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Mutational analysis of SATB1 gene in hepatocellular carcinomas

Authors

  • Chang J. Kim,

    1. Biomedical Research Center, Ulsan University Hospital, College of Medicine, University of Ulsan, Ulsan, Korea
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  • Gong R. Lee,

    1. Biomedical Research Center, Ulsan University Hospital, College of Medicine, University of Ulsan, Ulsan, Korea
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  • Jung W. Shin,

    1. Department of Internal Medicine and Biomedical Research Center, Ulsan University Hospital, College of Medicine, University of Ulsan, Ulsan, Korea
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  • Seok W. Jung,

    1. Department of Internal Medicine and Biomedical Research Center, Ulsan University Hospital, College of Medicine, University of Ulsan, Ulsan, Korea
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  • Bo R. Park,

    1. Department of Internal Medicine and Biomedical Research Center, Ulsan University Hospital, College of Medicine, University of Ulsan, Ulsan, Korea
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  • Neung H. Park

    1. Biomedical Research Center, Ulsan University Hospital, College of Medicine, University of Ulsan, Ulsan, Korea
    2. Department of Internal Medicine and Biomedical Research Center, Ulsan University Hospital, College of Medicine, University of Ulsan, Ulsan, Korea
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To the Editor

To the Editor

The Special AT-rich Sequence-Binding protein 1 (SATB1) is a cell-type-specific organizer of the genome [1]. SATB1 regulates gene expression in thymocytes and pre B-cells by binding to matrix attachment region (MAR) of DNA [2]. MAR is a specific DNA sequence that binds to nuclear matrix in vitro and these sequences have been posited to form the base of chromosomal loops [3]. It plays an important role in the organization of higher-order chromatin structure [4]. SATB1 as a MAR-binding protein regulates the genes by folding chromatin into loop domain [5]. Recent studies have shown that SATB1 promotes tumor growth and metastasis through chromatin gene recombination in breast cancer [6]. Furthermore, SATB1 is not only overexpressed in a large number of human cancers, but is also an excellent prognostic marker in some cancers [7-10]. All of these findings strongly imply that SATB1 is a possible candidate oncogene that it may contribute to carcinogenesis.

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world with the highest incidence in Southeast Asia and Africa. In Korea, it accounts for an estimated 12.2% of all malignancies, with 16.4% in the male population and 6.5% in the female population [11]. The pathogenesis of HCC has been studied extensively, and molecular changes during malignant transformation have been identified. Hepatocarcinogenesis is considered as a multistep process originating from hepatic stem cells or mature hepatocytes [12]. Genetic and epigenetic alterations leads to an activation of oncogenes and inhibition of tumor suppressor genes accompanied by an escalation of genetic instability and the disruption of signaling pathways related to the main promoters of hepatocarcinogenesis, namely cell proliferation and angiogenesis [13]. Until now, mutation of the SATB1 gene has not been described in HCC. To investigate whether or not genetic alterations of SATB1 are involved in hepatocellular carcinogenesis, we searched for somatic mutation of the SATB1 gene in HCC.

Hepatocellular carcinoma samples and their corresponding non-cancerous liver tissues of 38 patients were evaluated. This study was approved by the Institutional Review Boards at the Ulsan University Hospital. Frozen tissue samples were ground to a very fine powder in liquid nitrogen. Genomic DNA was prepared using a procedure based on a protocol described previously [14]. Genomic DNA samples from cancer cells and corresponding non-cancerous liver tissues were amplified with 15 sets of primers covering the entire coding region (10 exons) of the SATB1 gene (Table 1). Numbering of DNA of the SATB1 was done in respect to the ATG start codon according to the genomic sequence of Genbank accession no. NM_002971. All PCR products in exons 2–11 of the SATB1 gene were screened by single strand conformation polymorphism (SSCP) analysis (Mutation Detection Enhancement; FMC BioProducts, Rockland, ME, USA) with 10% glycerol and sequencing analysis. We repeated the experiments three times to ensure the specificity of the results, and found that the data were consistent.

Table 1. Primer sequence for amplifying the coding region of the SATB1 gene
Name of primerNucleotide SequenceProduct size (bp)
E2A-F5′-GACAACAGGGCTTTCAGCTTTTCA-3′206
E2A-R5′-ACAATGTGAGTGATCCGAAGGGTC-3′ 
E2B-F5′-TGTACTCCCAAGCCTTCCTCTTCC-3′217
E2B-R5′-CGTAGGTCCCCGCCCAACTA-3′ 
E3-F5′-TCCAACCCCACGTACACAT-3′245
E3-R5′-GATTGTTGGTTTTCCATCTCTTCT-3′ 
E4-F5′-GACCAGGTAAGAAGAATGTTTC-3′220
E4-R5′-CTGAGGGTAAAATATTTAGTGACA-3′ 
E5-F5′-CCATCAAACCTCAAATCAG-3′230
E5-R5′-AATCTTGTAATAATCCCCTTTCTA-3′ 
E6-F5′-GGTATGTAGCCACTGCTGCAATGT-3′202
E6-R5′-TTCCATCTTGTGCTGCTGTGTATT-3′ 
E7A-F5′-CAGAACCACTTATGAAACACAACT-3′249
E7A-R5′-CAGCAGTATGCAGTGAATAGACT-3′ 
E7B-F5′-TGACAGGGGGAGGGTGGTTCAAGT-3′239
E7B-R5′-TCCAGGGAACACAGCCGAGCAG-3′ 
E7C-F5′-GCAGGTTTGGAAGAGGTGTC-3′197
E7C-R5′-TGTTGGCTGAATGTGATTTTACTC-3′ 
E8A-F5′-AAAGCTAAACAAAGAAGGGCAAGG-3′231
E8A-R5′-AAGGAAGAGGACCCCAAGACTG-3′ 
E8B-F5′-CTGCATAGCCCGAAGGTTTA-3′194
E8B-R5′-TGCCTCTGACTGTTCATTTTCTAA-3′ 
E9-F5′-ACAGATAAACATTCTCAATGCCTA-3′232
E9-R5′-CAATGAAAAATGACAGCCA-3′ 
E11A-F5′-CAAGGTGGACTTTTCATTTTGTTA-3′224
E11A-R5′-AGAGGTCGATGTGGCAGAATA-3′ 
E11B-F5′-CCAAATCCTTCAGCAGCTCCTCT-3′235
E11B-R5′-ATTTCAGTGGAAGCCTTGGGAATC-3′ 
E11C-F5′-AGAGTCTGGATGGCCTCTTCGTCA-3′226
E11C-R5′-AGCAGCAGCAGCAGGCACC-3′ 

In this study, we found one (2.6%) mutation in 38 HCC cases. The mutation was missense mutation: a AGT to AAT transition at codon 354 (S354N). S354N was found in exon 7A located within the MAR domain of the SATB1 gene. It has been reported that SATB1 recruits histone deacetylase complex to the MAR site inside the interleukin-2 receptor α gene [5]. In addition, SATB1 regulates the expression of fetal globin genes by binding to MAR in the β-globin cluster [15]. Thus, it is likely that the SATB1 mutation identified in cancer cells may contribute to the development of HCC by defect of protein-protein interaction. There was no mutation in corresponding normal DNAs of these tissues, indicating that the mutation detected in the cancer cells had arisen somatically. SSCP gels of these cases with mutation showed aberrantly migrating mutant bands with remaining wild-type bands, suggesting hemizygous mutations (Fig. 1). Thus, these results suggest that the mutation of the SATB1 gene maybe a rare event in the development of HCC in Korean population.

Figure 1.

Mutation of the SATB1 gene detected in HCCs. (A) SSCPs of DNAs from cancer cells shows one aberrantly migrating band (arrow) with wild-type bands. (B) Sequencing data showed G to A transition at codon 354, which changed an amino acid from serine to asparagine in case no.25. (N, normal; T, tumor).

Recent study revealed that SATB1 plays an important role in the development and progression of liver cancer through increase of cell cycle progression and defect in apoptosis [16]. As SATB1 promotes tumor growth and metastasis in liver cancer, we presumed that mutation of SATB1 gene might affect not only cell cycle progression but also apoptosis pathway in liver cancer. Functional analysis of the mutation identified in this study will broaden our understanding of the pathogenesis of HCCs.

This work was supported by Priority Research Center Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012-0717).

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