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

: 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.