title: [MicroRNA 144 negatively regulates Toll-like receptor 2 expression in rat macrophages].
abstract: OBJECTIVE
  To investigate the relationship between miR-144 and Toll-like receptor 2 (TLR2).
  
  
  METHODS
  RT-qPCR was used to determine the expression of TLR2 and its downstream inflammatory cytokine TNF-α in rat macrophage cell line NR8383 transfected by a mimic miR-144 or miR-144 inhibitor. The fragments of 3'UTR region of rat TLR2 mRNA including wild or mutant miR-144 binding site obtained by PCR using rat liver cDNA were ligated to pmirGLO report gene vector digested with SacI and XbaI to construct the recombinant vectors of pmir-TLR2-3'UTR and pmir-mutant-TLR2-3'UTR. The miR-144 targeting TLR2 was further determined by dual luciferase reporter assay and miR-144 mimics.
  
  
  RESULTS
  TLR2 and TNF-α in NR8383 cells were decreased after transfection with 100 nmol/L mimic miR-144 for 24 h and increased after transfection with 100 nmol/L miR-144 inhibitor. PCR and double-enzyme digestion with SacI and XbaI confirmed successful insertion of the target fragments. Dual luciferase reporter assay suggested the binding of miR-144 to the 3'UTR of rat TLR2 mRNA.
  
  
  CONCLUSIONS
  miR-144 negatively regulates the expression of TLR2 and its down-stream cytokine TNF-α by targeting TLR2 in NR8383 cells.
authors.affiliation: Department of Biochemistry and Molecular Biology, Xi'an Jiaotong University Health Science Center, Xi'an 710061, China.baroquewang@gmail.com.
authors.forename: Xuan
authors.initials: X
authors.lastname: Wang
authors.forename: Xi
authors.initials: X
authors.lastname: Lan
authors.forename: Li
authors.initials: L
authors.lastname: Liu
authors.forename: Jing
authors.initials: J
authors.lastname: Yi
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Yue
authors.initials: Y
authors.lastname: Li
authors.forename: Meichen
authors.initials: M
authors.lastname: Wang
authors.forename: Jiaxi
authors.initials: J
authors.lastname: Li
authors.forename: Liu-Mei
authors.initials: LM
authors.lastname: Song
authors.forename: Dongmin
authors.initials: D
authors.lastname: Li
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D005822:Genetic Vectors; D008156:Luciferases; D008264:Macrophages / Q000378:metabolism*; D035683:MicroRNAs / Q000378:metabolism*; D012333:RNA, Messenger; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction; D051195:Toll-Like Receptor 2 / Q000378:metabolism*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D020413:3' Untranslated Regions; C555034:MIRN144 microRNA, rat; D035683:MicroRNAs; D012333:RNA, Messenger; C495343:Tlr2 protein, rat; D051195:Toll-Like Receptor 2; D014409:Tumor Necrosis Factor-alpha; D008156:Luciferases