Examination of Artificial MiRNA Mimics with Centered-Site Complementarity for Gene Targeting

Abstract

Background:MiRNA primarily acts to repress gene expression at the post-transcriptional level through imperfect complementarity of its 5′ region to the "seed site" in the 3′ untranslated region of target mRNAs, with its "3′-supplementary site" and "center site" also playing important roles under certain circumstances. The aim of this study was to test if artificial miRNA mimics (miR-Mimics) that are designed to target the "centered sites" without "seed sites" complementarity are able to repress gene expression as natural miRNAs.Methods:We designed miR-Mimics carrying centered-site matches (CS-miR-Mimics) or seed-site matches (SS-miR-Mimics) and siRNA to two antiapoptotic genes BCL2 and AKT1. We tested the gene targeting of these constructs using real-time RT-PCR and Western blot to quantify mRNA and protein levels of BCL2 and AKT1, respectively, luciferase reporter gene assay to investigate the interaction between miR-Mimics and their target sites, and cell survival assay to study the functional outcomes of the miR-Mimics.Results:We found that CS-miR-Mimic, SS-miR-Mimic and siRNA, all down regulated the mRNA and protein levels of their cognate target BCL2 or AKT1 in a concentration-dependent manner. Luciferase reporter gene assay further confirmed the functional interactions of CS-miR-Mimic, SS-miR-Mimic and siRNA with their target sites. We then observed that the miR-Mimics and siRNAs were all able to induce cell death, as indicated by the reduced survival rate of cells.Conclusions:We have provided evidence for the feasibility of CS-miR-Mimics for post-transcriptional repression of genes, which can be designed to have reduced numbers of seed type off-target sites compared to the number of target sites from an average endogenous seed-site miRNA. CS-miR-Mimics may be a novel approach for miRNA research requiring miRNA gain-of-function. © 2013 Zhang et al.