Lentivirus-mediated antagomir expression for specific inhibition of miRNA function.
Lentivirus-mediated antagomir expression for specific inhibition of miRNA function.
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DOI：
10.1093/nar/gkm971
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发表时间：
2007
影响因子：
14.9
通讯作者：
Eder, Matthias
中科院分区：
文献类型：
--
作者：
Scherr, Michaela;Venturini, Letizia;Battmer, Karin;Schaller-Schoenitz, Michael;Schaefer, Daniel;Dallmann, Iris;Ganser, Arnold;Eder, Matthias
微小RNA（miRNA）通过多蛋白复合物与互补mRNA靶序列的杂交和募集来调节基因表达。miRNA的功能可以通过与单个miRNA互补的经化学修饰的寡核苷酸来瞬时拮抗。在这里，我们描述了通过慢病毒介导的miRNAs表达诱导稳定的功能丧失表型。慢病毒表达的miRNAs由位于慢病毒3′LTR内的H1启动子转录，并针对多顺反子miR 17 -92转录物上编码的miRNA。相应的miR-18 a、miR-19 b和miR-20 a的功能性沉默特异性地解除miRNA介导的报告基因抑制。通过miRNA特异性定量RT-PCR，miRNA功能的抑制与“miRNA”扩增的减少相关。此外，E2 F-1（一种已知的miR-20靶标）的蛋白质表达通过慢病毒表达的抗miR-20 miR-20 a以剂量依赖性方式增强，而miR-20 a的过表达降低E2 F-1水平。最后，特定miRNA和miR-20的组合过表达揭示了miR-18 a和miR-20 a的个体和互补功能，并在细胞培养模型中证明了特定miRNA对细胞增殖的影响。
Micro RNAs (miRNA) regulate gene expression by hybridization and recruitment of multi-protein complexes to complementary mRNA target sequences. miRNA function can transiently be antagonized by antagomirs—chemically modified oligonucleotides complementary to individual miRNAs. Here, we describe the induction of stable loss-of-function phenotypes for specific miRNAs by lentivirus-mediated antagomir expression. Lentivirally expressed antagomirs are transcribed from a H1-promoter located within the lentiviral 3′LTR and were directed against miRNAs encoded on the polycistronic miR17-92 transcript. Functional silencing of miR-18a, miR-19b and miR-20a by the corresponding antagomirs specifically relieves miRNA-mediated reporter gene repression. Inhibition of miRNA function correlates to reduction of ‘miRNA’ amplification by miRNA-specific quantitative RT-PCR. Furthermore, protein expression of E2F-1, a known miR-20 target, is enhanced by lentivirally expressed anti-miR-20 antagomirs in a dose-dependent manner, whereas over-expression of miR-20a reduces E2F-1 levels. Finally, combined over-expression of specific miRNAs and antagomirs reveals individual and complementary functions of miR-18a and miR-20a and demonstrates specific miRNA impact on cell proliferation in a cell culture model.