| Objective: Persistent high-risk HPV infection is not only one of the important causes of cervical cancer,which is the most common malignant tumor in women worldwide,but also has been a research hotspot.Although HPV preventive vaccines have been put into use and achieved good results,the prevalence and mortality of cervical cancer in developing countries and economically disadvantaged areas are still high due to geographical location,transportation conditions and economic development.Therefore,it is important to study the causative factors of HPV causing cervical cancer.The life cycle of high-risk HPV can be synchronized with the differentiation of host cells,during which viral DNA is amplified and integrated with the host cell genome.In view of this feature,the differentiation culture of cell models simulating HPV infection was used to study the growth changes of HPV16 infected cell models,the changes of HPV virus appendator integration status,and the effects of HPV DNA integration status on DNA damage and repair pathways.Although we have constructed HPV16-infected cells,the study of differentiation culture of HPV-infected cell models is critical because the status of viral genome integration is not yet clear.Methods: The recovered HaCaT cells were passaged,and when the confluency reached80%,the cells were collected to detect their basal-like state by RT-PCR and agarose gel electrophoresis.Then,the cells of the HPV16 infected group were resuscitated,and when their confluency reached 80%,they were inoculated in the prepared 1.5%methylcellulose semi-solid complete medium,in which the cells were placed in a semi-adherent and semi-suspended state,and the cells were differentiated for 96 hours,and then the cells were collected.Firstly,the RNA of the cells of the differentiated HPV16-infected group was extracted,and the transcriptional level of keratin was detected by RT-qPCR.Then,the total protein of the cells of the differentiated HPV16-infected group was extracted,and the translation level of its keratin was detected by Western Blot.In addition,flow cytometry was used to detect the cell cycle changes of cells in the HPV16-infected group after differentiation.On the other hand,the ratio of E2/E6 in HPV16-infected cells before and after differentiation was detected by RT-qPCR.In addition,RT-qPCR was also used to detect the expression of DNA damage repair related indicators before and after differentiation.Results: We verified the expression of K5 and K14 in HaCaT cells by RT-PCR and agarose gel electrophoresis,and proved that the host cell is in a basal-like state,that is,the cell can be differentiated and cultured.RT-qPCR and Western Blot verified that keratin IVL and KLF4 expression levels were increased and P63 expression levels decreased,which could prove that HPV16-infected cells could differentiate in 1.5%methylcellulose semi-solid complete medium.On the other hand,the detection of the E2/E6 ratio in the cells of the undifferentiated HPV16 infected group by RT-qPCR showed that the ratio of E2/E6 in the cells of the undifferentiated HPV16 infected group was equal to 1,which could prove that the episome state of the viral appendage existed outside the chromosomes of the host cells.The ratio of E2/E6 in the cells of the differentiated infected group can be detected that the ratio of E2/E6 in the cells of the differentiated HPV16 infected group is equal to 0,that is,the HPV16 DNA has been integrated with the host cell genome.Subsequently,the relevant indicators of DNA damage repair pathway were detected by RT-qPCR and it was found that the process of differentiation culture activated the DNA damage repair pathway.Conclusion: Differentiation culture can successfully induce differentiation of HPV16 infected cells,and the differentiation process can not only significantly accelerate the cell cycle of the infected group,but also activate the DNA damage repair pathway,thereby promoting the genome integration of HPV16 virus appendages with host cells. |
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