title: Transcriptional regulation of miR-224 upregulated in human HCCs by NFκB inflammatory pathways.
abstract: OBJECTIVE
  miR-224 is up-regulated in human HCCs as compared to both paired peri-tumoral cirrhotic tissues and cirrhotic livers without HCC. Here, we have cloned the miR-224 regulatory region and characterized its transcriptional regulation by the NFκB-dependent inflammatory pathways.
  
  
  METHODS
  Mature miRNA expression was evaluated by a 2 step stem-loop real-time RT-PCR. The recruitment of polymerase II and transcription factors on the pre-miR-224 promoter has been assessed by ChIPSeq and ChIP.
  
  
  RESULTS
  We found miR-224 levels strongly up-regulated in both peri-tumoral cirrhotic livers and HCC samples as compared to normal livers. In silico analysis of the putative miR-224 promoter revealed multiple NFκB sites. We showed that LTα and TNFα activate transcription from the miR-224 promoter and of endogenous miR-224 expression in HCC cell lines, whereas the expression of miR-224 target API5 was reduced. Exogenously expressed p65/RelA activates the miR-224 promoter and a dominant negative form of IκBα (IκBSR) represses it. ChIP analysis showed that p65/NFκB is recruited on the miR-224 promoter and that its binding sharply increases after exposure to LPS, TNFα, and LTα. Altogether these findings link the inflammatory signals to NFκB-mediated activation of miR-224 expression. An antago-miR specific for miR-224 blocked LPS and LTα stimulated HCC cells migration and invasion. Conversely, the IKK inhibitor BMS-345541 blocks pre-miR-224-induced cellular migration and invasion.
  
  
  CONCLUSIONS
  Our results identify p65/NFκB as a direct transcriptional regulator of miR-224 expression and link miR-224 up-regulation with the activation of the LPS, LTα, and TNFα inflammatory pathways and cell migration/invasion in HCC.
authors.affiliation: EAL Inserm U785, Sapienza University of Rome, Rome, Italy.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Scisciani
authors.forename: Stefania
authors.initials: S
authors.lastname: Vossio
authors.forename: Francesca
authors.initials: F
authors.lastname: Guerrieri
authors.forename: Valeria
authors.initials: V
authors.lastname: Schinzari
authors.forename: Rossana
authors.initials: R
authors.lastname: De Iaco
authors.forename: Paolo
authors.initials: P
authors.lastname: D'Onorio de Meo
authors.forename: Melchiorre
authors.initials: M
authors.lastname: Cervello
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Montalto
authors.forename: Teresa
authors.initials: T
authors.lastname: Pollicino
authors.forename: Giovanni
authors.initials: G
authors.lastname: Raimondo
authors.forename: Massimo
authors.initials: M
authors.lastname: Levrero
authors.forename: Natalia
authors.initials: N
authors.lastname: Pediconi
mesh_terms: D000368:Aged; D006528:Carcinoma, Hepatocellular / Q000473:pathology / Q000503:physiopathology*; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D005260:Female; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008099:Liver / Q000473:pathology / Q000502:physiology; D008103:Liver Cirrhosis / Q000473:pathology / Q000503:physiopathology; D008113:Liver Neoplasms / Q000473:pathology / Q000503:physiopathology*; D008233:Lymphotoxin-alpha / Q000494:pharmacology; D008297:Male; D035683:MicroRNAs / Q000502:physiology*; D008875:Middle Aged; D016328:NF-kappa B / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D015854:Up-Regulation / Q000502:physiology*
chemical_list: D008070:Lipopolysaccharides; D008233:Lymphotoxin-alpha; C546140:MIRN224 microRNA, human; D035683:MicroRNAs; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha