Lentivirus-mediated antagomir expression for specific inhibition of miRNA function.
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.
Scherr
Michaela
M
Department of Hematology, Hemostasis, and Oncology, Hannover Medical School, Hannover, Germany. m.scherr@t-online.de
Venturini
Letizia
L
Battmer
Karin
K
Schaller-Schoenitz
Michael
M
Schaefer
Daniel
D
Dallmann
Iris
I
Ganser
Arnold
A
Eder
Matthias
M
D049109:Cell Proliferation; D050687:E2F1 Transcription Factor / Q000378:metabolism; D005456:Fluorescent Dyes / Q000032:analysis; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D016086:Lentivirus / Q000235:genetics*; D035683:MicroRNAs / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism*
D050687:E2F1 Transcription Factor; D005456:Fluorescent Dyes; D035683:MicroRNAs; D009841:Oligonucleotides; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins