MYEOV Increases HES1 Expression And Promotes Pancreatic Cancer Progression By Enhancing SOX9 Transactivity

Pancreatic ductal adenocarcinoma(PDAC)is a deadly gastrointestinal malignancy,and it is the most common type of pancreatic malignant tumor.The prognosis for patients with PDAC is typically dismal.Emerging evidence indicates that myeloma overexpressed(MYEOV)is an oncogene and plays crucial roles in multiple human cancers.However,its roles in the development of PDAC remain elusive.In this study,we explored the effect of this gene on PDAC,and sought to determine the underlying molecular mechanisms.The subject consisted of four parts.The specific contents are as follows:Chapter 1:The expression level of MYEOV and its clinical translational significance in PDACObjective:To research the expression level of MYEOV in PDAC,and its clinical translational significance in PDAC.Methods:We used immunohistochemistry to detect the expression levels of MYEOV protein in 71 paired primary PDAC and adjacent normal pancreatic tissue specimens from a tissue microarray,and analysed its relationship with clinical feature and survival of PDAC patients.Additionally,we detected the expression levels of MYEOV protein in pancreatic cancer cell lines and human pancreatic duct epithelial cell line by western blot.We also investigated MYEOV mRNA expression in PDAC by using bioinformatic databases(TCGA and GEO),and analysed its relationship with clinicopathologic characteristics of PD AC.Results:MYEOV was found to be overexpressed in PD AC.MYEOV overexpression was associated with advanced T classification of PDAC.High MYEOV protein expression was an independent prognostic factor that predicts poor survival of patients with PDAC.Chapter 2:The regulation mechanism of MYEOV expression in PDACObjective:To research the mechanism underlying the increased expression level of MYEOV in PDAC.Methods:We used the public databases(MethHC and cBioPortal)to investigate the MYEOV promoter methylation status in normal pancreatic tissues PDAC tissues and PDAC tissues,and analysed the relationship between MYEOV mRNA expression and its promoter methylation.We performed pyrosequencing to dectected the methylation level of MYEOV promoter in pancreatic cell lines We performed qPCR to dectected the MYEOV mRNA in cell lines expressed low MYEOV level with or without demethylation.Results:The rate of MYEOV promoter methylation was lower in PDAC.Additionaly,our results showed a negative association between MYEOV promoter methylation and its mRNA expression in PDAC.The results of qPCR revealed that demethylation could restore MYEOV expression level.Chapter 3:The impact of MYEOV on the biological functions of PDACObjective:To investigate the effect of MYEOV on proliferation and metatasis of PDAC.Methods:Cell transfection,wound healing assays,transwell migration assays,and Matrigel invasion assays,CCK8,colony formation assays,liver metastases and subcutaneous tumorigenesis in nude mice were performed to investagate the impact of MYEOV on metatasis and proliferation of PDAC.Results:MYEOV knockdown decreased metastatic potential and proliferative ability of PDAC cells both in vitro and in vivo,whereas its overexpression had the opposite effect.Chapter 4:The molecular mechanisms of the pro-tumor effect of MYEOV in PDACObjective:To determine the molecular mechanisms underlying the pro-tumor function of MYEOV in PDAC.Methods:We performed immunoprecipitation,mass spectrometry,Co-IP and co-immunofluorescence to screen the interational proteins of MYEOV.We investigated the impact of MYEOV on SOX9-mediated transcriptional activation of HES1 by using qPCR?luciferase assays and ChIP.We performed rescue expeiments by knockdowned HES1 and SOX9 in PDAC cells with or without MYEOV overexpression.Results:Immunoprecipitation and mass spectrometry indicated that SOX9 may interact with MYEOV,and the results of Co-IP and co-immunofluorescence substantiated this finding.The results of qPCR?luciferase assays and ChIP indicated that MYEOV promoted SOX9 DNA-binding activity in pancreatic cancer and thus increased the expression of HES1 gene.Rescue expreiments indicated that HES1 or SOX9 knockdown led to a decrease in the oncogenic potential of MYEOV overexpressing cells.Taken together,we demonstrate that MYEOV overexpression is due to promoter hypomethylation in pancreatic cancer.MYEOV plays a pivotal oncogenic role through interacting with SOX9 and increasing its DNA-binding ability,consequently activating HES1 transcription in pancreatic carcinogenesis.Therapeutic intervention of MYEOV function might be a novel strategy for arresting uncontrolled growth and metastasis in PDAC.Additionally,MYEOV has the potential to be an independent factor to predict the survival of patients with PDAC.