The Mechanism Of High Risk HPV16 Oncoprotein E6 Inhibit Autophagy Of Cervical Cancer

Background:Cervical cancer is the fourth most common cancer between global women.The average survival time of advanced cervical cancer was16.8 months,five years total survival rate is 68 3%,which suggests that the treatment effect is still insufficient.Autophagy,generally existing in eukaryotic cells,response to external stimuli,maintain the steady state of cells,may play a role in the process of cervical tumor genesis.Many studies have shown that,after HPV integration into the host genome,can inhibit the key factor of the autophagy pathway,such as BECN1,Atg5,Atg8 expression of molecules,which suggests that autophagy may as a potential therapeutic targets.At present there is no research systematically explore how high-risk HPV supresse autophagy and how can be used as a target to save lives of patients with advanced cervical cancer.Aims:To explore the basic situation of autophagy in cervical squamous cell carcinomas and by which means that E6 oncoprotein influence of cervical cancer autophagy.Methods:1.Collecting cervical cancer tissue adjacent to carcinoma and carcinoma specimens of HPV positive patients and use q RT-PCR to explore the expression of key factors of autophagy pathway in cervical cancer and the expression differences in tissue adjacent to carcinoma;Then do some bioinformatics analysis of autophagy factor through GEPIA including bladder cancer,breast cancer,cervical cancer and ovarian cancer,HPV16 E6 drawing survival curve;2.Konckdown the expression of HPV 16 E6oncoprotein called Si Ha E6-si and highly expression of HPV 16 E6oncoprotein C33a-the E6 cells,after treatment of Rapamycin to induce cell autophagy,chloroquine treatmentt to inhibit autophagy,compare each cell proliferation,oxidative stress injury,comparison between groups of autophagy,autophagy induction and autophagy inhibition effect on cisplatin drugs;3.By targeting lysosome and autophagosome,study how E6 oncoprotein impact the autophagy.Results:The low expression of ATG12,ULK1,and BCL2 may indicate that autophagy was inhibited by HPV.Survival analysis showed that the lower expression of ATG5,ATG12,the better of the prognosis of patients with cervical cancer.Whether HPV negative or positive,excessive autophagy suppresses cervical cancer cell proliferation,inducing autophagy may promote pharmacological effect of cisplatin.But for HPV16 positive cervical cancer cells,inhibiting autophagy decrease the proliferation of cells and the increase of P62 and LC3BII/I suggest that autophagy may be blocked.Western blot suggested the expression of Stx17 reduced in E6 positive cancer cells(P<0.05),and laser confocal showed Si Ha and Si Ha E6-si after induced with Rapamycin in cell lines,the colocalization rates of lysosome and autophagosome average 33.83%and 46.46%,respectively.(P<0.05).Cytoflow also showed that fluorescence interval in 10~3-10~4in E6positive cells,compared with negative groups of 10~4-10~5.But the lysosome HPV16 E6 surface markers LAMP1 did not have obvious difference(P>0.05)Conclusion:E6 oncoprotein may mainly inhibit the expression of Stx17 to inhibit autophagy,and may disturb the acid environment to decrease the infusion of lysosome and autophagosome or block the way to degrade the substrates.