Prognostic Significance And Immune Cell Infiltration Associate To LILRB1 Expression In Glioma

Research BackgroundGlioma is the most common primary malignant tumor of the central nervous system.Gliomas are highly malignant and aggressive.Despite aggressive surgery,temozolomide chemotherapy,electric field therapy and radiotherapy,the average survival period is still very short and the prognosis is poor.According to the latest World Health Organization(WHO)Guidelines for the Classification of Central Nervous System Tumors,Version 5(2021),tumor types are determined by histological,biological and molecular pathological characteristics.Gliomas are classified as glioblastoma,IDH wild-type,astrocytoma,IDH mutant,and oligodendroglioma with IDH mutation accompanied by 1p/19q combined deletion.Therefore,there is an urgent need to explore new effective molecular mechanisms for the pathogenesis of glioma,in order to better explore effective treatment methods to treat glioma.Leukocyte immunoglobulin like receptor subfamily B1(LILRB1)is a transmembrane glycoprotein and a key receptor for human leukocyte antigen G.LILRB1 is an immunosuppressive receptor that is expressed on several different types of human immune cells,including dendritic cells,monocytes,T cells,B cells,NK cell subsets,and macrophages.LILRB1 inhibits the immune system by binding to molecules of the typical and atypical human major histocompatibility complex class I.LILRB1 has been proved to play a crucial role in promoting tumor development and metastasis,such as in breast cancer,gastric cancer and pancreatic cancer.However,little is known about the possible biological role of LILRB1 in gliomas.To achieve this goal,we used bioinformatics to explore the potential biological role of LILRB1 in glioma,and validated the prognostic relationship and potential biological role of LILRB1 in glioma through in vitro experiments.Research Purposes1.To explore the correlation between LILRB1 expression levels and clinical characteristics and prognosis in patients with glioma.2.To elucidate the relationship between the expression level of LILRB1 and DNA methylation,tumor mutation burden and microsatellite instability,immune infiltrating cells,M2 macrophages and immune checkpoints in glioma3.To verify the effects of LILRB1 on the malignant phenotype of glioma cells.Research MethodThis study used data from the UCSCXENA database,the Cancer Genome Atlas(TCGA)and the Genotype Tissue Expression(GTEx)project.After classification and collation based on WHO CNS5 in 2021,R software was used to explore the correlation between the expression level of LILRB1 and the clinicopathological characteristics and prognosis of glioma patients.We conducted genetic difference analysis on the collected data and used ggplot2 R package to draw heat maps and volcanic map.KEGG-GO analysis was performed using a clustering analysis software package.Using R packets to perform gene set enrichment analysis on glioma related data from the TCGA database,using STRING database for protein protein interaction analysis and using MEXPRESS database for DNA methylation analysis.The TCGA database was used for tumor mutation burden and microsatellite instability analysis and the TIMER and TIMER2.0databases were used to determine the correlation between the expression of LILRB1and tumor immune infiltrating cells in glioma patients.We collected 38 tumor and normal tissue samples from the neurosurgery department of the Second Affiliated Hospital of Guangzhou Medical University to conduct immunohistochemical experiments.We measured the expression of LILRB1 using Image-Pro Plus and explored the correlation between the expression level of LILRB1 and the clinicopathological characteristics and prognosis of glioma patients.Proteins extracted from tumor and adjacent tissue samples from 24 patients were selected for protein western blot experiments.The expression of LILRB1 and GAPDH were measured using Image J software and the ratio of LILRB1 and GAPDH expression was used to draw a matching map.We followed up patients by phone and conducted statistical analysis based on the follow-up results,and drawn a survival curve.HMC cells transfected with lentivirus were co-cultured with U251 and U87 cells.Cell proliferation was detected by CCK-8 assay,and the ability to invade and migrate was detected by Transwell assay.SPSS25 software was used for statistical analysis.T-test or Wilcoxon rank sum test were used to compare the two groups and one-way or two-way ANOVA tests were used to evaluate multiple comparisons between the two groups.Using R software version 3.6.3 for evaluation and visualization of the analysis process,a P value<0.05 is considered to have a statistical significance.Research resultsBioinformatics analysis showed that the expression of LILRB1 in glioma tissue was higher than that in normal brain tissue and the expression of LILRB1 in WHO grade 4 glioma was higher than that in WHO grade 2 and WHO grade 3.Glioma patients with high expression of LILRB1 had a poor survival prognosis.GO and KEGG enrichment analysis showed that neutrophil activation in BP,the secretory granular membrane in CC,receptor ligand activity in MF,the chemokine signaling pathway and NOD-like receptor signaling pathway in KEGG were positively correlated with LILRB1.Gene set enrichment analysis showed that LILRB1 was positively correlated with JAK/STAT signaling pathway,NOD-like receptor signaling pathway,chemokine signaling pathway,Tol-like receptor signaling pathway and B-cell receptor signaling pathway.High expression of LILRB1 was positively correlated with hypomethylation.LILRB1 combined with tumor mutation burden(TMB)and microsatellite instability(MSI)may be an indicator of the effectiveness of immunotherapy in patients with glioma.TIMER2.0 analysis showed that the high expression of LILRB1 was positively correlated with the infiltration level of B cells,CD4~+T cells,M2 macrophages,neutrophils and myeloid dendritic cells.High expression of LILRB1 is positively correlated with increased expression levels of immune checkpoint molecules and M2macrophages markers.Univariate regression analysis showed OS(HR=1.042,95%CI=1.031,1.053,P<0.001),DSS(HR=1.044,95%CI=1.032,1.055,P<0.001)and PFI(HR=1.040,95%CI=1.029,1.050,P<0.001);Multivariate Cox regression analysis showed OS(HR=1.021,95%CI=1.007,1.035,P=0.003),DSS(HR=1.022,95%CI=1.008,1.037,P=0.002),and PFI(HR=1.022,95%CI=1.009,1.035,P<0.001).Univariate and multivariate Cox regression analysis confirmed that overexpression of LILRE1 was an independent factor for glioma.From our experimental analysis of 38 clinical samples,it can be found that the expression of LILRB1 in glioma tissue was higher than that in normal brain tissue.The expression of LILRB1 in WHO grade 4 glioma was higher than that in WHO grade 2 and WHO grade3.Glioma patients with high expression of LILRB1 have a poor survival prognosis.Finally,through cell experiments,it was found that knockdown of LILRB1 can inhibit the proliferation,migration,and invasion of glioma cells.Research ConclusionPatients with glioma with high expression of LILRB1 have a poor prognosis.LILRB1 is an oncogene of gliomas and an independent pathogenic factor for glioma.