title: miR-675 mediates downregulation of Twist1 and Rb in AFP-secreting hepatocellular carcinoma.
abstract: BACKGROUND
  Alpha-fetoprotein (AFP)-secreting hepatocellular carcinomas (HCC) represent a genetically distinct subset of tumors often associated with a worse prognosis. However, the molecular mechanisms that underlie these phenotypic differences remain poorly understood.
  
  
  METHODS
  HCC tumor samples from 27 patients were profiled using the Affymetrix 133 Plus 2.0 GeneChips. GeneGO Metacore software was used to identify altered biologic pathways. Expression validation was confirmed by RT-PCR. Manipulation of miR-675 by overexpression and antagomir-mediated knockdown was carried out with subsequent evaluation of effects on cell behavior by cell cycle, proliferation, invasion, and growth in soft agar assays.
  
  
  RESULTS
  We identified a strong relationship between primary tumor H19 gene expression and elevated serum AFP. H19 has recently been identified to encode microRNA-675 (miR-675), and we confirmed the relationship in an independent sample of patients. Pathway analyses of the effect of miR-675 overexpression in hepatoma cells revealed a predominant upregulation of cell adhesion and cell cycle initiation pathways. We have demonstrated that miR-675 mediates increases in proliferation and an accumulation of cells with tetraploid DNA content associated with a repression of Rb. We also demonstrated that overexpression of miR-675 alters cellular morphology, reduces invasive potential, and increases anchorage-independent growth capacity. These findings are consistent with a mesenchymal-to-epithelial transition, associated with a reduction in the expression of the key EMT mediator, Twist1.
  
  
  CONCLUSIONS
  Expression of the miR-675 in hepatocellular carcinoma links a dramatic upregulation of proliferative and growth capacity with inhibition of motility in HCC cells.
authors.affiliation: Department of Gastrointestinal Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Hernandez
authors.forename: A
authors.initials: A
authors.lastname: Elahi
authors.forename: C W
authors.initials: CW
authors.lastname: Clark
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: L A
authors.initials: LA
authors.lastname: Humphries
authors.forename: B
authors.initials: B
authors.lastname: Centeno
authors.forename: G
authors.initials: G
authors.lastname: Bloom
authors.forename: B C
authors.initials: BC
authors.lastname: Fuchs
authors.forename: T
authors.initials: T
authors.lastname: Yeatman
authors.forename: D
authors.initials: D
authors.lastname: Shibata
mesh_terms: D000368:Aged; D017209:Apoptosis; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002448:Cell Adhesion; D002465:Cell Movement; D049109:Cell Proliferation; D058750:Epithelial-Mesenchymal Transition; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008156:Luciferases / Q000378:metabolism; D008297:Male; D035683:MicroRNAs / Q000235:genetics*; D008875:Middle Aged; D060787:Neoplasm Grading; D009367:Neoplasm Staging; D009687:Nuclear Proteins / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D011379:Prognosis; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D016160:Retinoblastoma Protein / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015996:Survival Rate; D014407:Tumor Cells, Cultured; D051879:Twist-Related Protein 1 / Q000378:metabolism*; D000509:alpha-Fetoproteins / Q000378:metabolism*
chemical_list: C097353:AFP protein, human; D014408:Biomarkers, Tumor; C548379:MIRN675 microRNA, human; D035683:MicroRNAs; D009687:Nuclear Proteins; D012333:RNA, Messenger; D016160:Retinoblastoma Protein; C103173:TWIST1 protein, human; D051879:Twist-Related Protein 1; D000509:alpha-Fetoproteins; D008156:Luciferases