MYEOV Reduces Prognosis Of Pancreatic Cancer Patients By Promoting Cell Proliferation And Recruiting Neutrophil Infiltration

Pancreatic cancer is an insidious,rapidly progressive,and highly lethal malignancy.Its inferior prognosis is associated with deep anatomical location,lack of reliable early screening,and ineffective therapeutic measures,making pancreatic cancer increasingly one of the leading causes of cancer-related deaths.Therefore,it is important to investigate the key genes that can affect the prognosis of pancreatic cancer,so as to clarify their roles in the development and prognosis of pancreatic cancer,and then to explore the corresponding molecular markers for diagnosis and candidate therapeutic targets.In recent years,bioinformatics has played an important role in uncovering disease-related therapeutic targets,and to some extent,has provided direction for researchers.Based on this,we have used bioinformatics to identify MYEOV,a gene with the most prognostic value for pancreatic cancer patients,which is known as the myeloma overexpressed gene.This gene has been reported to have prognostic value in a variety of tumors,but its specific molecular mechanism is not yet clear.This study aimed to uncover the molecular mechanism that affects the prognosis of pancreatic cancer patients.The subject consists of three parts.Part I: Expression of MYEOV and its correlation with clinicopathological features and prognosis in pancreatic cancerObjective: This part of the study aims to clarify the expression of MYEOV in pancreatic cancer,compare the differences in MYEOV expression between tumor cells and non-tumor cells,and explore the correlation between MYEOV expression and clinicopathological characteristics of patients,and reveal the significance of MYEOV in prognosis.Methods: The correlation between individual genes and the overall survival of pancreatic cancer patients in TCGA database was analyzed by bioinformatics methods,and the genes with the most significant differences were screened.The expression of MYEOV in cancer and non-cancerous tissues were analyzed,followed by single-cell sequencing data to analyze the expression of MYEOV in different cells within the tumor.Western blot and tissue microarray immunohistochemistry were used to verify the expression of MYEOV.Clinical pathological data of patients were collected,and the correlation between MYEOV expression and various parameters of clinicopathological features in TCGA database and tissue microarrays were analyzed.Prognostic data of patients in tissue microarrays were collected,Kaplan-Meier survival curves were plotted according to MYEOV expression,and the prognostic value of MYEOV in pancreatic cancer was explored by univariate and multivariate COX regression analysis.Finally,preoperative peripheral blood routine and biochemical indicators were collected for analysis,and the influence of MYEOV expression on these indicators was explored by grouping them according to MYEOV expression level.Results: MYEOV was identified as a key gene with predictive value for pancreatic cancer prognosis by bioinformatics screening.MYEOV was highly expressed in cancer tissues,and single-cell sequencing showed that MYEOV was mainly expressed in pancreatic cancer cells to perform the corresponding function.High expression of MYEOV was correlated with gender,tumor histological grade,and may be related to tumor size.COX regression analysis showed that high expression of MYEOV was a significant independent predictor of poor prognosis in pancreatic cancer.In addition,high expression of MYEOV was associated with an increase in the percentage of neutrophils and a decrease in the percentage of lymphocytes in the peripheral blood of patients,as well as an increase in the neutrophil-to-lymphocyte ratio.Conclusion: MYEOV is highly expressed in pancreatic cancer compared with adjacent tissues.The expression of MYEOV is correlated with gender,tumor cell differentiation,and tumor size.Its high expression is an independent risk factor for poor prognosis in pancreatic cancer.MYEOV may play a pro-tumor progressive role through neutrophils.Part II: Effects of MYEOV on the biological function of malignant cells in pancreatic cancer cellsObjective: To observe whether altered MYEOV expression can have direct effects on the malignant biological behavior of pancreatic cancer cells.Methods: Western blot and q RT-PCR were performed to detect the baseline expression of MYEOV in pancreatic cancer cell lines and the immortalized normal ductal epithelial cell line HPNE.Stable knockdown and overexpression of MYEOV in pancreatic cancer cell lines were constructed,and transfection efficiency was verified by western blot and q RT-PCR.The effects of MYEOV on the proliferation of pancreatic cancer cells in vitro were observed by performing CCK-8 assay and clone formation assay;the effects of MYEOV on the migration and invasion ability of pancreatic cells were observed by cell scratch assay and transwell invasion assay;the changes of apoptosis rate after knocking down MYEOV were observed by apoptosis assay;the subcutaneous tumorigenesis assay was performed in nude mice to explore the effect of altered MYEOV expression on pancreatic cancer cell growth in vivo.The subcutaneous tumorigenic samples were stained with H&E to observe the microscopic morphology of each group.Using Ki-67 immunohistochemical staining to observe the cell proliferation level,and LY6 G immunohistochemical staining to observe the alteration of tumor-infiltrating neutrophils.Results: MYEOV expression was significantly higher in pancreatic cancer cell lines than in HPNE,and the increased level of MYEOV m RNA was not completely consistent with the protein expression.Stable transfection of pancreatic cancer cell lines with lentiviral-packed sh RNA or overexpression plasmids significantly knocked down or increased the expression of MYEOV.Knockdown of MYEOV expression in pancreatic cancer cell lines significantly reduced cell proliferation,migration and invasion and increased apoptosis,while overexpression of MYEOV significantly enhanced cell proliferation,migration and invasion.Subcutaneous tumorigenesis assay in nude mice showed that the tumorigenic ability of pancreatic cancer cells in mice was reduced after knockdown of MYEOV,while tumorigenic ability was increased after overexpression of MYEOV.H&E staining of tumor tissues showed no significant changes in cell morphology in all groups,while Ki-67 index decreased in the knockdown group and increased in the overexpression group.Immunohistochemical staining for LY6 G showed fewer neutrophils infiltrating the periphery of the control tumor group,while the level of infiltrating neutrophils decreased further in the knockdown group and increased significantly in the overexpression MYEOV group.Conclusion: MYEOV significantly increased the proliferation,migration,invasion ability of pancreatic cancer cells in vitro and tumorigenic ability in vivo,and inhibited cell apoptosis.Altered MYEOV protein expression had no significant effect on the morphology of subcutaneous tumorigenic cells.MYEOV increased the proliferation index of tumorigenic cells in vivo and promoted the recruitment of neutrophils.Part III: Molecular mechanism of MYEOV promotes tumor cell proliferation and recruits neutrophil infiltrationObjective: To explore the molecular mechanisms by which MYEOV promotes tumor cell proliferation and recruits neutrophil infiltration.Methods: Cell membrane,cytoplasmic,and nucleus proteins were extracted,and detected by western blot to determine the subcellular localization of MYEOV proteins.Then laser confocal microscopy was used for validation.MYEOV-interacting proteins were screened and validated by immunoprecipitation and immunofluorescence staining.The altered signaling pathways associated with the interacting protein were analyzed by western blot.In addition,bioinformatics methods were used to investigate the effect of MYEOV on immune cell infiltration in the tumor microenvironment,and the alteration of the immune microenvironment was verified by immunohistochemistry and multiplex immunofluorescence staining.The chemokines causing this alteration were explored based on the GEO database and q RT-PCR,and the venn diagram was drawn to find the key chemokine undergoing alteration.Finally,the alteration of chemokine was verified by ELISA assay and the chemotactic function was validated by chemotaxis assay.Results: Western blot and laser confocal microscopy showed that MYEOV was mainly located in the nucleus,and the protein levels in the nucleus were significantly reduced after knockdown of MYEOV expression.STAT1 was identified as a potential interacting protein of MYEOV,and was validated by co-IP experiment and immunofluorescence staining.Western blot was used to detect the expression and activation of STAT1,which showed that MYEOV changed the phosphorylation level of STAT1 without affecting its upstream pathway,and jointly affected cell proliferation and apoptosis by interacting with STAT3.TIMER2.0 analysis of the tumor immune microenvironment showed a positive correlation between MYEOV and neutrophil infiltration,which was validated by immunohistochemistry and multiplex immunofluorescence staining.Analysis of the GEO dataset and q RT-PCR datas revealed that the downregulation of chemokine CXCL1 may be responsible for this alteration.Finally,it was confirmed by ELISA that MYEOV alteration could affect CXCL1 expression levels and the chemotactic ability towards neutrophils.Conclusion: MYEOV protein is mainly localized in the nucleus and can promote the malignant phenotype of tumors by interacting with STAT1 and inhibiting the activation level of STAT1 protein,subsequently causing alterations in proliferation and apoptosis-related pathways.In addition,MYEOV can also play a pro-cancer role by promoting the expression of chemokine CXCL1 to recruit neutrophils to infiltrate into the tumor microenvironment.