| Aims Cervical cancer is one of the most common cancers in women, its incidence ranks second among female cancers. The epidemiological results indicate that human papillomavirus infection is the cause of cervical cancer, some study also suggest that HPV E7-induced cell cycle re-replication can contribute to the occurrence of cervical cancer. However, the cellular changes that have experienced from HPV infection to cancer have not been fully elucidated, the mechanism of HPV E7 induced re-replication is also unclear. In addition, succinylation, one of post-translational modifications that was discovered in recent years, has been proved to play an important role in cellular metabolism, proliferation and carcinogenesis. Therefore, we examined whether succinylation have some positive effects on the carcinogenesis of cervical epithelial cells. In this study, we selected HPV immortalized cell line and SiHa cell line, respectively, to represent HPV infected cell and cervical cancer cell, to analyze the differences of protein expression between the two cell lines so that we can learn the changes from HPV infection to cancer. In addition, we explored the mechanism by which E7 induces DNA re-replication, and we focused on proteins that are related to DNA replication or cell cycle.Methods We used Trypsin to digest the proteins into peptides, then we utilized high performance liquid chromatography (HPLC) and mass spectrometry (MS) technique to identified the total proteins and succinylation. Then, we did bioinformatic analysis to the data in order to fully understand molecular changes from HPV infected cell to cancer. In addition, we used the siRNA approach combined with Western Blot technique to detect cell cycle-related protein expression changes. We also checked the changes of cell cycle profiles by flow cytometry.Results From the two cell lines used, we identified 10,691 proteins and 6,342 succincylation sites by HPLC-MS. After analyzing by label-free quantity software, we identified 572 differentially expressed proteins. The differential expression of one of the proteins was confirmed by Western blot. The identified proteins were then analyzed by bioinformatics approaches. We found that 273 proteins in HPV immortalized cells are mostly associated with diseases pathway, while 299 proteins in SiHa cells are mostly involved in metabolic and ribosomal pathway. The total number of succincylation between HPV immortalized cells and SiHa cells has no big difference, but we found a reliable succincylational site on SGK1 at K41, which has not been reported. Cell cycle results showed that HPV E7 can induce re-replication after treating with bleomycin in RPE1 E7 cells. The results of Western Blot showed that HPV E7 can up-regulate Emil, Cdtl, CDC6. After transfecting the Emil siRNA in RPE1 E7 and RPE1 vector cells, the expression of CDC6 and Skp2 goes down. We also observed re-replication in HeLa cells upon transfection with the Emil siRNA.Conclusion Comparing with the HPV-immortalized cells, we found there is a higher level of metabolism and stronger protein-synthesis-related proteins in the SiHa cells, while most up-regulated proteins in HPV immortalized cell are associated with particular diseases. SGK1 K41 succincylation is likely to be a new protein post-translational modification site. Emil plays an important role in HPV E7 induced re-replication; the occurrence of cell cycle/re-replication induced by E7 may due to the effect of the APC ubiquitination pathway that was inhibit by Emi1, and some cell cycle-related proteins such as CDC6 and Cdt1, may play a role.Innovation and significanceThis is the first study comparing the total proteins between HPV immortalized cell line and SiHa cell line; we made the novel observations that the level of metabolism/protein-synthesis is higher in cancer cells than in HPV-infected cells. We further explored the mechanism by which HPV E7 induces re-replication. These studies laid the foundation for a better understanding of the pathogenic mechanism of cervical cancer. |
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