| BackgroundEsophageal squamous cell carcinoma(ESCC)is one of the most common malignant tumors with high incidence and mortality rates,and low five-year survival rate.The prognosis of ESCC is generally poor,and there is a lack of existing biomarkers for diagnosis and prognosis.Surgery remains the best treatment option for this type of cancer,and clinical therapy is particularly important for the treatment of this disease.However,the molecular mechanisms underlying the development of esophageal squamous cell carcinoma are still poorly understood.Better understanding of the molecular mechanisms underlying the development of ESCC is crucial for the clinical diagnosis and treatment of patients with this disease.Apelin is a regulatory peptide and ligand of the APJ receptor,which belongs to the G protein-coupled receptor family.It was first isolated from the bovine stomach.The apelin precursor protein is cleaved into multiple subtypes,including Apelin-12,Apelin-13,Apelin-17,and Apelin-36.Among these subtypes,Apelin-13 has the most biological activity and is the most commonly used.Apelin plays an important physiological and pathological role in our body by binding to its receptor APJ.Studies have shown that endogenous Apelin and its receptor APJ are overexpressed in tumor tissues,and endogenous Apelin promotes tumor development.Its overexpression in tumor tissues can be used to evaluate the marker of tumor cancer.The Apelin system is an endogenous physiological regulator and is becoming a potential therapeutic target for many diseases.According to the TCGA database,Apelin is upregulated in esophageal squamous cell carcinoma tissues.However,the mechanism by which Apelin is involved in the development of ESCC is still unclear.Our study aims to explore whether Apelin can serve as a new prognostic biomarker for ESCC and the mechanism by which Apelin affects the development of ESCC through the ERK signaling pathway.ObjectiveThis project aims to investigate whether Apelin can serve as a novel prognostic biomarker for esophageal squamous cell carcinoma through esophageal squamous cell carcinoma-related bioinformatics analysis,ECA109 cells,and studies of esophageal squamous cell carcinoma and adjacent samples.Additionally,we aim to investigate the mechanism by which Apelin affects the development of esophageal squamous cell carcinoma through the ERK signaling pathway.Methods1..Analyze the expression of Apelin between cancerous and normal samples using the GEPIA and TIMER databases.2.Conduct survival analysis of various cancers using the Kaplan-Meier(K-M)database and TIMER database.3.Establish a survival rate risk model using the TIMER database and analyze the correlation between Apelin expression and MMP9 and PCNA protein expression.4.Analyze the expression level of Apelin in clinical samples using the UALCAN database.5.Conduct functional enrichment analysis of Apelin-related genes using the Linked Omics database,KEGG,and GO analysis.6.Construct a protein-protein interaction network related to Apelin using the STRING database and conduct KEGG enrichment analysis of the main signaling pathways involved in Apelin.7.Collect clinical samples of esophageal squamous cell carcinoma and adjacent cancer tissue,and detect the expression of Apelin,MMP9,and PCNA proteins using Western Blot,q RT-PCR,and immunohistochemistry.8.Transfect p CMV-Apelin plasmid into ECA109 cells and extract RNA and protein 24 hours later to detect the expression levels of Apelin,MMP9,and PCNA proteins using Western Blot and q RT-PCR.9.Use si RNA to knock down the Apelin gene in ECA109 cells and extract total RNA and protein48 hours later to detect the expression levels of Apelin,MMP9,and PCNA proteins using Western Blot and q RT-PCR.10.Use 0.1 μmol/L Apelin-13 inducer to stimulate ECA109 cells and use scratch assays,Transwell invasion assays,and colony formation assays to detect the migration,invasion,and proliferation ability of ECA109 cells.11.ECA109 cells were stimulated with Apelin-13 inducer for a short period,and protein extraction was performed to detect the expression of p-ERK1/2.Prior to stimulation with Apelin-13 inducer,ECA109 cells were pretreated with PD98059,and protein extraction was performed to detect the expression of Apelin-induced MMP9 and PCNA proteins using Western Blot.12.Stimulate ECA109 cells with the inducer Apelin-13 for a short period of time,and extract protein to detect the expression of p-ERK1/2.Prior to stimulating ECA109 cells with the Apelin-13 inducer,pre-treat the cells with PD98059,extract protein,and use Western Blot to detect the expression of Apelin-induced MMP9 and PCNA proteins.Results1.The GEPIA and TIMER databases show increased expression of Apelin in various tumors such as COAD,ESCC,GBM,HNSC,KIRC,LIHC,READ,and STAD.The Kaplan-Meier(K-M)database and TIMER database indicate that high Apelin expression is associated with poor prognosis in CESC,ESA,ESCC,KICH,KIRP,LGG,LIHC,MESO,SARC,SKCM,and UVM tumors.2.The UALCAN database shows that Apelin expression is significantly higher in esophageal squamous cell carcinoma than in the normal control group,based on analysis of various clinical pathological parameters such as age,gender,race,disease stage,esophageal cancer subtype,TP53 mutation,smoking habit,and lymph node metastasis.Apelin expression is higher in cancer tissue than in adjacent tissue.Multivariate Cox proportional hazards analysis shows that Apelin acts as an independent prognostic factor in esophageal squamous cell carcinoma in the presence of various infiltrating immune cells.3.The Linked Omics database was used to analyze co-expressed genes with Apelin in esophageal squamous cell carcinoma.The GO functional enrichment results in GSEA show that genes co-expressed with Apelin are mainly involved in collagen metabolism,mitochondrial gene expression,extracellular structural organization,angiogenesis,protein translation,cell aggregation,and endocrine processes.The KEGG pathway enrichment results in GSEA show that genes co-expressed with Apelin are mainly concentrated in signaling pathways such as protein hydrolysis,DNA replication,focal adhesion formation,ribosomes,and IL-17,while signaling pathways such as insulin and physiological cycle movement are inhibited.The correlation between Apelin and PCNA and MMP9 proteins was also analyzed and validated using the TIMER database and esophageal squamous cell carcinoma patient tissue samples.4.In ECA109 cells,transfection with p CMV-Apelin plasmid was performed,and total RNA and protein were extracted 24 h later.Western Blot and q RT-PCR results show that overexpression of Apelin increases MMP9 and PCNA expression in ECA109 cells.5.In ECA109 cells,Apelin gene was knocked down using si RNA,and total RNA and protein were extracted 48 h later.Western Blot and q RT-PCR results show that knocking down Apelin decreases MMP9 and PCNA expression in ECA109 cells.6.ECA109 cells were stimulated with 0.1 μmol/L Apelin-13 inducer,and scratch test,Transwell experiment,and colony formation experiment results show that the proliferative,migratory,and invasive abilities of ECA109 cells were enhanced after addition of Apelin-13 inducer..7.The STRING database was used to construct a protein interaction network,and KEGG pathway enrichment analysis was performed on the proteins that interact closely with Apelin.The results show that Apelin is mainly involved in the Apelin/APJ signaling pathway.Treatment of ECA109 cells with the ERK phosphorylation inhibitor PD98059 significantly inhibits the increase in MMP9 and PCNA protein expression induced by Apelin.Conclusion1.In esophageal squamous cell carcinoma,the expression of Apelin is increased compared to the normal control group.High expression of Apelin is associated with poor prognosis in esophageal squamous cell carcinoma patients,and Apelin can serve as an independent prognostic biomarker for esophageal squamous cell carcinoma.2.Apelin can regulate the expression of MMP9 and PCNA by participating in the ERK signaling pathway,thereby promoting the proliferation,migration,and invasion of esophageal squamous cell carcinoma cells. |
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