C1为对照,并稳定转染MCF-7人乳腺癌细胞.通过RT-PCR和Western印迹分析结果显示p-Mat01-1稳定转染克隆中c-myc/c-MYC的表达降低.采用2-DE及LC-ESI-MS/MS等方法,研究了p-Mat01-1与pEGFP-C1稳定转染克隆的蛋白组表达差异.结果显示,p-Mat01-1稳定转染克隆中有47个c-myc非调控蛋白点表达升高或降低,约占423个随机检测蛋白点的11.1%.这些蛋白涉及细胞骨架、代谢、增殖、信号传导、分子伴侣、氧化还原等多条途径.实验结果表明,质粒介导靶向c-myc的siRNA在MCF-7细胞中存在明显的"off-target"效应,提示在设计siRNA实验及应用研究时应系统考察其靶向特异性.

The siRNA off-target effects is a great concern in the applications of RNA interference.To investigate specificity of siRNAs,siRNA eukaryotic expression plasmid p-Mat01-1 targeting to c-myc oncogene and its mismatch plasmid p-Mis09-1 were constructed,and stably transfected into MCF-7 human breast cancer cells.Proteomic analyses for the off-target effects on the plasmid-mediated c-myc siRNA were performed using a pEGFP-C1 plasmid as the control.The RT-PCR and Western blot results showed that the c-MYC expressions were decreased in the p-Mat01-1 transfected MCF-7 cell pool.2-DE and LC-ESI-MS/MS analyses on p-Mat01-1 and pEGFP-C1 transfected stable clones revealed 47（about 11.1%） non-target proteins of the total 423 up-or down-regulated spots,including proteins of cytoskeleton,metabolism,proliferation,signal transduction,molecular chaperones and detoxification/redox proteins.The results showed that plasmid-mediated c-myc siRNA resulted in a substantial off-target effect in MCF-7 cells.Our findings suggested that nonspecific effects on siRNA inhibition of mammalian protein expressions should be considered in the siRNA designs and the experimental applications.