Integrin Beta 5 Is A Prognostic Biomarker And Potential Therapeutic Target In Glioblastoma

Objective:Glioblastoma(GBM)is the most lethal cancer of the central nervous system.Integrin beta5(ITGβ5)is thought to be involved in intercellular signal transduction and regulation of tumor initiation and progression.However,the function of ITGβ5 in GBM is not known.Elevated ITGβ5 expression was highly correlated with glioma malignant progression and a mesenchymal subtype and poor survival in GBM patients.Further research verified that ITGβ5 participates in the immune response and angiogenesis in the GBM microenvironment.Furthermore,it will indicate that ITGβ5 can serve as a predictive biomarker for GBM patient survival and is a potential therapeutic target in GBM treatmentMethods:In this study,we analyzed the expression of genes encoding integrin family members in glioma using CGGA and TCGA RNAseq datasets and found that ITGβ5 showed the greatest difference in expression between LGG and GBM.We investigated the ITGβ5 copy number variation(CNV)frequency by GISTIC2.0 analysis in glioma using TCGA RNAseq and TCGA 4502A microarray GBM datasets We compiled a list of EV-related genes from intercellular and secretory vesicle genes,and used these gene sets for principal component analysis(PCA)with CGGA and TCGA RNAseq datasets.We examined the correlation between extracellular vesicle-related genes and tumor purity as well as immune and stromal scores in CGGA RNAseq and TCGA RNAseq and TCGA 4502A microarray GBM datasets in order to determine the relationship between ITGβ5 and the above three factorsWe analyzed ITGβ5 expression in different glioma grades with CGGA and TCGA RNAseq datasets.We performed western blot and immunohistochemical experiments using clinical specimens to detect the expression difference of ITGβ5 in different glioma grades.We analyzed ITGβ5 expression differences in different GBM subtypes using CGGA RNAseq and TCGA RNAseq and TCGA 4502A microarray GBM datasets.Moreover,according to data from the Ivy GBM database,we analyzed ITGβ5 expression differences in areas of microvascular proliferation relative to other regions of the tumor.We analyzed ITGβ5 expression differences in isocitrate dehydrogenase(IDH)1-wild-type as compared to IDH1-mutant GBM,and we explored whether ITGβ5 is differentially expressed between GBM patients with and those without 06-methylguanine DNA methyltransferase promoter methylationWe used CGGA RNAseq and TCGA RNAseq datasets and TCGA 4502A mRNA microarray GBM datasets to evaluate survival distributions using Kaplan-Meier curves,and then used log-rank tests to assess differences between stratified groups,which were used to verify the correlation between ITGβ5 expression and survival of glioma patients.We used 53 clinical GBM specimens to further verify that the high ITGβ5 expression can affect the GBM patients survival through immunohistochemical experiments.To assess the correlation between ITGβ5 expression and the clinical characteristics in GBM,we performed univariate and multivariate Cox regression analyses with the clinical characteristics of age,IDH1 mutation status,radiotherapy,and chemotherapy as variables by using CGGA RNAseq and TCGA RNAseq GBM datasetsWe compiled a list of 1,043 genes whose expression was correlated with a high ITGβ5 expression level based on CGGA and TCGA RNAseq GBM datasets.The gene ontology analysis(GO)was used to study the function of ITGβ5 in GBM.Then,we performed gene enrichment analysis(GSEA),principal component analysis(PCA),and gene set variation analysis(GSVA)to study the function of ITGβ5 and the signalling involved in GBM.To examine the functions of ITGβ5 in GBM in greater detail,we used two ITGβ5-specific siRNAs to knock down ITGβ5 expression in a stable glioma cell line(LN229)and a primary glioma cell line derived from a clinical GBM specimen(PGC1228).The effect of ITGβ5 on GBM migration and invasion was studied by Transwell experiment,the effect of ITGβ5 on GBM proliferation was measured by MTS proliferation experiment,the effect of ITGβ5 on endothelial cell tube formation ability was studied by angiogenesis experiment.GSEA revealed that EMT signaling was increased in association with high ITGβ5 expression by using CGGA and TCGA RNAseq datasets.Consistent with this observation,western blot experiments were performed using GBM specimens to verify the effect of ITGβ5 on the expression of EMT-related markers.We evaluated the association between ITGβ5 and the immune and stromal cell populations in the GBM microenvironment with the MCP-counter method by using CGGA and TCGA RNAseq GBM datasetsResults:we analyzed the expression of genes encoding integrin family members in glioma using CGGA and TCGA RNAseq datasets and found that ITGβ5 showed the greatest difference in expression between LGG and GBM,then we investigated the ITGβ5 copy number variation(CNV)frequency in glioma,and these data indicated that no mutations were present in ITGβ5 in GBM.We compiled a list of EV-related genes from intercellular and secretory vesicle gene sets,and a principal component analysis(PCA)revealed that these genes can distinguish GBM from LGG.We examined the correlation between extracellular vesicle-related genes and tumor purity as well as immune and stromal scores in CGGA and TCGA RNAseq GBM datasets and determined that ITGβ5 is the only gene that is significantly correlated with all three factors along with unfavorable survival of GBM patientsWe analyzed ITGβ5 expression in different glioma grades,and we found that ITGβ5 levels increased with tumor grades.Western blot and immunohistochemical analyses of clinical specimens showed that ITGβ5 was overexpressed in GBM as compared to non-tumor and grades Ⅱ and Ⅲ glioma tissue.We then analyzed ITGβ5 expression in different GBM subtypes using CGGA and TCGA RNAseq GBM datasets and found that ITGβ5 was more closely associated with the mesenchymal phenotype in GBM.Moreover,according to data from the Ivy GBM database,ITGβ5 was overexpressed in areas of microvascular proliferation relative to other regions of the tumor.Also,ITGβ5 was overexpressed in isocitrate dehydrogenase(IDH)1-wild-type as compared to IDH1-mutant GBM by using CGGA,TCGA RNAseq GBM datasets and TCGA 4502A mRNA microarray GBM datasets However,there was no significant difference in ITGβ5 expression between GBM patients with and those without O6-methyl guanine DNA methyl transferase promoter methylation using TCGA RNAseq and TCGA 4502A mRNA microarray GBM datasets.We examined the correlation between ITGβ5 expression and the survival of glioma patients using CGGA,TCGA RNAseq datasets and TCGA 4502A mRNA microarray GBM datasets.Elevated ITGβ5 expression was associated with an unfavorable outcome for glioma patients.We also analyzed the relationship between ITGβ5 expression and the survival of 53 GBM patients and determined that patients with higher ITGβ5 levels had poorer outcomes compared to those with a lower ITGβ5 expression level.Furthermore,among GBM cases in CGGA,TCGA RNAseq and TCGA 4502A mRNA microarray that underwent radiotherapy and chemotherapy,those with higher ITGβ5 expression had a significantly shorter survival time than those with lower expression.We performed univariate and multivariate Cox regression analyses with the clinical characteristics of age,IDH1 mutation status,radiotherapy,and chemotherapy as variables.The results showed that ITGβ5 was an independent prognostic factor for overall survival in GBMWe used databases to show that ITGβ5 participates in the immune response and angiogenesis in the GBM microenvironment through gene ontology analysis(GO),gene over-enrichment analysis(GSEA),principal component analysis(PCA),and gene set variation analysis(GSVA).To examine the functions of ITGβ5 in GBM in greater detail,we used two ITGβ5-specific siRNAs to knock down ITGβ5 expression in GBM.Researches showed that GBM cell migration and invasion as well as the tube formation capacity of endothelial cells induced with glioma cell-conditioned medium were inhibited,however,knockdown ITGβ5 expression did not significantly change the proliferation capacity of GBM cell.The expression of matrix metalloproteinase(MMP)2 and MMP9 was also attenuated by ITGβ5 knockdown.In addition,GSEA revealed that EMT signaling was increased in association with high ITGβ5 expression,and the results showed that knockdown ITGβ5 can down-regulate the expression of EMT-related markers.We evaluated the association between ITGβ5 and the immune and stromal cell populations in the GBM microenvironment with the MCP-counter method by using CGGA and TCGA RNAseq GBM datasets.The results showed that CD8+T cell,NK cell,fibroblast,B cell,monocyte,and myeloid dendritic cell numbers were positively associated with ITGβ5 expression,providing further evidence for the regulatory role of ITGβ5 in the tumor microenvironment Conclusions:In summary,our study shows that a high expression level of ITGβ5 is an indicator of progressive malignancy in glioma and predicts unfavorable outcome in GBM patients.ITGβ5 has a strong ability to predict mesenchymal phenotype in GBM,and can be used as a diagnostic marker for this subtype.ITGβ5 not only influences the migration and invasion of glioma cells,but is also involved in regulating the immune response and angiogenesis in the tumor microenvironment.Thus,ITGβ5 is a useful prognostic biomarker and potential therapeutic target in GBM.