The Mechanism Of HMGB Regulating TFAP2A/PD-L1 Signaling Pathway To Promote The Proliferation And Metastasis Of Cervical Cancer

Objective: Cervical cancer is the fourth mortality rate cancer among women worldwide,which seriously affects women’s health.Invasive metastasis is the main cause of death in cervical cancer patients,TFAP2 A has been found to be highly expressed in a variety of malignant tumors,and is closely related to the invasion and migration of cancer cells.However,its role in cervical cancer is still unknown.Therefore,this study aims to clarify the role of TFAP2 A in cervical cancer and explore the specific regulatory mechanism of the increased expression of TFAP2 A in cervical cancer.Methods:(1)The gene set data of cervical cancer patients were obtained from GEPIA,GEO,TCGA,Oncomine and HPA databases to analyze the mRNA expression level of TFAP2 A in cervical cancer tissues.The tissue samples of cervical cancer and normal cervical cancer were collected and the TFAP2 A protein expression level in cervical cancer tissues was investigated by immunohistochemical staining.The clinicopathologic parameters related to TFAP2 A expression were selected by single factor regression analysis of clinical data of cervical cancer patients in TCGA,GEO database and clinical samples.Finally,UCSC Xena,cBioPortal and Xiantao Academic databases were used to analyze the relationship between TFAP2 A and PD-L1 online.(2)Lentivirus transfection technology was used to construct SiHa and HeLa cervical cancer cell lines with stable overexpression and silencing of HMGB1.Western Blot was used to detect the effects of different HMGB1 expression levels on TFAP2 A and PD-L1 protein expressions.Then,after overexpression and silencing of TFAP2 A,the influence of different TFAP2 A expression levels on PD-L1 protein expression was detected by Western Blot.Meanwhile,the changes of TFAP2 A expression levels in cervical cancer cell lines with overexpression and silencing of PD-L1 were detected.The interaction between HMGB1,TFAP2 A and PD-L1 was elucidated.In addition,the proliferation,migration,invasion and apoptosis of cervical cancer cells were detected by CCK8,Transwell,wound healing,clonogenesis and flow cytometry in cervical cancer cell lines that silenced TFAP2 A and overexpressed PD-L1 simultaneously.To elucidate the effect of downregulation of TFAP2 A and PD-L1 on biological behavior of cervical cancer cells.(3)Dual luciferase reporter gene and chromatin immunoprecipitation assay were used to elucidate the interaction mechanism between TFAP2 A and PD-L1.The effects of downregulation of TFAP2 A on tumor growth of cervical cancer were investigated by constructing subcutaneous tumor transplantation model of cervical cancer in nude mice and mapping tumor growth curve.Results:(1)TFAP2A is overexpressed in cervical cancer,and is correlated with clinicopathological features and prognosis of patients with cervical cancer: Cervical cancer data in GEPIA,GEO,TCGA,Oncomine,HPA and other databases were analyzed.It was found that compared with normal cervical tissues,TFAP2 A mRNA level in cervical cancer tissues was up-regulated.The positive expression of TFAP2 A protein in cervica l cancer tissue was found by immunohistochemistry,indicating that the expression of TFAP2 A protein in cervical cancer tissue was up-regulated.Subsequently,through the analysis of clinicopathologic data,it was found that the expression level of TFAP2 A was correlated with FIGO stage,pathological stage,tumor size,HPV infection,vascul ar invasion,lymph node metastasis and contraceptive history of patients with cervical cancer.Most importantly,the group with high TFAP2 A expression showed poorer surv ival.Finally,the analysis of UCSC Xena,cBioPortal and Xiantao Academic databases found a positive correlation between TFAP2 A and PD-L1.(2)The protein level of HMGB1 affected the protein expressions of TFAP2 A and PDL1: After the downregulation of HMGB1,the protein levels of TFAP2 A and PD-L1decreased;conversely,after the overexpression of HMGB1,the protein levels of TFAP2 A and PD-L1 increased.(3)The interaction between TFAP2 A and PD-L1: the expression of PD-L1 decreased after TFAP2 A silencing,whereas the expression of PD-L1 was up-regulated after TFAP2 A overexpression.Similarly,after PD-L1 silencing,TFAP2 A protein expression was downregulated,while after PD-L1 overexpression,TFAP2 A protein level was up-regulated.(4)TFAP2A/PD-L1 axis promoted the proliferation and migration of cervical cancer cells,and inhibited apoptosis: in cervical cancer cell lines SiHa and HeLa,silting TFAP2 A inhibited the proliferation and migration of cervical cancer cells,and promoted apoptosis,which could be reversed by the overexpression of PD-L1.In a mouse model of cervical cancer xenotransplantation,silencing TFAP2 A significantly inhibited tumor growth rate and reduced tumor volume.(5)The mechanism of action between TFAP2 A and PD-L1: The results of double luciferase reporter gene and chromatin immunoprecipitation experiment suggest that TFAP2 A can be used as a transcription factor to directly bind to the promoter region of PD-L1,causing the increase of PD-L1 protein level,thus promoting the proliferation,migration and inhibiting apoptosis of cervical cancer cells.Conclusion: In cervical cancer tissues and cells,the mRNA and protein levels of the oncogene TFAP2 A are significantly highly expressed,and promotes the proliferation and migration of cervical cancer cells and inhibits apoptosis.The mechanism is mainly that TFAP2 A regulated by HMGB1 can be used as a transcription factor to bind to the PD-L1 promoter to promote its protein expression.Therefore,the key protein TFAP2 A can be used as a prognostic biomarker and potential therapeutic target for cervical cancer.