The Mechanism Of Attenuated XPC Expression And Transcriptional Regulation Study In Human Bladder Cancer

Xeroderma pigmentosum group C (XPC) is a DNA damage recognition protein that plays an important role in the nucleotide excision repair (NER) process. The XPC protein binds tightly with an HR23B protein to form a stable XPC-HR23B complex . Studies indicate that the XPC-HR23B complex is the first protein component that recognizes and binds to the damaged sites . The XPC protein might also play an important role in other DNA damage-induced cellular responses, including cell cycle checkpoint regulation and apoptosis. It has been found that XPC expression is significantly attenuated in bladder cancer. Therefore, the characterization of the elements that regulate the XPC promoter is of crucial interest to a better understanding the mechanisms of XPC gene expression. However, very little is known about the regulation of the expression of XPC gene itself. To better understand the molecular mechanisms that regulate the expression of XPC gene, we carried out this study and the major results are summarized below:1. The paraffin wax-embedded tissues of bladder papillary urothelial carcinoma were collected from patients who underwent transurethral resection of bladder tumor,and XPC protein levels were evaluated by immunohistochemistry. Kaplan-Meier analysis demonstrated that the median survival of patients with lower XPC protein levels was shorter compared with patients with higher XPC levels . Cox regression analysis further indicated that XPC protein level may act as an independent prognostic factor for bladder cancer patients (P=0.043).2. After prediction of CpG island in promoter of XPC gene using"MethPrimer"software, the status of methylation of CpG island in promoter of XPC gene was analyzed using bisulfite sequencing PCR (BSP) and sequence assay. The results revealed that the CpG island in promoter of XPC was partly methylated only in bladder cancer but not in normal bladder tissues.3. The minimal promoter region of the human XPC gene were identified in this study.The 5′flanking region of the human XPC gene was amplified and analyzed in detail for minimal promoter localization utilizing a series of 5′and 3′deletions of the XPC 5′flanking region fused upstream of the firefly luciferase reporter gene. These experiment identified the minimal promoter region, -43/-27bp, that is necessary for the expression of human XPC gene.4. Chromatin immunoprecipitation (ChIP) analysis and electrophoretic mobility shift assay (EMSA) revealed that transcription factor CREB interacted with the–43 ~ -27bp promoter region of the human XPC gene in vivo and in vitro.5. The results of the co-transfection experiments showed that the transcriptional activity of human XPC promoter was found to be regulated negatively by transcription factors CREB, through binding the putative binding sites within XPC promoter region. Western blot further demonstrated that PKA inhibitor H89 decreases DNA damage-inducible expression of XPC following UV-C irradiation in HEK293 cells.6. The SssI methylation of XPC promoter-luciferase plasmid extemely decreased the luciferase activities ,indicating that Site specific DNA methylation may play a role in interfering with the binding of CREB1 to DNA and silencing transcription. It was found CREB 1 is sensitive to the methylation status of its cognate binding sites in vitro.In summary, the minimal promoter region of the humanXPC gene was mapped in the region from nucleotides -43 to -27bp. The transcriptional activity of human XPC promoter was found to be regulated by transcription factor CREB, through binding the CREB putative binding sites within XPC promoter region. In addition, we also investigated the epigenetic regulation of XPC gene. The results from this study suggest that hypermethylation of CpG island in promoter of XPC is probably responsible for XPC expression silencing in bladder cancer, and the methyltransferase inhibitor such as 5-Aza-dc can effectively inactivate the expression of XPC. The expression of XPC was reduced with increasing invasive potential in bladder cancer patients. Reduced XPC expression was correlated with tumor aggressiveness and poor patient survival.The information generated from this study provides a solid base for further study of the transcriptional regulation of XPC gene and a novel insight in bladder cancer diagnosis and treatment.