Cuproptosis-Related Genes To Predict Prognosis And Immune Response In Lung Adenocarcinoma

Background and objectivesAt present,lung cancer remains the leading cause of cancer deaths worldwide.Lung adenocarcinoma(LUAD)remains the predominant histological type of lung cancer.Cell death is involved in the tumorigenesis and progression.Exploring the role of cell death-related genes in tumors can provide theoretical basis and new perspectives for finding targets for tumor therapy.Cuproptosis has been implicated as a novel copper ion-dependent programmed cell death type that plays an important function in the regulation of tumor progression and immune microenvironment.However,whether Cuproptosis-related genes(CRGs)are associated with the prognosis of LUAD and their impact on tumor immune environment remains unknown.Therefore,the role CRGs expression in the prognosis of LUAD and the correlation with immune infiltration should been investigated for searching potential biomarkers.This provides a new perspective for prognostic assessment and immunotherapy in LUAD patients.Methods1.We downloaded clinical information and RNA-seq data of LUAD tumor and adjacent tissues from The Cancer Genome Atlas(TCGA).Download RNA microarray data of LUAD tumor and adjacent tissues from the Gene Expression Omnibus(GEO).TCGA and GSE32863 data were merged to compare the differential expression of CRGs in LUAD tumor tissues with adjacent tissues.The CRGs protein-protein interaction network(PPI)was constructed with the STRING database,imported into Cytoscape software and then screened for hub genes.Kaplan-Meier(K-M)and COX univariate analyses were performed in the TCGA-LUAD cohort.LUAD samples were clustered into cuproptosis genes related subgroups A and B based on the expression of CRGs in the TCGA-LUAD cohort.Biological functions and pathways were further enriched for differential genes between the two molecular subgroups,and differences in the abundance of immune cell infiltration between the two groups were compared.2.Kaplan-Meier and COX analyses were performed in the LUAD cohort to screen for for the lipoyltransferase 1(LIPT1),which is associated with prolonged overall survival.Differential expression of LIPT1 in LUAD tumor and adjacent tissues was further analysed by using TCGA and GSE32863 data.Quantitative Real-time Polymerase Chain Reaction(qRT-PCR)experiments were performed to verify the expression of LIPT1 using tissue specimens and cell lines,respectively.The TCGA-LUAD cohort was divided into two groups,high and low LIPT1 expression.Gene set variation analysis(GSVA),Gene Ontology(GO)and the Kyoto encyclopedia of genes and genomes(KEGG)were also applied between the LIPT1 high and low expression groups for biological function and pathway enrichment analysis.Further,based on the expression of LIPT1 gene at the RNA level and protein level,TCGA-LUAD patients and clinical cohorts were classified into high and low expression groups,to investigate the correlation between LIPT1 and clinicopathological factors and prognostic value of LUAD patients.Finally,the relationship between LIPT1 expression and the immune characteristics of LUAD and its predictive value for the efficacy of immunotherapy were evaluated.Results1.A comprehensive literature study progressed to define CRGs,containing a total of 18 genes(NFE2L2,NLRP3,ATP7B,ATP7A,SLC31A1,FDX1,LIAS,LIPT1,DLD,DLAT,PDHA1,PDHB,MTF1,GLS,CDKN2A,DBT,GCSH,DLST).Based on the expression of CRGs at the RNA level,a combination analysis of the TCGA and GSE32863 databases showed that 11 CRGs were differentially expressed between LUAD tumor and adjacent samples.Of these,five were highly expressed in LUAD tumors and six were lowly expressed.A total of 9 CRGs were obtained by screening.KM and COX univariate analysis showed that LIPT1 and NLRP3 had significant predictive value(P=0.027,HR=0.617;P=0.029,HR=0.735)for the prognosis of LUAD patients.2.Based on the expression of CRGs in TCGA-LUAD,the cohort was ultimately divided into A and B subgroups.Biological function and pathway enrichment analyses revealed that CRGs were involved in tumor development and immune-related pathways.Immune cell infiltration abundance was significantly higher in subgroup B compared to subgroup A.K-M and univariate COX analyses in the TCGA-LUAD cohort,based on the expression of CRGs at the RNA level,showed that LIPT1 had the most significant predictive value for the prognosis of LUAD patients.LIPT1 was verified to be significantly less expressed in LUAD tumor tissue compared to adjacent tissue by public databases,LUAD tissue specimens and cell line experiments.Biological functional and pathway enrichment results showed that differential LIPT1 expression was associated with the development of multiple tumors.Analysis of LIPT1 expression in relation to the clinicopathological characteristics of LUAD patients showed that LIPT1 expression correlated with the maximum tumor diameter in LUAD,with smaller tumor diameters in the high expression group.In TCGA-LUAD samples,LIPT1 expression correlated with T-stage and clinical stage,and LUAD patients in the high expression group had earlier T-stage and clinical stage.Survival analysis of the TCGA-LUAD cohort showed that high LIPT1 expression could be considered as a molecular indicator of good prognosis in LUAD(p<0.05).Meanwhile,immune characteristics analysis revealed increased CD8+T lymphocyte infiltration and the expression of Cytotoxic T-lymphocyte-associated protein 4(CTLA-4)in the high LIPT1 expression group.Increased infiltration of CD8+T cells in the high LIPT1 expression group was verified in clinical samples using Immunohistochemistry(IHC).A retrospective analysis of a cohort of 33 LUAD patients receiving immunotherapy revealed that Partial Response(PR)patients had the highest LIPT1 expression,suggesting that LIPT1 is a molecular marker of a good response to immunotherapy.At the same time,the difference in Disease Control Rate(DCR)between the high and low LIPT1 expression groups was statistically significant(P<0.05).The higher expression of LIPT1,the more LUAD patients will benefit from immunotherapy.ConclusionCRGs are involved in the development and immune regulation of LUAD.The cuproptosis-associated gene LIPT1 was closely associated with the development of LUAD,and its expression was significantly reduced in LUAD.High expression of LIPT1 is associated with small tumor diameter,early staging and favorable prognosis.Meanwhile,high LIPT1 expression was related to sensitivity to tumor related immunotherapy.Therefore,LIPT1 could be identified as a potential biomarker to predict the prognosis of LUAD patients and provide new research insights for the immunotherapy in LUAD.