Investigating the Promoter of FAT10 Gene in HCC Patients

Abstract

FAT10, which is also known as diubiquitin, has been implicated to play important roles in immune regulation and tumorigenesis. Its expression is up-regulated in the tumors of Hepatocellular Carcinoma (HCC) and other cancer patients. High levels of FAT10 in cells have been shown to result in increased mitotic non-disjunction and chromosome instability, leading to tumorigenesis. To evaluate whether the aberrant up-regulation of the FAT10 gene in the tumors of HCC patients is due to mutations or the aberrant methylation of CG dinucleotides at the FAT10 promoter, sequencing and methylation-specific sequencing of the promoter of FAT10 was performed. No mutations were found that could explain the differential expression of FAT10 between the tumor and non-tumorous tissues of HCC patients. However, six single nucleotide polymorphisms (SNPs), including one that has not been previously reported, were identified at the promoter of the FAT10 gene. Different haplotypes of these SNPs were found to significantly mediate different FAT10 promoter activities. Consistent with the experimental observation, differential FAT10 expression in the tumors of HCC patients carrying haplotype 1 was generally higher than those carrying haplotype II. Notably, the methylation status of this promoter was found to correlate with FAT10 expression levels. Hence, the aberrant overexpression of the FAT10 gene in the tumors of HCC patients is likely due to aberrant methylation, rather than mutations at the FAT10 promoter.

Keywords: FAT10; SNPs; expression; methylation; promoter.

Figure 1

Study Design. Workflow of the project is presented, including the source of the patient samples and the techniques employed.

Figure 2

Primers for the generation of the different haplotypes of single nucleotide polymorphisms (SNPs) at the FAT10 promoter. Top: Schematic representation of the location of the various primers. Bottom: Table showing the sequence of the various primers. Bold underlined letters represent the SNP allele.

Figure 3

Haplotype of SNPs at the FAT10 promoter region in three ethnic populations. The six single nucleotide polymorphisms are schematically represented at the top panel. TSS: transcription start site. The frequencies of the haplotype of SNPs at the FAT10 promoter region as predicted in silico are shown at the lower panel. CH: Chinese, EA: European-American, and AA: African-American.

Figure 4

Haplotype of SNPs at the FAT10 promoter region in HCC patients and non-HCC individuals, and the FAT10 promoter activity mediated by the various haplotypes. (A) The frequencies of the haplotype of SNPs at the FAT10 promoter region, as predicted in silico. HCC: hepatocellular carcinoma patients, non-HCC: individuals of a similar age who have not been diagnosed with HCC; (B) Normalized β-galactosidase activity of the various FAT10 promoters carrying the different haplotypes in Hep3B cells. Data represent the mean and standard errors from four independent experiments. ** denotes significant difference (p < 0.01) between the various FAT10 promoter haplotypes and wild-type haplotype (I); (C) Fold difference in the normalized FAT10 transcript expression (as determined using reverse-transcription, real-time PCR) in the tumour versus non-tumorous liver tissues of HCC patients with haplotypes I and II.

Figure 5

Correlation between the methylation status at the FAT10 promoter and FAT10 transcript expression in HCC patient tissues. The seven CG dinucleotides are schematically represented at the top panel. TSS: Transcription Start Site. The bottom panel shows the correlation between the methylation status and the fold difference in FAT10 transcription expression between the tumor and non-tumorous liver tissue of each HCC patient. The last column shows the sum of the scores of either the fully methylated (score = 2) denoted by ●, half methylated (score = 1) denoted by ◎, and unmethylated (score = 0) denoted by ○.

Figure 6

Activity of the FAT10 promoter that has been methylated in vitro versus the promoter that has not been methylated. The FAT10 promoter that has either been methylated with M.SssI or mock methylated (unmethylated) is fused with the enhanced green fluorescent protein (EGFP) reporter protein and transfected into Hep3B cells. The FAT10 promoter activity expressed through EGFP protein levels is determined. Figure shows the mean and standard error from four (methylated) and five (unmethylated) independent experiments. *** denotes a significant difference (p < 0.001) in activity between the unmethylated and methylated FAT10 promoter.