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 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.