| Background and Objective: Persistent infection with high-risk types of Human Papillomavirus(HPV)is the main cause of cervical cancer.E6 is a crucial oncogene of HPV,which can integrate into the host cell genome and influence the occurrence and development of cervical cancer through multiple mechanisms.However,the precise mechanisms by which HPV16 E6 remodels the tumor microenvironment to promote cervical cancer formation remain to be elucidated.Cervical cancer cells have the ability to secrete exosomes that mediate intercellular communication by carrying various substances such as proteins and nucleic acids which can also target endothelial cells to promote angiogenesis in the tumor microenvironment.Endothelial cell-specific molecule 1(ESM1)has been found to be highly expressed and impact tumor angiogenesis in various cancers,but its role in the cervical cancer microenvironment remains unknown.This study aims to explore the correlation between ESM1 expression and cervical cancer at clinical,bioinformatics,and cellular levels,as well as to investigate the role of ESM1 in mediating HPV16 E6 regulated cervical cancer angiogenesis,laying a foundation for identifying new targets for cervical cancer prevention,diagnosis,and treatment,as well as elucidating the pathogenesis of cervical cancer.Methods: 1.The correlation between ESM1 expression and prognosis,occurrence and clinical characteristics in cervical cancer was analyzed and validated utilizing the TCGA and GEO databases,as well as the online analysis platforms GEPIA and UALCAN.Immunohistochemical was conducted to assess the differential expression of ESM1 in a total of 140 specimens,including normal cervical tissue,cervical intraepithelial neoplasia and clinical cervical cancer tissues,subsequently,statistical analysis was performed.2.Si Ha cells were transfected with lentivirus or si RNA to knock down the expression of HPV16 E6 or ESM1 expression,the effects of HPV16 E6 and ESM1 on the proliferation,invasion,and migration ability of cervical cancer cells were evaluated by CCK8,scratch wound,and Transwell invasion assays respectively.3.Exosomes were isolated and characterized by differential ultracentrifugation,NTA,TEM,and Western Blot(WB),respectively.The correlation between HPV16 E6 and the expression of ESM1 in Si Ha cells and their exosomes was validated by WB.4.si RNAs were transfected to downregulate ESM1 expression in human umbilical vein endothelial cells(HUVEC),and the effects and mechanisms of ESM1 on HUVEC angiogenesis were verified by WB,CCK8,scratch wound,and tube formation assays.5.Laser confocal microscopy was used to examine the uptake of PKH26-labeled exosomes by HUVEC,and changes in ESM1 expression,downstream signaling pathways and angiogenesis of HUVEC were assessed utilizing WB,CCK8,scratch wound,and tube formation assays after co-culturing with exosomes.Results: 1.Bioinformatics database analysis reveals significant upregulation of ESM1 in cervical cancer tissues,which is correlated with a poor prognosis.Immunohistochemical indicates that the expression level of ESM1 protein in cervical tissues increases with the severity of cervical lesions,suggesting a correlation between ESM1 expression and the occurrence and development of cervical cancer.2.Knockdown of HPV16 E6 and ESM1 both reduce the malignant phenotype of Si Ha cells.3.HPV16 E6 positively regulates the expression of ESM1 protein in both intracellular and exosomes of Si Ha cells.4.Suppression of ESM1 diminishes the angiogenesis of HUVEC by modulating the MEK/ERK/Cyclin D1 signaling pathway.5.HUVEC efficiently uptake exosomes derived from Si Ha cells into the cytoplasm,leading to an increased expression level of ESM1 protein and promoting the angiogenesis of HUVEC,which is positively regulated by HPV16 E6.Conclusion: The remodeling of the cervical cancer angiogenic microenvironment by HPV16 E6 through exosomal ESM1 protein offers a novel perspective to enhance our understanding of cervical cancer pathogenesis. |
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