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

Background: MiRNA primarily acts to repress gene expression at the post-transcriptional level through imperfect complementarity of its 59 region to the ‘‘seed site’ ’ in the 39 untranslated region of target mRNAs, with its ‘‘39– 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