HPV16 E6 Promotes Cervical Cancer Cells Proliferation By Upregulating TKT Activity

BackgroundCervical cancer is one of the most common cancers in women.The incidence of cervical cancer worldwide ranks third among common cancers in women.More than 99%of precancerous lesions and cervical cancer are related to HPV infection,especially hrHPV 16,18,31,33,and 45,which are detected in approximately 97%of cervical cancer patients.Infection with HPV 16 and 18 has been reported to be present in more than 70%of cervical cancer patients worldwide.Currently,the development and application of HPV related vaccines can effectively control HPV infection.However,the application and popularization of HPV vaccine is still limited.In other countries,such as the United States,less than 40%of women of the right age are vaccinated with HPV vaccine,while the vaccination rate of men is almost zero.In addition,many developing countries cannot afford the price of the vaccine.More importantly,the vaccine only prevents HPV infection,and there is no effective treatment for patients who are already infected or who have already developed cervical cancer.Therefore,HPV-related diseases are still a major public health problem facing China and even the world.The growth of HPV-positive cancer cells depends on the continuous expression of oncogene E6.Many studies have shown that E6 oncoprotein is the key factor to maintain the malignant phenotype of HPV-positive cancer cells.E6 can avoid cell checkpoints by degrading p53,thus causing uncontrolled cell malignant proliferation,and finally transforming cells into cancer cells.However,p53 degradation can not fully explain the malignant transformation induced by HPV16 E6.Therefore,it is necessary to search for new targets of HPV carcinogenic mechanism.Metabolic abnormalities promote the occurrence and development of various tumors.In tumor cells,glycolysis is often overactivated.High throughput glycolysis not only provides energy for malignant tumor proliferation,but also provides raw materials for biosynthesis within tumor cells.The pentose phosphate pathway(PPP)is a branch of glycolysis that is divided into oxidation and non oxidation stages.R5P is a biological precursor for the synthesis of nucleic acids and amino acids,and plays an important role in regulating the metabolism and proliferation of cancer cells,as well as DNA damage reactions;NADPH provides raw materials for tumor biosynthesis and can reduce the level of oxidative stress in tumor cells.Therefore,the activation of non oxidative PPP is crucial for rapidly proliferating tumor cells.ObjectiveTo clarify the role of HP V16 E6 activation to upregulate TKT activity to promote cell malignant proliferation,and to explore the possible mechanism of action;Meanwhile to clarify the role of TKT inhibitors in treating HPV-positive tumors and overcoming chemoresistance.Methods1.HPV16 E6 promotes the proliferation of cervical cancer cells by activating TKT(1)Construction of cervical cancer cell lines ectopically expressing HPV16 E6 The cervical cancer cell line with ectopic expression of HPV16 E6 was constructed by lentivirus vector,and the protein and mRNA levels of HPV16 E6 were detected by Western blot and RT-qPCR.(2)HPV16 E6 promotes the proliferation of cervical cancer The effect of HPV16 E6 on the proliferation of cervical cancer cells was detected by CCK8 and clonogenesis test,and the effect of HPV16 E6 on the growth of cervical cancer was further verified in vivo by nude mice tumorigenesis test.(3)HPV16 E6 activates non-oxidizing PPP Metabolites significantly altered after HPV infection were identified by untargeted metabolomics;The differentially expressed genes in HPV positive cervical cancer tissues and HPV negative paracervical tissues were screened out by bioinformatics,and the biological functions played by these differentially expressed genes were further determined by go analysis and KEGG enrichment analysis.(4)HPV 16 E6 up-regulates TKT enzyme activity The effects of HPV 16 E6 on TKT protein and mRNA levels were detected by Western blot and RT-qPCR;The effect of HPV 16 E6 on TKT enzyme activity was detected by spectrophotometry.(5)HPV16 E6 promotes cervical cancer proliferation by up-regulating TKT enzyme activity CCK8,clonogenesis and EdU were used to detect the effect of the addition of TKT inhibitor OT on the proliferation and DNA synthesis of cervical cancer cells,and the effect of OT treatment on tumor growth was further tested by nude mice tumorigenesis experiment.2.HPV16 E6 promotes the proliferation of cervical cancer cells by activating AKT and up-regulating TKT enzyme activity(1)HPV16 E6 activates AKT The effect of HPV16 E6 on AKT and p-AKT protein levels was detected by Western blot.(2)HPV16 E6 upregulates TKT enzyme activity by activating AKT Western blot was used to detect the effect of HPV16 E6 on AKT,p-AKT and TKT protein levels after adding AKT inhibitor TCN;The effect of HPV16 E6 on TKT enzyme activity after adding TCN was detected by spectrophotometry.(3)HPV16 E6 promotes cervical cancer cell proliferation by activating AKT and upregulating TKT enzyme activity The effects of HPV16 E6 on the proliferation and DNA synthesis of cervical cancer cells after adding TCN were detected by CCK8,cloning,and EdU.3.The role of TKT inhibitor in the treatment of HPV-positive cervical cancer and overcoming chemotherapy resistance(1)TKT inhibitors inhibit the proliferation of cervical cancer cells and overcome chemoresistance in vitro CCK8 was used to detect the effect of OT or DDP alone or combined with OT and DDP on the viability of cervical cancer cells.(2)The antitumor effect of TKT inhibitors in vivo and overcoming chemoresistance The effect of OT or DDP alone or combined with OT and DDP on tumor growth was detected by nude mice tumorigenesis experiment;The effects of different administration schemes on the levels of PCNA and Ki67 protein in transplanted tumor tissue were detected by IHC staining.Results1.HPV16 E6 promotes the proliferation of cervical cancer cells by activating TKT(1)Western blot and RT-qPCR showed that the level of HPV16 E6 protein and mRNA in C33A cells of ectopic surface group increased significantly.(2)Through CCK8 and clonogenesis experiments,it was found that HPV16 E6 enhanced the viability and clonogenesis of cervical cancer cells;Through the tumorigenic experiment in nude mice,it was found that HPV16 E6 promoted tumor growth.(3)Through non targeted metabolomics,it was found that HPV infection resulted in a significant increase in non oxidative PPP metabolites;Through TCGA database screening,it was found that in the selected dataset,there were 91 differentially expressed genes in HPV positive cervical cancer tissues and HPV negative cervical paracancerous tissues,and these differentially expressed genes were mainly enriched in metabolic pathways such as DNA synthesis,nucleotide metabolism,and purine synthesis.(4)Western blot and RT-qPCR showed that HPV16 E6 did not change the level of TKT protein and mRNA;It was found by spectrophotometry that HPV16 E6 significantly promoted TKT enzyme activity.(5)Through CCK8,cloning,and EdU,it was found that the addition of OT inhibited the proliferation and DNA synthesis of cervical cancer cells induced by ectopic expression of HPV16 E6;Through the tumorigenesis experiment in nude mice,it was found that OT treatment inhibited the tumor growth promoted by ectopic expression of HPV16 E6.2.HPV 16 E6 promotes the proliferation of cervical cancer cells by activating AKT and up-regulating TKT enzyme activity(1)Western blot showed that HPV16 E6 increased the level of p-AKT protein.(2)Western blot showed that adding TCN inhibited the increase of p-AKT protein level caused by ectopic expression of HPV16 E6,but did not change the level of TKT protein;Through spectrophotometry,it was found that the addition of TCN inhibited the increase in TKT enzyme activity caused by ectopic expression of HPV16 E6.(3)Through CCK8,cloning,and EdU,it was found that the addition of TCN inhibited the proliferation and DNA synthesis of cervical cancer cells induced by ectopic expression of HPV 16 E6.3.The role of TKT inhibitor in the treatment of HPV-positive cervical cancer and overcoming chemotherapy resistance(1)Through the CCK8 experiment,it was found that both OT and DDP alone could inhibit the activity of cervical cancer cells,and the combined treatment of the two could further inhibit the activity of cervical cancer cells.(2)Through the tumorigenesis experiment in nude mice,it was found that both OT and DDP alone could inhibit tuMor growth,and the combination of the two could further inhibit tumor growth;Through IHC staining,it was found that the expression of PCNA and Ki67 protein in transplanted tumor tissue could be inhibited by single administration,and the expression of PCNA and Ki67 protein could be further inhibited by combined administration.Non-oxidizing PPP is mainly responsible for the production of ribose 5-phosphate(R5P),and can also indirectly produce nicotinamide adenine dinucleotide phosphate(NADPH).However,no studies have yet elucidated the role of non-oxidative PPP in HPVrelated cell transformation and tumor development.Conclusions1.HPV 16E6 promotes the proliferation of cervical cancer by activating AKT and upregulating TKT enzyme activity.2.OT can inhibit the growth of HPV-positive cervical cancer to a certain extent,and combined use with DDP can further enhance the anti-tumor effect of DDPValueThis experiment clarified the role and mechanism of HPV infection leading to the proliferation of cervical cancer,and clarified that the change of metabolism after HPV infection can promote the malignant proliferation of cervical cancer cells,which not only provides a new strategy for the research of HPV-related diseases and the treatment of cervical cancer,but also provides a theoretical basis and experimental basis for the development of effective drugs for the treatment of cervical cancer.