title: SIP1 is downregulated in hepatocellular carcinoma by promoter hypermethylation.
abstract: BACKGROUND
  Smad interacting protein-1 is a transcription factor that is implicated in transforming growth factor-β/bone morphogenetic protein signaling and a repressor of E-cadherin and human telomerase reverse transcriptase. It is also involved in epithelial-mesenchymal transition and tumorigenesis. However, genetic and epigenetic alterations of SIP1 have not been fully elucidated in cancers. In this study, we investigated mutations and promoter hypermethylation of the SIP1 gene in human hepatocellular carcinomas.
  
  
  METHODS
  SIP1 expression was analyzed in HCC cell lines and primary tumors in comparison to normal and non-tumor liver tissues by using semi-quantitative RT-PCR, quantitative real-time RT-PCR and immunohistochemistry. Mutation and deletion screening of the SIP1 gene were performed by direct sequencing in HCC-derived cells. Restoration of SIP1 expression was sought by treating HCC cell lines with the DNA methyl transferase inhibitor, 5-AzaC, and the histone deacetylase inhibitor, TSA. SIP1 promoter methylation was analyzed by the combined bisulfite restriction analysis assay in in silico-predicted putative promoter and CpG island regions.
  
  
  RESULTS
  We found that the expression of SIP1 was completely lost or reduced in five of 14 (36%) HCC cell lines and 17 of 23 (74%) primary HCC tumors. Immunohistochemical analysis confirmed that SIP1 mRNA downregulation was associated with decreased expression of the SIP1 protein in HCC tissues (82.8%). No somatic mutation was observed in SIP1 exons in any of the 14 HCC cell lines. Combined treatment with DNA methyl transferase and histone deacetylase inhibitors synergistically restored SIP1 expression in SIP1-negative cell lines. Analysis of three putative gene regulatory regions revealed tumor-specific methylation in more than half of the HCC cases.
  
  
  CONCLUSIONS
  Epigenetic mechanisms contribute significantly to the downregulation of SIP1 expression in HCC. This finding adds a new level of complexity to the role of SIP1 in hepatocarcinogenesis.
authors.affiliation: Bilkent University, Department of Molecular Biology and Genetics, Ankara, Turkey.
authors.forename: Tolga
authors.initials: T
authors.lastname: Acun
authors.forename: Emin
authors.initials: E
authors.lastname: Oztas
authors.forename: Tamer
authors.initials: T
authors.lastname: Yagci
authors.forename: Mustafa C
authors.initials: MC
authors.lastname: Yakicier
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001374:Azacitidine / Q000494:pharmacology; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors; D004252:DNA Mutational Analysis; D015536:Down-Regulation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D015183:Restriction Mapping; D014158:Transcription, Genetic / Q000187:drug effects; D055815:Young Adult; D000074165:Zinc Finger E-box Binding Homeobox 2
chemical_list: D056572:Histone Deacetylase Inhibitors; D018398:Homeodomain Proteins; D006877:Hydroxamic Acids; D012097:Repressor Proteins; C492363:ZEB2 protein, human; D000074165:Zinc Finger E-box Binding Homeobox 2; C012589:trichostatin A; D015254:DNA Modification Methylases; D001374:Azacitidine