| Objective:Bioinformatics methods were used to analyze the gene chip data of esophageal squamous cell carcinoma to find the markers BUB1B,UBE2C and NDC80 that are related to prognosis.And then immunohistochemical methods were used to analyze the expression of BUB1B,UBE2C and NDC80 in ESCC,and to explore the relationship between the expression of BUB1B,UBE2C and NDC80 in ESCC and clinicopathological parameters.Methods:1.Use the GEO2R online tool to analyze the differentially expressed genes of esophageal squamous cell carcinoma and normal tissues in the datasets GSE20347,GSE38129,GSE77861,use the Venn tool to draw the intersection plot of the three datasets,and use the DAVID database to compare the differentially expressed genes(DEGs)GO analysis and KEGG pathway analysis were performed.Then,a PPI network was constructed for the differentially expressed genes through the STRING database.Finally,the top 10 hub genes were screened by Cytoscape,and the target gene-miRNA network and gene transcription factor regulatory network were constructed using the Cytargetlinker plug-in of Cytoscape.Then,we used GEPIA 2 to analyze the expression of Hub gene in esophageal cancer online and the correlation of BUB1B,UBE2C,and NDC80.Finally,we analyzed the overall survival and recurrence-free survival of Hub gene in the Kaplan-Meier Plotter database.2.Collect and randomly select paraffin tissue specimens diagnosed with esophageal squamous cell carcinoma and undergo surgical resection in the Affiliated Hospital of North Sichuan Medical College from May 2020 to May 2021.The expression of BUB 1B,UBE2C and NDC80 protein was detected by immunohistochemical methods.The comparison of the expression of the above proteins between the experimental group and the control group and the relationship between the expression and the clinicopathological characteristics were performed using the χ2 test and the continuously corrected χ2 test.Correlation analysis of BUB1B,UBE2C and NDC80 proteins was performed using Spearman rank correlation analysis.All statistical data were analyzed with SPSS 25.0 software,and P<0.05 indicated that the difference was statistically significant.Results:1.A total of 471 differentially expressed genes were identified from the three datasets.Furthermore,we build a PPI network with 254 nodes and 1094 edges.Functional enrichment analysis showed that differentially expressed genes clustered in DNA replication,cell division,cell cycle,and ECM-receptor interactions.Then through the CytoHubba plugin in Cytoscape software,we screened out 10 Hub genes,including AURKB,TTK,AURKA,UBE2C,BUB1,NDC80,CDC45,TPX2,TOP2A,BUB1B.An important module was found from the protein interaction network of DEGs using the MCODE plug-in.The enrichment analysis of this module indicated that this module is related to cell cycle,DNA replication,and cell cycle pathways,and is involved in cell division,DNA replication,and chromosome segregation.and other biological processes.The target gene-miRNA network and gene transcription factor regulatory network were constructed by Cytargetlinker plug-in.Through the GEPIA 2 database,we verified that the TOP 10 Hub genes are highly expressed in esophageal cancer,and there may be correlations between BUB1B,UBE2C,and NDC80.Through the Kaplan-Meier Plotter database,we found that the expression of AURKA,BUB1,BUB IB,CDC45,TOP2A,TPX2,TTK genes was closely related to the overall survival(OS)of esophageal squamous cell carcinoma(P<0.05).The expression of BUB 1,BUB1B,NDC80,TOP2A,TPX2,TTK and UBE2C genes was closely related to recurrence-free survival(RFS)of esophageal squamous cell carcinoma(P<0.05).2.It was found by immunohistochemistry that there were differences in the expression of BUB1B protein in esophageal squamous cell carcinoma and paracancerous tissues(P<0.05),which were 58.93%(33/56)and 9.09%(2/22)respectively;UBE2C protein was expressed in esophageal squamous cell carcinoma and paracancerous tissues There were differences in the expression of NDC80 protein in esophageal squamous cell carcinoma and adjacent tissues(P<0.05),respectively,which were 73.21%(41/56)and 68.18%(15/22),respectively17.86%(10/56),0.00%(0/22).3.It was found by immunohistochemistry that in esophageal squamous cell carcinoma,BUB1B protein was correlated with tumor T stage(P>0.05),but not with gender,age,tumor N stage,degree of differentiation,vascular and nerve invasion(P>0.05)There was no correlation between UBE2C protein and patients’ gender,age,tumor TN stage,degree of differentiation,vascular and nerve invasion(P>0.05).NDC80 protein was correlated with tumor nerve invasion(P>0.05),but not with patient gender,age,tumor T stage,N stage,degree of differentiation,,vascular invasion or not(P>0.05).4.It was found by immunohistochemistry that the expression of UBE2C protein and BUB 1B protein in esophageal squamous cell carcinoma was positively correlated(r=0.315,P<0.05),while UBE2C and NDC80 protein(r=0.071,P>0.05)and BUB 1B and NDC80 protein(r=0.105,P>0.05)had no correlation.Conclusions:1.Using GEO database,the Top 10 Hub genes screened by bioinformatics analysis are AURKB,TTK,AURKA,UBE2C,BUB1,NDC80,CDC45,TPX2,TOP2A,BUB 1B.Among them,BUB 1B,UBE2C and NDC80 are involved in the regulation of cell cycle,and the three may also be related in expression.In addition,BUB1B,UBE2C and NDC80 are closely related to the poor prognosis of esophageal squamous cell carcinoma,and are expected to be markers for esophageal squamous cell carcinoma screening,treatment and prognosis evaluation.2.Immunohistochemical results showed that BUB 1B and UBE2C were highly expressed in esophageal squamous cell carcinoma tissues,and BUB1B protein was correlated with tumor T stage.In addition,NDC80 protein was correlated with nerve invasion of tumor(P<0.05),but its expression difference in esophageal squamous cell carcinoma tissues and adjacent tissues was not statistically significant.3.Immunohistochemical results showed that BUB 1B was positively correlated with UBE2C expression in esophageal squamous cell carcinoma. |
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