Transcriptional Regulatory Mechanism Of DEK Gene In Human Hepatocellular Carcinoma

PURPOSE:DEK protein is overexpressed in multiple human invasive tumors. DEK gene has been identified as an oncogene and is closely related to occurrence and development of malignant tumors. The previous results showed that the expression level of DEK gene in human hepatocellular carcinoma (HCC) was obviously higher than that in normal liver cells, which indicated that DEK might exert an important role in pathogenesis of HCC; meanwhile, the methylation level of DEK core promoter (-167bp～+35bp) showed significant differences between HCC cell lines and normal liver cells, and it was generally in low methylation status in HCC cell lines. Currently, the transcriptional regulatory mechanism of DEK gene in HCC remains unclear. Hence, based on the previous work, this DNA region was further analysed with following-up studies in order to determine the important transcription factors which can regulate DEK gene expression, thus revealing the transcriptional regulatory mechanism of DEK gene which is overexpressed in HCC.Method:Based on the previous research, this study predicted the transcription factors which might be combined with DEK promoter CpG island region and their binding sites by online software TFSEARCH. Combined with the differences analysis of DNA methylation, we initially screened out the transcription factor and its binding site that played an important regulatory role in the transcription activity of DEK core promoter. Then, we verified the predicted transcription factor binding site as an important regulational region that maintained DEK gene promoter activity through point mutation and deletion mutation assays. Finally, we used siRNA interference, transcription factor overexpression assays and chromatin immunoprecipitation (ChIP) to verify the regulation of the predicted important transcription factor to DEK promoter activity and its transcription activity as well as its interaction with DEK promoter in HCC cell lines. Results:By dual luciferase reporter gene experimental analysis, results showed that among the truncated DEK promoter sequence, CpG2-2, which was near to the transcription start site, had the most influence on DEK promoter activity. Further, predicted results using TFSEARCH showed that CpG2-2 region had several transcription factor binding sites, such as AP-2a. Combined with the analysis of methylation differences in normal liver cells and tissues and HCC cell lines, we found that the methylation differences of AP-2a binding site was in the largest, so we predicted that the AP-2a may be one of the important transcription factors, which could regulate the activity of DEK promoter. Then, the results of point mutation and deletion assays showed that the AP-2a binding site could obviously regulate the transcription activity of DEK core promoter. Furthermore, siRNA interference, transcription factor overexpression assays and chromatin immunoprecipitation (ChIP) showed that transcription factor AP-2a could interact with DEK promoter in HCC cell lines and regulate DEK gene expression positively.Conclusion:Based on the above experimental results, we draw the conclusion: AP-2α was one of the important transcription regulatory factors which associated with DEK gene overexpression in human hepatocellular carcinoma (HCC). Through interaction with its binding site in DEK core promoter region CpG2-2, AP-2α could promote expression of DEK gene in HCC cell lines.