title: Chemokine receptor CCR6 expression is regulated by miR-518a-5p in colorectal cancer cells.
abstract: BACKGROUND
  Recently, involvement of the chemokine/receptor system CCL20/CCR6 in colorectal cancer (CRC) progression was shown. Here, we analyzed the functional interaction of miRNA-518-5p (miR-518a-5p) with CCR6 and its impact on CCR6 expression in CRC cells.
  
  
  METHODS
  MiR-518a-5p was identified by computer software to potentially interact with CCR6. Hence, functional implications of miR-518a-5p with the 3'UTR of CCR6 were analyzed using the Dual Luciferase Reporter assay system. Confirmation of the predicted target site for miR-518a-5p was achieved by site-directed mutagenesis of the seed sequence in the 3'UTR of CCR6 and subsequent application of the mutated seed sequence in a luciferase assay with miR-518a-5p mimics. Accordingly, two CRC cell lines (Caco-2 and HT-29) were transfected with miR-518a-5p miRNA mimics and gene and protein expression of CCR6 was monitored using qRT PCR and immunocytochemistry, respectively.
  
  
  RESULTS
  Addition of miR-518a-5p led to significant down-regulation of luciferase activity (P < 0.05), which was significantly reversed in a reporter test system containing the mutated seed sequences in the 3'UTR of CCR6. Following transfection of CRC cell lines with miR-518a-5p mimics and subsequent monitoring of CCR6 expression showed significant down-regulation of CCR6 mRNA and CCR6 protein expression in both CRC cell lines under investigation (P < 0.05).
  
  
  CONCLUSIONS
  We have shown that miR-518a-5p functionally interacts with CCR6 and that transfection of CRC cells with miR-518a-5p leads to significant CCR6 down-regulation. Consequently, CCR6 expression is regulated by miR-518a-5p in CRC cells indicating that regulation of CCR6 expression by miR-518a-5p might be a regulatory mechanism involved in CRC pathogenesis.
authors.affiliation: Department of General -, Visceral-, Vascular - and Paediatric Surgery, University of the Saarland, 66421 Homburg/Saar, Germany. claudia.rubie@uks.eu.
authors.forename: Claudia
authors.initials: C
authors.lastname: Rubie
authors.forename: Bianca
authors.initials: B
authors.lastname: Kruse
authors.forename: Vilma Oliveira
authors.initials: VO
authors.lastname: Frick
authors.forename: Kathrin
authors.initials: K
authors.lastname: Kölsch
authors.forename: Pirus
authors.initials: P
authors.lastname: Ghadjar
authors.forename: Mathias
authors.initials: M
authors.lastname: Wagner
authors.forename: Friedrich
authors.initials: F
authors.lastname: Grässer
authors.forename: Stefan
authors.initials: S
authors.lastname: Wagenpfeil
authors.forename: Mathias
authors.initials: M
authors.lastname: Glanemann
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D001483:Base Sequence; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics*; D015536:Down-Regulation / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D054399:Receptors, CCR6 / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D020413:3' Untranslated Regions; C106311:CCR6 protein, human; C571457:MIRN518 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; D054399:Receptors, CCR6; D008156:Luciferases