Multi-target inhibition by four tandem shRNAs embedded in homo- or hetero-miRNA backbones

Abstract

The functional influence of microRNA (miRNA) backbone selection remains unclear with respect to multiplexing miRNA-based short hairpin RNAs (shRNAmiRs), due to a lack of comparative studies. To this end, a pair of shRNAmiR tetramers were designed in the present study that targeted four genes with a shared miR30a backbone (homo-BB) or four miRNA backbones (hetero-BB). A PBLT+ 293A cell line overexpressing four targets was established, which permitted simultaneous dissection of individual gene knockdown. Multi-target inhibition was confirmed by a decrease in positive cell populations of the relative gene and mean fluorescence intensities, with almost comparable activities of homo- and hetero-BB tetramers. Of note, this multi-inhibition was sustained over a 1-month period, with no notable difference, particularly in the late-phased inhibitory effects between homo- and hetero-BB tetra-shRNA miRs. These preliminary data may indicate little influence of scaffold substitution in the functionalities of multiplexed shRNAmiRs and little recombination-depleted risk of repetitively adopting the same miRNA backbone in this artificial in vitro system. More comparative studies are further required to explore extended repertoires of scaffold-paralleled multi-shRNAmiRs in more physiologically relevant models.