SIP1 is downregulated in hepatocellular carcinoma by promoter hypermethylation

Acun, T.
Oztas, E.
Yagci, T.
Yakicier, M.C.
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BMC Cancer
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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. © 2011 Acun et al; licensee BioMed Central Ltd.

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azacitidine, bisulfite, DNA methyltransferase inhibitor, histone deacetylase inhibitor, messenger RNA, Smad interacting protein 1, transcription factor, trichostatin A, unclassified drug, DNA methyltransferase, histone deacetylase inhibitor, homeodomain protein, hydroxamic acid, repressor protein, trichostatin A, ZEB2 protein, human, article, cancer cell culture, cancer tissue, computer model, controlled study, CpG island, DNA methylation, down regulation, drug potentiation, epigenetics, exon, gene deletion, gene mutation, gene sequence, human, human tissue, immunohistochemistry, liver, liver carcinogenesis, liver cell carcinoma, major clinical study, primary tumor, promoter region, protein expression, restriction mapping, reverse transcription polymerase chain reaction, somatic mutation, adult, aged, down regulation, drug antagonism, drug effect, female, gene expression regulation, genetic transcription, genetics, liver cell carcinoma, liver tumor, male, metabolism, middle aged, molecular genetics, nucleotide sequence, pathology, promoter region, tumor cell line, Adult, Aged, Aged, 80 and over, Azacitidine, Base Sequence, Carcinoma, Hepatocellular, Cell Line, Tumor, DNA Methylation, DNA Modification Methylases, DNA Mutational Analysis, Down-Regulation, Female, Gene Expression Regulation, Neoplastic, Histone Deacetylase Inhibitors, Homeodomain Proteins, Humans, Hydroxamic Acids, Liver Neoplasms, Male, Middle Aged, Molecular Sequence Data, Promoter Regions, Genetic, Repressor Proteins, Restriction Mapping, Transcription, Genetic, Young Adult
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