Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark.
RNA. 2013 Feb;19(2):280-93. doi: 10.1261/rna.034850.112. Epub 2012 Dec 18.
MicroRNAs (miRNAs) are key regulators of gene expression and modulators of diverse biological pathways. Analyses of miRNA function as well as therapeutic managing of miRNAs rely on cellular administration of miRNA inhibitors which may be achieved by the use of viral vehicles. This study explores the miRNA-suppressive capacity of inhibitors expressed intracellularly from lentivirus-derived gene vectors. Superior activity of two decoy-type inhibitors, a "Bulged Sponge" with eight miRNA recognition sites and a hairpin-shaped "Tough Decoy" containing two miRNA recognition sites, is demonstrated in a side-by-side comparison of seven types of miRNA inhibitors transcribed as short RNAs from an RNA Pol III promoter. We find that lentiviral vectors expressing Tough Decoy inhibitors are less vulnerable than Bulged Sponge-encoding vectors to targeting by the cognate miRNA and less prone, therefore, to reductions in transfer efficiency. Importantly, it is demonstrated that Tough Decoy inhibitors retain their miRNA suppression capacity in the context of longer RNA transcripts expressed from an RNA Pol II promoter. Such RNA Pol II-transcribed Tough Decoy inhibitors are new tools in managing of miRNAs and may have potential for temporal and spatial regulation of miRNA activity as well as for therapeutic targeting of miRNAs that are aberrantly expressed in human disease.
微小 RNA（miRNAs）是基因表达的关键调控因子，也是多种生物途径的调节剂。miRNA 功能的分析以及 miRNA 的治疗管理都依赖于 miRNA 抑制剂的细胞内给药，这可以通过使用病毒载体来实现。本研究探讨了从慢病毒衍生的基因载体中细胞内表达的抑制剂对 miRNA 的抑制能力。在对七种类型的 miRNA 抑制剂进行并排比较时，两种诱饵型抑制剂——具有八个 miRNA 识别位点的“膨出海绵”和含有两个 miRNA 识别位点的发夹状“坚韧诱饵”——表现出更高的活性，这些抑制剂是由 RNA Pol III 启动子转录为短 RNA 的。我们发现，表达坚韧诱饵抑制剂的慢病毒载体比编码膨出海绵的载体更不容易受到同源 miRNA 的靶向，因此，转移效率降低的可能性更小。重要的是，证明了坚韧诱饵抑制剂在由 RNA Pol II 启动子表达的较长 RNA 转录本的情况下保留其 miRNA 抑制能力。这种由 RNA Pol II 转录的坚韧诱饵抑制剂是 miRNA 管理的新工具，可能具有 miRNA 活性的时空调节潜力，以及对人类疾病中异常表达的 miRNA 的治疗靶向潜力。
