| BackgroundLung cancer is one of the tumors with the highest morbidity and mortality in the world.Lung adenocarcinoma is the most common subtype of lung cancer,accounting for more than 50%.The clinical symptoms of lung adenocarcinoma are not obvious,and most of them are diagnosed in the middle and advanced stage,often accompanied by distant metastasis,and the prognosis of patients is poor.In recent years,the treatment methods and means of lung adenocarcinoma have been continuously improved,but the clinical survival of patients is still facing great challenges.Therefore,it is very important to establish a lung adenocarcinoma biological model to evaluate the prognosis of patients and optimize clinical medication,and to find and identify pathogenic genes related to the tumor microenvironment.ObjectiveThis study used the lung adenocarcinoma dataset from The Cancer Genome Atlas(TCGA)and GEO(Gene Expression Omnibus)to perform bioinformatics analysis,validate the screened genes by in vitro cellular assays,and then construct a clinical prognostic model for lung adenocarcinoma and investigate the role of CCT3(chaperonin containing TCP1 subunit 3),a molecular chaperone protein,in shaping the tumor microenvironment of lung adenocarcinoma.Methods(1)535 lung adenocarcinoma cases and 59 control samples were selected from the TCGA database,and the combined GSE30219 dataset(containing data from 307 lung cancer cases)was used to screen for immune-related genes(IRGs)and validated using the combined GSE68465,GSE101929,GSE37745 and GSE83845 datasets.A risk model based on IRGs was developed for risk assessment of lung adenocarcinoma patients.Gene set enrichment analysis(GSEA)and gene set variation analysis(GSVA)were used to explore the biological process(BP)and signaling pathway(SP)associated with the risk model.The m RNA expression levels of IRGs were detected by reverse transcription real-time fluorescence quantitative PCR(RT-q PCR)(2)The expression of CCT3 in lung adenocarcinoma was analyzed using the UALCAN database,Human Protein Atlas(HPA)and TCGA data.Wilcoxon rank sum test and regression model were used to analyze the relationship between clinicopathological characteristics and CCT3 expression in lung adenocarcinoma patients.Cox regression model,Kaplan-Meier method and Nomogram prediction model were used to assess the predictive value of CCT3 on the prognosis of lung adenocarcinoma patients.Secondly,we searched for genes associated with CCT3 Hub by Gene MANIA and String databases and determined the immune cell correlation of CCT3 with infiltration by single sample gene set enrichment analysis(ss GSEA).Gene set enrichment analysis(GSEA)was performed to explore CCT3-related signaling pathways.Finally,the role of CCT3 on proliferation and apoptosis of lung adenocarcinoma A549 cells was verified by knocking down CCT3 expression by small interfering RNA(si RNA).Results(1)Two immune-related genes highly associated with plasma cells,namely purinergic receptor P2RX1 and Purkinje cell protein 4(PCP4),were screened.RT-q PCR results showed that P2RX1 and PCP4 were highly expressed in plasmacytoma cells RPMI-8226,while no expression was seen in lung adenocarcinoma cells A549,H3255 and HCC827.A lung adenocarcinoma risk model based on P2RX1 and PCP4 had a risk score that was negatively correlated with immune cell infiltration of lung adenocarcinoma memory B cells(P < 0.05),plasma cells(P < 0.001),CD8+ T cells(P < 0.05)and activated CD4+ memory T cells(P < 0.05).Kaplan-Meier analysis showed that the high-risk group had significantly lower survival than the low-risk group(P = 0.004 for the TCGA training set;P = 0.023 for the GEO validation set).Moreover,the response rate to PD-L1/PD-1 blockade therapy was significantly lower in the high-risk group than in the low-risk group(P=0.012).The results of GSVA and GSEA-GO analysis showed that Toll-like receptor(TLR),T-cell receptor(TCR),Nod-like receptor(NLR),JAK-STAT pathway,Hedgehog,Gn RH(Gonadotropin-releasing hormone),FcεRI,chemokines,calcium receptors,B-cell receptors and other signaling pathways were enriched in the low-risk rated group,in contrast,higher risk scores were associated with signaling pathways such as Wnt pathway,PPAR(peroxisome proliferator-activated receptor),P53,m TOR pathway,insulin and Erb B pathway.(2)Bioinformatics analysis showed that CCT3 was significantly upregulated in both m RNA and protein levels in lung adenocarcinoma tissues(both P < 0.001).CCT3 overexpression correlated with clinicopathological characteristics such as gender,smoking,T-stage,N-stage and poor prognosis in lung adenocarcinoma patients.gene MANIA and String databases identified a set of genes associated with CCT3 that genes are associated with the maintenance of cytoskeletal microfilaments,DNA repair and the assembly and stabilization of methylation-related proteins.ss GSEA analysis revealed that CCT3 was positively correlated with infiltrating Th2 cells in the tumor microenvironment(r = 0.442,p < 0.01)and negatively correlated with mast cells(r =-0.49,p < 0.01)and immature dendritic cells(r =-0.401,p < 0.001)were negatively correlated.Functional analysis of pathways based on GSEA approach showed that cell cycle,protein export,proteasome and ribosome pathways were enriched in the CCT3 high expression group,while JAK/STAT,B-cell receptor pathway(BCR),T-cell receptor(BCR)and Toll-like receptor(TLR)pathways were enriched in the CCT3 low expression group.In addition,CCT3 knockdown significantly inhibited the proliferation of A549 cells and promoted apoptosis.Conclusions(1)The immune-related model constructed based on P2RX1 and PCP4 can be used to predict the prognosis of lung adenocarcinoma and the efficacy of PD-L1/PD-1blockade therapy.(2)CCT3 is a regulator of the formation of immunosuppressive tumor microenvironment,and its abnormal expression is closely related to the clinicopathological features of lung adenocarcinoma and the genetic and immunological features of tumor microenvironment. |
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