Identification Of High-risk Human Papillomavirus Integration Sites And The Potential Mechanism Research

Purpose:Human papillomavirus (HPV) is closely related to the carcinogenesis of cervical cancer. This study apply DIPS-PCR (Detection of integrated papillomavirus sequences by ligation-mediated PCR) to detect the integration sites of HPV16 and HPV18 in Siha cell line and Hela cell line。Method:Genomic DNA of Siha cell line and Hela cell line were extracted, and DIPS-PCR was performed to explore the integration sites of HPV in Genomic. Then fluorescence in situ hybridization (FISH) was performed to confirm the data of IIPV integration sites.Results:HPV16 was found to integrate at the site of 13q22.1 in the genomic DNA of Siha cell line. And HPV18 was found to integrate at the site of 8q24.21 in the genomic of Hela cell line. The integration sites of HPV in Siha cell line and Hela cell line were further confirmed by fluorescence in situ hybridization.Conclusion:DIPS-PCR is a reliable method to detect the integration sites of HPV, and the results of DIPS-PCR could be verified by fluorescence in situ hybridization. Purpose:The integration of human papillomavirus 16 (HPV16) into host gemomic DNA is positively related to the development of cervical cancer. In this study, DIPS-PCR was performed to detect the integration sites of HPV16 in clinical samples of cervical cancer.Method:Genomic DNA of cervical cancer samples were extracted, and HPV typing PCR-sequencing was performed to determine the subtype of HPV. In HPV16 positive samples, DIPS-PCR was applied to detect the integration sites of the virus. Further target region capture sequencing was performed to verify the data of DIPS-PCR.Results:In 125 cervical cancer tissues,65 samples were HPV 16 positive. Of the 65 HPV 16 positive samples,55 samples were found to be integrated by HPV16 virus. The virus was found to integrate into 3p14 site of host genomic in 14 samples and Xp22.1 site of host genomic in 8 samples. Moreover, in 6 samples that had the 3p14 virus integration,3 samples were confirmed to be 3p14 integration positive by target region capture sequencing.Conclusion:HPV16 were more prone to integrate into 3p14 and Xp22.1 sites of host genomic and cause the genomic instability of the host cells. Purpose:To illustrate the underlying molecular mechanism of HPV integration in cervical cancer, Zinc finger nuclease (ZFN) technology was developed to modify the genomic DNA sequence in cervical cancer cell lines.Method:Zinc finger nucleases targeting EGFP gene was designed and constructed. EGFP gene was cleaved by zinc finger nucleases in vitro and in Siha cell line. Moreover, ZFNs targeting OCT4 intron 1 was transfected into Hela cell line with Donor-466 to facilate the insertion of EGFP gene into the genomic.Results:ZFNs targeting EGFP gene successfully cleaved the EGFP DNA in vitro. And these ZFNs disrupt the EGFP plasmid in Siha cell line, thereby diminishing the EGFP positive rate of these cells in flow cytometry analysis. Further, by ZFNs targeting intron 1 of OCT4 gene, EGFP gene was successfully inserted into the specific site in Hela cell line.Conclusion:ZFN technology could knock-out and knock-in specific gene in cervical cancer line, thereby providing a powerful model for studying the underlying mechanism of IIPV integration.