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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 “off-target effects” of siRNAs are a hot issue in the field of RNAi applications.Proteomics-based siRNA can stably silence plasmid-mediated siRNA silencing of the “off- target ”effect, and lay a theoretical and experimental basis for the specific evaluation of siRNA targeting.Establishing siRNA eukaryotic expression plasmid p-Mat01-1 targeting c-myc and its mismatched plasmid p-Mis09-1 , Empty vector pEGFP-C1 as a control, and stably transfected into MCF-7 human breast cancer cells.The expression of c-myc / c-MYC in p-Mat01-1 stably transfected clones was confirmed by RT-PCR and Western blot analysis Decreased.The protein expression of p-Mat01-1 and pEGFP-C1 stably transfected clones was studied by 2-DE and LC-ESI-MS / MS.The results showed that p-Mat01-1 stably transfected the clones 47 c-myc non-regulatory protein spots were up-regulated or down-regulated, accounting for 11.1% of 423 randomly detected protein spots.These proteins involved in cytoskeleton, metabolism, proliferation, signal transduction, chaperones, redox Pathway.The experimental results show that plasmid-mediated c-myc siRNA has obvious “off-target” effect in MCF-7 cells, suggesting that Design and application of siRNA experiments should be systematically investigated its target specificity.