| Objective:1)Jointly analyze the bioinformatics database,extractmRNA sequencing information based on transcriptome sequencing,compare the expression of genes in cervical squamous cell carcinoma tissue and normal cervical tissue,screen out differentially expressed genes,and explore through relevant bioinformatics analysis Differential gene enrichment molecular signaling pathways and their relationship with prognosis;2)Further explore the clinical information and gene expression information of the TCGA database,and explore the biological significance of kinesin superfamily protein 14(KIF14)in cervical squamous cell carcinoma;3)To verify the expression of KIF14 in cervical squamous cell carcinoma samples through in vitro tissue and cell experiments,to explore the effect of KIF14 on the proliferation and migration of cervical squamous cell carcinoma cells and to explore its mechanism of action.Methods:1)Analyze four GEO databases and TCGA databases to screen differentially expressed genes in cervical squamous cell carcinoma,and conduct differential gene pathway enrichment analysis through Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)to establish protein-The protein interaction(PPI)network is used to study the protein interactions between differentially expressed genes,and the core differentially expressed genes are identified through Cytoscape software,and Combined analysis of differentially expressedmRNA and differentially expressed lncRNA and miRNA to construct a ceRNA network;The differentially expressed genes are subjected to KM survival analysis to evaluate the significant impact of differential genes on the prognosis of cervical squamous cell carcinoma;2)Compare the expression of KIF14 in the TCGA database with the clinicopathological characteristics of cervical squamous cell carcinoma or analyze the correlation,explore the influence of KIF14 expression on the clinical characteristics of tumor patients;use ROC curve to evaluate the prediction of KIF14mRNA on the 1-year survival of cervical squamous cell carcinoma patients Performance,calculate the best cut-off value of risk score,and divide patients into KIF14mRNA high expression group and low expression group.Kaplan-Meier survival curve method is used to evaluate the clinical characteristics of different patients and the clinical characteristics of patients with high and low KIF14mRNA expression.For survival,log-rank test is used to determine whether there is a difference in survival between high and low KIF14mRNA expression groups;COX regression analysis is used for univariate and multivariate analysis to further clarify the relationship between KIF14mRNA expression and the clinical prognosis of patients,using GSEA Perform gene set enrichment analysis on the obtained expression profile data,and comprehensively explore the biological processes involved in KIF14;3)Detection of KIF14 Gene expression in cervical squamous cell carcinoma tissues to verify the results of database analysis;inhibit the expression of KIF14 by transiently transfecting interference sequences,and detect the expression of KIF14 by qRT-PCR,Western blot,CCK-8,Transwell experiments,etc.The effect on the biological behaviors of cervical cancer cell line Hela and cervical squamous cell carcinoma cell line siHa,such as proliferation,migration and invasion,and its mechanism of action is preliminarily explored by detecting the expression of cell cycle-related proteins CDK1,cyclinB1 and P21.Results:1)By analyzing the two databases of GEO and TCGA,71 up-regulated genes,20 down-regulated genes,a total of 91 differential genes,as well as 238 differential lncRNAs and 139 miRNAs are obtained.The results of functional enrichment analysis showed that the differential genes were mainly enriched in the cell proliferation,biological death,morbidity or mortality of tumor cell lines.The PPI network analysis of differentially expressed genes revealed that there were 1778 interactions among the proteins encoded by 79 differentially expressed genes.Through the analysis of the Starbase database,there is an interaction relationship between 20mRNA,51 lncRNA and 17 miRNAs.Kaplan-Meier analysis showed that 10 genes with significant prognosis were significantly related to the difference in overall survival of patients with cervical squamous cell carcinoma,and the difference in KIF14 was the most obvious;2)The statistical analysis of clinical features and the difference in KIF14mRNA expression showed that the expression of KIF14mRNA was related to the KIF14mRNA expression in different grades,tumor size<3cm and≥3cm,during M0 and M1,between one-year survival and death groups,and during different clinical stages.The median of is positively correlated.Kaplan-Meier analysis showed that the difference in survival curves between N stage and clinical stage was statistically significant,which was positively correlated with patient death.COX regression analysis showed that KIF14mRNA≥3.100,tumor size≥3cm,G2 and G1 grades were independent risk factors for death in patients with cervical squamous cell carcinoma.GSEA results show that the signal pathways enriched in tumor samples with high expression of KIF14 mainly involve cell adhesion molecules,cell cycle,oxidative phosphorylation,P53 signaling pathways,cancer signaling pathways,primary immunodeficiency,ribosomes,and TGF-β signaling pathways;3)The results of qRT-PCR experiments showed that in 20 cases of cervical squamous cell carcinoma and 10 cases of normal cervical tissue,the expression of KIF14 in cervical squamous cell carcinoma tissue was significantly higher than that in normal cervical tissue.CCK8 and Transwell experiments show that when the expression of KIF14 is disturbed,the cell proliferation,migration and invasion ability are significantly inhibited.The results of Western Blot experiments showed that in Hela and siHa cells,the expression of CDK1 and cyclinB1 were significantly reduced after the siKIF14-2 interference sequence,and the P21 protein was significantly increased in Hela cells,but there was no significant difference in siHa cells.Conclusion:1)The key genes in cervical squamous cell carcinoma were screened by bioinformatics methods,and it was found that KIF14 can be used as a prognostic molecular marker for cervical squamous cell carcinoma;2)The analysis of clinical data in the TCGA database shows that KIF14 is an independent risk factor for the clinical prognosis of cervical squamous cell carcinoma,suggesting that KIF14 can be used as a target for potential prognostic diagnosis;3)KIF14 may affect the invasion,migration and proliferation of cells by participating in the process of regulating the cell cycle,and its detailed mechanism still needs further study. |
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