Studies On The Methy Lation Of DEK Promoter In Hepatocellular Carcinoma

PURPOSE:DEK was initially discovered as a fusionprotein with CAN in acute myeloid leukemia(AML) patients. It is found that DEK is overexpressed in a variety kinds of tumor cells and its function concerned with tumor growth and survival attracts more and more attention. The level of DEK expression in hepatocellular carcinoma (HCC) is significantly upregulated compared with that of the normal tissue. It suggests DEK may play an important role in the pathogenesis of HCC. The experiment is to clarify the epigenetic mechanism of DEK overexpression in HCC by exploring the relationship between the expression level and the methylation status of CpG islands in promoter region.METHODS:We used seven different HCC cell lines (HuH-7, HepG2,7402,7721,97-L,97-H, LM3), one normal liver cell line (L02) and one normal tissue as the experiment materials. Real-time quantitative PCR and western blotting were used to detect the mRNA and the protein expression level of DEK. DNA methylation status was examined by Bisulfite genome sequencing (BGS). We analyzed both the methylated sites of the CpG islands and the binding sequences of the transcription factors in DEK promoter region in detail. Dual luciferase reporter gene assays were used to check the activities of4methylated and unmethylated DNA fragments in DEK promoter region.RESULTS:The level of DEK mRNA and protein expression was upregulated in all the7HCC cell lines, whereas the CpG islands in DEK promoter of these cell lines were shown hypomethylated. Combined with binding sequences of the transcription factors, we found that most of the methylated sites locate at these binding sequences. Dual luciferase reporter gene assays showed that the activity of the methylated sequences was suppressed significantly. Compare with the upstream CpG island fragment (CpG1), the fragment CpG2-1, which is adjacent to transcription start site, showed a remarkable activity. The activity of the fragment (CpG2-2) inside the CpG2-1was not significantly declined, which concentrates lots of methylated sites. It indicates that CpG2-2may be a crucial sequence in DEK promoter.CONCLUSION:Our finding indicated that DEK overexpression in HCC was significantly correlated with DNA hypomethylation in its promoter region. It could be presumed that methylation patters lead to transcriptional suppression of DEK by interfering with binding of transcription factors to the promoter. The CpG island fragment nearly transcription start area may be a pivotal sequence in relation to DEK transcriptional regulation.