| Objective:Malignant melanoma remains a significant contributor to skin cancer mortality.The infiltration depth of melanoma cells is a crucial predictor of patient prognosis,and integrin dysregulation has been implicated in this process.we used public gene data to perform prognosis analyses on integrin-associated genes and develop a model to predict clinical outcomes.Additionally,we conducted validation studies and functionally explored the expression and immune infiltration of ITGAL in clinical tissue samples.We further investigated the relationship between ITGAL expression,immune infiltration,clinical prognosis,and specific T cell subsets in melanoma tissue.Methods:1.Obtain gene expression data and clinical data for melanoma from The Cancer Genome Atlas(TCGA)and gene expression data for normal human tissues from Genotype-Tissue Expression(GTEx).2.Use the Wilcoxon test to filter the TCGA melanoma dataset and GTEX data,obtain differentially expressed genes using R software(limma package),intersect with integrin genes,and perform survival analysis and univariate Cox regression analysis again.3.Perform enrichment analysis and functional analysis(Gene Ontology,GO)and pathway analysis(Kyoto Encyclopedia of Genes and Genomes,KEGG)on the obtained integrin gene set.4.Build a prognostic risk model using multiple Cox regression analysis and Lasso regression analysis.5.Use Kaplan-Meier survival analysis and time-dependent ROC curve to validate the prognostic model.6.Use real-time quantitative PCR,protein blotting experiments,and immunofluorescence staining techniques to study the relationship between the expression of key genes ITGAL in melanoma,immune infiltration,and specific types of T cells predicted by the model.Results:1.A total of 6462 differentially expressed genes were identified,including 2541 upregulated genes and 3921 downregulated genes,meeting the criteria of p<0.05 and |log2 FC)|>1.0.2.A total of 12 integrin-related genes that were differentially expressed and correlated with prognosis were screened out(meeting the criteria of Kaplan-Meier survival analysis p<0.05 and univariate Cox regression analysis hazard ratio HR>1 or<1 and p<0.05).High expression of ITGAL,ITGB3,and ITGB2 was associated with better survival prognosis,while high expression of ITGB6 and ITGB4 was associated with poorer survival prognosis.3.Functional enrichment analysis was performed on these 12 differentially expressed integrin-related genes.The results of GO analysis showed that these genes were mainly enriched in cell adhesion.The results of KEGG analysis showed that these genes were mainly enriched in the regulation of actin cytoskeleton pathway,which could maintain the homeostasis of actin cytoskeleton and respond to extracellular stimuli.4.A prognostic risk model composed of two integrin-related genes was obtained by screening(with the minimum AIC value),and the risk score was calculated.Further analysis showed that the high-risk group had significantly poorer prognosis than the low-risk group(p<0.05).Moreover,ROC curve analysis showed that the model had high accuracy and reliability.5.The ITGAL gene was highly expressed in clinical tissues of melanoma in multiple samples,but only in the early stage of melanoma,while the expression of ITGAL did not increase significantly with the development and metastasis of the tumor.Therefore,this can explain why the high expression of ITGAL is associated with a better prognosis.6.ITGAL was significantly correlated with most immune cells(3A),including CD8+T cells(rho=0.322),CD4+T cells(rho=0.487),B cells(rho=0.218),monocytes(rho=0.791),neutrophils(rho=0.666),regulatory T cells(rho=0.38),NK cells(rho=0.77),and dendritic cells(rho=0.695).These results suggest that ITGAL plays an important role in immune infiltration in melanoma.7.Clinical tissue samples from multiple immune fluorescence staining were divided into ITGAL high expression group and ITGAL low expression group based on the MEDIAN expression level of ITGAL.Compared with the ITGAL low expression group,CD4+T cell and CD8+T cell infiltration was significantly increased in the ITGAL high expression group(P<0.Conclusion:The integrin-related prognostic risk model constructed in this study can be used to predict and evaluate the prognosis of melanoma patients.The high expression of the key gene ITGAL in the model is significantly positively correlated with CD4+T cell and CD8+T cell infiltration in melanoma.The high expression of ITGAL is involved in the T cell receptor signaling pathway and plays an important role in the occurrence and progression of melanoma... |
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