title: Functional microRNA high throughput screening reveals miR-9 as a central regulator of liver oncogenesis by affecting the PPARA-CDH1 pathway.
abstract: BACKGROUND
  Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths, reflecting the aggressiveness of this type of cancer and the absence of effective therapeutic regimens. MicroRNAs have been involved in the pathogenesis of different types of cancers, including liver cancer. Our aim was to identify microRNAs that have both functional and clinical relevance in HCC and examine their downstream signaling effectors.
  
  
  METHODS
  MicroRNA and gene expression levels were measured by quantitative real-time PCR in HCC tumors and controls. A TargetScan algorithm was used to identify miR-9 downstream direct targets.
  
  
  RESULTS
  A high-throughput screen of the human microRNAome revealed 28 microRNAs as regulators of liver cancer cell invasiveness. MiR-9, miR-21 and miR-224 were the top inducers of HCC invasiveness and also their expression was increased in HCC relative to control liver tissues. Integration of the microRNA screen and expression data revealed miR-9 as the top microRNA, having both functional and clinical significance. MiR-9 levels correlated with HCC tumor stage and miR-9 overexpression induced SNU-449 and HepG2 cell growth, invasiveness and their ability to form colonies in soft agar. Bioinformatics and 3'UTR luciferase analyses identified E-cadherin (CDH1) and peroxisome proliferator-activated receptor alpha (PPARA) as direct downstream effectors of miR-9 activity. Inhibition of PPARA suppressed CDH1 mRNA levels, suggesting that miR-9 regulates CDH1 expression directly through binding in its 3'UTR and indirectly through PPARA. On the other hand, miR-9 inhibition of overexpression suppressed HCC tumorigenicity and invasiveness. PPARA and CDH1 mRNA levels were decreased in HCC relative to controls and were inversely correlated with miR-9 levels.
  
  
  CONCLUSIONS
  Taken together, this study revealed the involvement of the miR-9/PPARA/CDH1 signaling pathway in HCC oncogenesis.
authors.affiliation: Division of Hematology/Oncology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA. adrakaki@mednet.ucla.edu.|Laboratory of Tumor Biology, Department of Medical Oncology, University Hospital of Heraklion, Heraklion, Crete, Greece. adrakaki@mednet.ucla.edu.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Drakaki
authors.affiliation: Center for Systems Biomedicine, Division of Digestive Diseases, David Geffen School of Medicine, University of California, Los Angeles, 650 Charles E. Young Dr., CHS 44-133, Los Angeles, CA, 90095-7278, USA. MHatziapostolou@mednet.ucla.edu.
authors.forename: Maria
authors.initials: M
authors.lastname: Hatziapostolou
authors.affiliation: Center for Systems Biomedicine, Division of Digestive Diseases, David Geffen School of Medicine, University of California, Los Angeles, 650 Charles E. Young Dr., CHS 44-133, Los Angeles, CA, 90095-7278, USA. cpolytarchou@mednet.ucla.edu.
authors.forename: Christos
authors.initials: C
authors.lastname: Polytarchou
authors.affiliation: Center for Systems Biomedicine, Division of Digestive Diseases, David Geffen School of Medicine, University of California, Los Angeles, 650 Charles E. Young Dr., CHS 44-133, Los Angeles, CA, 90095-7278, USA. cvorvis@ucla.edu.
authors.forename: Christina
authors.initials: C
authors.lastname: Vorvis
authors.affiliation: Department of Surgery, Stanford School of Medicine, Stanford University, Palo Alto, CA, USA. gpoultsides@stanford.edu.
authors.forename: George A
authors.initials: GA
authors.lastname: Poultsides
authors.affiliation: Laboratory of Tumor Biology, Department of Medical Oncology, University Hospital of Heraklion, Heraklion, Crete, Greece. johnsougl@gmail.com.
authors.forename: John
authors.initials: J
authors.lastname: Souglakos
authors.affiliation: Laboratory of Tumor Biology, Department of Medical Oncology, University Hospital of Heraklion, Heraklion, Crete, Greece. georgouliasv@gmail.com.
authors.forename: Vassilis
authors.initials: V
authors.lastname: Georgoulias
authors.affiliation: Center for Systems Biomedicine, Division of Digestive Diseases, David Geffen School of Medicine, University of California, Los Angeles, 650 Charles E. Young Dr., CHS 44-133, Los Angeles, CA, 90095-7278, USA. diliopoulos@mednet.ucla.edu.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Iliopoulos
mesh_terms: D020413:3' Untranslated Regions; D015703:Antigens, CD; D015820:Cadherins / Q000235:genetics*; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement; D019295:Computational Biology / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D056945:Hep G2 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D047493:PPAR alpha / Q000235:genetics*; D015398:Signal Transduction
chemical_list: D020413:3' Untranslated Regions; D015703:Antigens, CD; C513384:CDH1 protein, human; D015820:Cadherins; C541874:MIRN92 microRNA, human; D035683:MicroRNAs; D047493:PPAR alpha