Nucleic Acids Research Advance Access originally published online on November 19, 2007
Nucleic Acids Research 2007 35(22):e149; doi:10.1093/nar/gkm971
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Nucleic Acids Research, 2007, Vol. 35, No. 22 e149
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Lentivirus-mediated antagomir expression for specific inhibition of miRNA function
Department of Hematology, Hemostasis, and Oncology, Hannover Medical School, Hannover, Germany
*To whom correspondence should be addressed. Tel: +49 511 532 9207; Fax: +49 511 532 9242; Email: m.scherr{at}t-online.de Correspondence may also be addressed to Matthias Eder. Tel: +49 511 532 9207; Fax: +49 511 532 9242; Email: eder.matthias{at}mh-hannover.de
Received July 22, 2007. Revised September 20, 2007. Accepted October 18, 2007.
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.