Lentivirus-mediated antagomir expression for specific inhibition of miRNA function

TLDR

MicroRNAs regulate gene expression by binding target mRNAs and recruiting protein complexes, and their activity can be transiently blocked by chemically modified antagomirs. The study aims to generate stable loss‑of‑function phenotypes for specific miRNAs via lentivirus‑mediated antagomir expression. Lentiviral vectors expressing antagomirs under an H1 promoter in the 3′LTR target miRNAs from the miR17‑92 cluster. Lentiviral antagomirs effectively silenced miR‑18a, miR‑19b, and miR‑20a, relieving reporter repression, reducing miRNA amplification, increasing E2F‑1 protein in a dose‑dependent manner, and together with miRNA overexpression clarified distinct and complementary roles of miR‑18a and miR‑20a in regulating cell proliferation.

Abstract

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.