| Object: Quantitative analysis of N-glycosylation expression levels in cancer tissues and adjacent tissues of 5 cervical cancer patients with HPV infection,and screen out the significant difference factor ITGA3 and ITGB1,the factor most correlated with the prognosis of cervical cancer patients.To analyze and verify its correlation with the clinicopathological prognosis and immune infiltration of cervical cancer patients,and to provide a theoretical basis for finding valuable potential new therapeutic targets for cervical cancer.Methods:1、5 pairs of cancer tissues and adjacent tissues from cervical cancer patients with HPV infection were sent to BGI for quantitative N-glycosylation analysis(item number: F21FTSNWKF6133),and the return data were analyzed to look for the proteins corresponding to the modified peptides with significant differences in N-glycosylation in the metabolic pathways associated with HPV infection.2、The Kaplan-Meier Plotter,GEPIA 2 and c Bio Portal databases were used to screen the significant proteins,and the proteins ITA3,ITB1,and their corresponding genes,ITGA3 and ITGB1,were found for analysis and study.3、The HPA database was used to analyze the expression of ITGA3 and ITGB1 in cervical cancer.Immunohistochemical(IHC)method was used to detect the expression level of ITGA3 and ITGB1 in cancer cells in cervical cancer tissue(CCCCs)of 120 cases and epithelial cells of normal cervical tissue(ECNCs)of 25 cases who underwent total hysterectomy for benign uterine diseases.Correlation between ITGA 3 and ITGB 1 was analyzed.4、The clinicopathological data of 120 patients with cervical cancer,including age,pathological type,FIGO stage,differentiation,tumor diameter,deep stromal invasion(DSI),lymph node metastasis(LNM),and lymph ovascular space infiltration(LVSI),were collected.The correlation of the expression level of ITGA 3 and ITGB 1 in CCCCs with the clinicopathological characteristics of the patients was analyzed.5、120 patients with cervical cancer were followed up,and,progression-free survival(PFS)and overall survival(OS)were calculated.COX regression analysis and Kaplan-Meier survival curves was used to analyze the correlation between the expression levels of ITGA3 and ITGB1 and the prognosis of cervical cancer patients.The expanded sample size was further verified with data from the TCGA-CESC dataset.6、The Receiver Operating Characteristic Curve(ROC curve)of ITGA3 and ITGB1 was drawn based on TCGA＿GTEx-CESC dataset.Time-dependent ROC curves and prognosis prediction model were drawn based on data from TCGA-CESC dataset.The prognostic model was visualized using the nomogram diagram and its correction curve(Calibration plot)and the clinical decision curve(Decision Curve Analysis,DCA)were drawn.7 、The Linked Omics database was detected the related genes expressed in ITGA3 and ITGB1 in cervical cancer and the related KEGG pathway and GO enrichment analysis.The Gene MANIA database and STRING database was constructed the relevant action network of ITGA3 and ITGB1 related top 50 genes.8、The TIMER,TISIDB and Kaplan-Meier Plotter databases were used to analyze the correlation between the expression level of ITGA3 and ITGB1 and the level of tumor infiltrating immune cells(TIICs)in cervical cancer.Results:1 、 Results of N-glycosylation analysis: 173 N-glycosylation modified peptides with significant differences were identified in 5 pairs of quantitative N-glycosylation analysis of cervical cancer and adjacent tissues,of which 139 were significantly up-regulated and 34 were significantly down-regulated.The GO and KEGG databases were used to enrich the protein sequence corresponding to the identified modified peptide segment and analyze 26 Pathway metabolic pathways and GO entries with significantly enriched differential proteins.2 、Screen of significant difference factors: The metabolic pathways related to HPV infection was analysised.The factors corresponding to the 10 N-glycosylation modifications enriched in this pathway,such as ATP6AP1,EGFR,TNC,ITGA3,LAMA2,FRRS1,ITGAV,ITGB1,TENM4,LAMA4,was analyzed in Kaplan-Meier Plotter database and GEPIA 2 database.The factor ITGA3,which was significantly related to the prognosis of cervical cancer patients and was highly expressed in cervical cancer tissue,was screened as the factor for our research group.Because ITGA3 and ITGB1 were interacting heterodimers in the pathway,and their correlation was strong(Pearson: r=0.66,P<0.05),they were included in the study at the same time.3、Analysis of the expression level of ITGA3 and ITGB1 in cervical cancer tissues and normal tissues:According to the HPA database,the expression level of ITGA3 and ITGB1 in cervical cancer tissues was significantly higher than that in normal tissues.In addition,the immunohistochemical results showed that75 cases(62.50%)of 120 cervical cancer tissues had high expression of ITGA3,85 cases(70.83%)of ITGB1,and 25 normal cervical tissues had low expression of ITGA3 and ITGB1.The expression rate of ITGA3 and ITGB1 in cervical cancer tissue was significantly higher than that in normal tissue,with significant statistical difference(both P<0.001).There was a significant correlation between the expression levels of ITGA3 and ITGB1 in cervical cancer(Spearman: r=0.878,P<0.001).4、Correlation analysis between the expression level of ITGA3 and ITGB1 and the clinicopathological characteristics of cervical cancer patients: The expression level of ITGA3 and ITGB1 in cervical cancer cells was significantly different from FIGO stage,pathological grade and DSI(both P<0.05);The tumor diameter and the expression level of LNM and ITGA3 were significantly different(both P<0.05).However,LVSI,age and tumor pathological type were not significantly different from the expression level of ITGA3 and ITGB1(both P>0.05).5 、 Correlation analysis between the expression level of ITGA3 and ITGB1 and the prognosis of cervical cancer patients: The Kaplan-Meier survival curve showed that the expression level of ITGA3 and ITGB1 was significantly correlated with the prognosis of cervical cancer patients(both P<0.05).The higher the expression level of ITGA3 and ITGB1 was,the shorter the OS and PFS of cervical cancer patients would be.Univariate and multivariate COX regression analysis showed that clinical stage,the expression level of ITGA3 and ITGB1 were independent risk factors for poor prognosis of cervical cancer patients.Based on this,the prognosis prediction model was constructed to predict the prognosis of cervical cancer patients,and the nomogram diagram was used to visualize the prediction model.6、Correlation analysis between the expression level of ITGA3 and ITGB1 and the level of TIICs in cervical cancer: KEGG and GO enrichment analysis suggested that the expression level of ITGA3 and ITGB1 was related to the immune-related pathways such as leukocyte migration through endothelial cells,antigen processing and presentation,NK cell-mediated cytotoxicity,etc.In TIMER and TISIDB databases,it was found that the expression level of ITGA3 and ITGB1 in cervical cancer was related to the level of B cells(P<0.05),and the expression level of ITGA3 was related to the level of neutrophils(both P<0.05).Conclusion:1 、 There were significant differences in the expression of N-glycosylation in cervical cancer and adjacent tissues.2 、 The expression levels of ITGA3 and ITGB1 were correlated with some clinicopathologic characteristics and prognosis of cervical cancer patients.3、ITGA3 and ITGB1 may participate in the regulation of immune response of cervical cancer,and immune infiltration may be one of the reasons for poor prognosis of cervical cancer patients with high expression of ITGA3 and ITGB1. |
PDF Full Text Request