Studies On AEG-1 Promoter Activity Regulated By HPV-E6/E7 Associated Transcription Factors

Cervical cancer,one of the most common female malignant tumors,threatens women's health seriously,whose morbidity ranks second in all female cancers.In recent years,many studies demonstrate that the HPV-E6/E7 is the leading cause of cervical cancer,the pathogenesis of which involves variety of endogenous factors while the key molecule and its mechanism have not been clarified.In addition,it is reported that AEG-1 is highly expressed in almost all of human malignant tumors,and function as a “relay-station” of signal transduction in the development of tumor.But it remains unknown whether AEG-1 participates in tumorgenesis and progression caused by HPV-E6/E7 and what the concrete mechanism is.According to our previous experiments' results,the expression level of AEG-1 is paralleled with the expression of E6/E7 in cervical cancer cell lines,downregulation of E6/E7 was accompanied by the decreased expression of AEG-1 whereas overexpression of E6/E7 was accompanied by the increased expression of AEG-1,which suggest that AEG-1 may be the crucial factor in the carcinogensis concerning E6/E7.In this study,promoter analysis techniques will be used to investigate the transcription factors binding sites in the 5' end of AEG-1 promoter,and then find out the transcription factors that are associated with E6/E7 expression.We hope that our research will lay good foundation for illuminating the mechanism how E6/E7 regulates the expression of AEG-1,and will provide theoretical basis and experimental evidences for the future study.Objective: In this study,we will investigate the promoter activity of AEG-1 gene and find out the key transcription factors by both enhanced green fluorescent protein(EGFP)and double luciferase reporter system.We hope our research will provide theoretical and experimental basis for elucidating the mechanism how E6/E7 protein regulates the expression of AEG-1,and will further lay good foundation for exploring new targets of cervical cancer therapy.Methods:(1)The full length of AEG-1 gene promoter was amplified from genomic DNA of human cervical cancer cell line HeLa,pGL3-Basic/EGFP/AEG-1p and pGL3-Basic/LUC/AEG-1p vectors were then constructed to detect the promoter activity by transfecting HeLa cells and human umbilical vein endothelial cell ECV304 cells,the cells transfected with tumor-specific Stathmin promoter vector was used as positive control.(2)We analysed the transcription factor binding site on AEG-1 gene promoter and based on the scientific reports screened out four key transcription factor: c-Myc?SP1?NF-?B and E2 F,and we through RT-PCR and Western Blot experiments verified that the expression level of the above transcription factoes were associated with the expression of E6/E7.Then we performed Chip test to verify whether transcription factors c-Myc,SP1,NF-?B and E2 F were able to bind to the corresponding transcriptional regulatory factor binding sites on the AEG-1 promoter.The AEG-1 gene was truncated into different length according to the binding site of above transcription factors.We then constructed the correspondence promoter analysis vectors and detected their activity,promoter activity region was determined based the analysis result.(3)Each truncated AEG-1 gene promoters was mutated by transcriptional regulatory factor binding site mutation assay,and the activity of mutated promoters was also detected.Thus,the activating transcription factor and transcriptional repressor were screened out based the promoter mutation assay.(4)The interference vector against the activating transcription factor was designed and transfected into HeLa cells,the stable transfected cell line was selected by G418.In order to confirm that factor is an activating transcription factor of AEG-1 promoter,the stable transfected HeLa cell line was transfected by the recombinant reporter vector pGL3-Basi/LUC/AEG-1p.In order to confirmed that the transcriptional regulator of the selected inhibitory transcription factor plays an inhibitory effect on the AEG-1 promoter activity,the eukaryotic expression vector of that transcription factor was also designed and transfected into HeLa cells,stable overexpression cell line was obtained and transfected with the recombinant reporter vector pGL3-Basi/LUC/AEG-1 as well,the promoter activity was then tested as above.Results:(1)The full length of AEG-1 gene promoter was amplified from HeLa genomic DNA,and was then ligased into the promoter activity detection vector.The recombinant vector was transfected into both HeLa and ECV304 cells,AEG-1 gene promoter activity was then detected post transfection.The expression of high intensity green fluorescent protein was observed in HeLa cells,while ECV304 cells were only trace.(2)The results of RT-PCR and Western Blot indicated that the expression level of the transcription factoes c-Myc,SP1,NF-?B and E2 F was associated with the expression of E6/E7.The result of ChIP assay showed that all of c-Myc,SP1,NF-?B and E2 F can bind to the corresponding transcriptional regulator binding sites of AEG-1 promoter.Different kinds of truncated AEG-1 gene promoter vectors were then constructed based on the binding site locations of each transcription factor,and their activity were detected,respectively.The results showed that the negative control vector and the truncated promoter AEG-1pa have no activity,but the activity of promoters AEG-1b,AEG-1c and AEG-1d were almost at the same level.(3)Mutant AEG-1 promoters were constructed and the activity of each mutated promoter was detected.The results showed that c-Myc and SP1 transcription factor binding sites had no significant effect on the AEG-1 promoter activity,but the activity of AEG-1 promoter was decreased followed the mutation on the NF-?B binding site and was enhanced followed by the mutation on the E2 F binding site.(4)The promoter activity of HeLa/shNF-?B cells,which were then transfected with pGL3-Basic/LUC/AEG-1p vector,was decreased,and in the same time the promoter activity of HeLa/pcE2 F cells overexpressed E2 F,which were then transfected with pGL3-Basic/LUC/AEG-1p vector,was decreased also.Conclusion:(1)The AEG-1 gene promoter is a tumor-specific promoter with high activity.(2)All of the transcription factors c-Myc,SP1,NF-?B and E2 F can bind to the corresponding transcriptional regulator binding site of AEG-1 gene promoter.The region of truncated promoters which contains NF-?B and E2 F binding sites may be the key region that affecting the activity of AEG-1 promoter.(3)The activity of AEG-1 promoter will be decreased followed by NF-?B binding site,in contrast its activity will be enhanced followed by E2 F binding site mutation.(4)The transcriptional regulatory factor NF-?B will enhance the activity the AEG-1 gene promoter,and the transcriptional regulator E2 F will decrease the activity of AEG-1 gene promoter.In summary,all of the above result showed that,the HPV-E6/E7 related transcription factor NF-?B and E2 F are key regulater of the AEG-1 gene promoter.These results provide a theoretical and experimental basis for elucidating the molecular mechanism of E6/E7 protein involved in the regulation of AEG-1 expression and lay the foundation for the targeted therapy of cervical cancer.