title: Examination of artificial MiRNA mimics with centered-site complementarity for gene targeting.
abstract: 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.
authors.affiliation: Department of MICU, the First Affiliated Hospital, Zhengzhou University, Zhengzhou, P. R. China.
authors.forename: Shu-Guang
authors.initials: SG
authors.lastname: Zhang
authors.forename: Chun-Yan
authors.initials: CY
authors.lastname: Liu
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Tong-Wen
authors.initials: TW
authors.lastname: Sun
authors.forename: Yong-Gang
authors.initials: YG
authors.lastname: Luo
authors.forename: Wen-Jing
authors.initials: WJ
authors.lastname: Yun
authors.forename: Jin-Ying
authors.initials: JY
authors.lastname: Zhang
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D035683:MicroRNAs / Q000235:genetics*; D018716:Molecular Mimicry; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats
chemical_list: D020413:3' Untranslated Regions; D035683:MicroRNAs; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; D008156:Luciferases; C494918:AKT1 protein, human; D051057:Proto-Oncogene Proteins c-akt