Relationship Of Gene Silencing Effects And The Structure And Targeting Sites Of Intronic Spliced Artificial MiRNAs

RNA interference (RNAi), a sequence-specific RNA degradation mechanism mediated by double-stranded RNA (dsRNA), can be used not only as a common research tool but also, as a therapeutic strategy. With the increasing discovery of hundreds of natural microRNAs (miRNAs) and the progressed research about miRNA biogenesis and function, a new strategy is rising, which employs a natural miRNA precursor sequence to express artificial miRNA for RNAi. Research indicates that compared with traditional shRNA vector, this artificial miRNA vector could achieve a higher silencing efficiency. Artificial miRNAs expression vectors such as pre-miRNA30-and pre-miRNA155-designed shRNAs vectors, splicing pre-miRNA155 (pSM155) and splicing pre-miRNA30 (pSM30) vectors which were designed to targets in ORF of endogenous and exogenous genes have been developed and used for RNA interference. However, up to now, it has been not yet clear to design rational stem-loop structure of miRNA155 (miR155)- pre-miRNA including position of bulge and mismatch in its stem seeing that duplex structures of the native miR155 precursors on which atificial miRNAs design should be depended are different according to species, and the defferent synthetic constructs were used in RNA interference.In the present study, we studied the inhibition efficiencies by differently designed atificial miRNAs against targets in 5'-UTR,3'-UTR and ORF of exogenous gene in 293T cells. The results indicated that SM155-miRNAs with a mismatch at the+1 position and a bulge at the+11,+12 positions in its stem could have seed-match effects and more efficiently suppress against the targets, in 3'-UTR of exogenous gene, and suggested that interferon response could be different according to stem-loop structures of SM155-miRNAs transfected in 293T cells for RNA interference.Avian influenza virus is the pathogen which causes the avian influenza. Meanwhile, avian influenza virus poses huge threat to the health of mankind. RNA interference using artificial miRNA had been used as an effective antiviral strategy for its specific silencing of viral gene expression in mammalian cells. To design artificial miRNA that remain effective despite antigenic drifts and shifts, we chose the two genes (including 5'-UTR and 3'-UTR) as the target sequence, which are essential to viral replication and host range restriction and contain highly conserved sequences in5'-UTR and 3'-UTR of them, and studied gene silencing by 6 single-, multiple-SM155-miRNAs designed for NP or PB2 genes. The results showed that the PB2-, NP-specific single-, multiple-SM155-miRNAs targeting 5'-UTR,3'-UTR and ORF of PB2 and NP gene could effectively down-regulate the expression of PB2 and NP gene, respectively, multiple-SM155-miRNAs simultaneously targeting PB2 and NPgene also could down-regulate the expression of PB2 and Np gene, efficiently.Our results confirmed that pSM155-miRNAs with newly designed stem structure could be a valuable artificial miRNA expression system for gene function study and antiviral RNA interference.