title: A quick and efficient approach for gene silencing by using triple putative microRNA-based short hairpin RNAs.
abstract: The RNA interference (RNAi) technique has been widely used in gene function studies. It is typical to screen for effective siRNAs by knocking down targeted genes since a single gene can be suppressed by several siRNAs to varying degrees. The miRNA-based short hairpin RNA (shRNA) is a natural inducer of RNAi and has been used in siRNA expression strategies. We investigated the potential application of multiple putative microRNA-based shRNAs for gene silencing and studied the inhibition efficiency of exogenous GFP and firefly luciferase (luc) by triple human mir155-based shRNA expression vectors. A total of three candidate siRNA sequences targeted against GFP or luc were selected based on an online prediction program. Single and triple miRNA-155-based shRNAs targeted against GFP or luc were transfected into HEK293 cells mediated by the pcDNA(3) vector with an RNA polymerase II-type CMV (cytomegalovirus) promoter. Comparisons with negative control shRNAs revealed that GFP levels were markedly reduced by the triple miRNA-155-based GFP shRNA by fluorescent microscopy. Consistent results from the dual luciferase assay and real-time quantitative RT-PCR revealed that the triple miRNA-155-based GFP shRNA significantly suppressed GFP expression (P < 0.01), without significant differences from the most effective single miRNA-155-based GFP shRNA (P > 0.05). Results from the dual luciferase assay and real-time quantitative RT-PCR revealed that the triple miRNA-155-based luc shRNA significantly suppressed luc expression as the most effective single miRNA-155-based luc shRNA (P < 0.05). These studies demonstrated the gene silencing efficiency mediated by the triple putative miRNA-155-based shRNAs. This suggested that multiple miRNA-based shRNAs are quick and valuable strategies for gene silencing.
authors.affiliation: Research Center of Medical Sciences, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, 510080, China.
authors.forename: Z X
authors.initials: ZX
authors.lastname: Shan
authors.forename: Q X
authors.initials: QX
authors.lastname: Lin
authors.forename: M
authors.initials: M
authors.lastname: Yang
authors.forename: C Y
authors.initials: CY
authors.lastname: Deng
authors.forename: S J
authors.initials: SJ
authors.lastname: Kuang
authors.forename: Z L
authors.initials: ZL
authors.lastname: Zhou
authors.forename: D Z
authors.initials: DZ
authors.lastname: Xiao
authors.forename: X Y
authors.initials: XY
authors.lastname: Liu
authors.forename: S G
authors.initials: SG
authors.lastname: Lin
authors.forename: X Y
authors.initials: XY
authors.lastname: Yu
mesh_terms: D000818:Animals; D002460:Cell Line; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D035683:MicroRNAs; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins