Construction Of Prognostic Model Of Lung Adenocarcinoma Based On Histone Phosphorylation Modified Regulatory Genes

Objective:1.A prognostic model was constructed to distinguish high-risk and low-risk populations and predict the survival time,tumor mutation burden and immune response of lung adenocarcinoma(LUAD)patients.2.Validate the expression of the key gene PBK in the LUAD cell line using CCK-8,scratch healing and Transwell experiments to verify the effect of PBK on the proliferation and migration ability of H1299 cells.Methods:1.Download the transcriptome data and clinical information of LUAD patients from the public data platform of The Cancer Genome Atlas(TCGA)for subsequent analysis;Collected and sorted out histone phosphorylation modification regulatory genes from published literature(see appendix).2.The differentially expressed genes(DEGs)of histone phosphorylation modification regulating genes in normal and tumor tissues were identified by differential analysis.3.Prognosis-related genes were obtained by single-factor Cox analysis.4.Based on prognostication-related genes,the least absolute shrinkage and selection operator(LASSO)was used to analyze and construct the prognosis model,and LUAD patients were divided into two groups according to the best median value: high and low risk.5.Download three LUAD datasets GSE30219(n=85),GSE31210(n=226)and GSE50081(n=127)from the Gene Expression Omnibus(GEO)database of the public data platform to verify the accuracy of the prognosis model.6.Gene ontology(GO)and the Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis were used to study the possible biological functions and signal pathways involved in the differential genes between different risk groups.7.Evaluated the infiltration of immune cells between high and low risk groups through CIBERSORT algorithm.8.Selected the key gene PDZ-binding kinase(PBK)in the model,and verified the expression of PBK in LUAD cell lines A549,NCI-H1975 and NCI-H1299 by Wilcoxon test,PCR and Western Blotting test.9.Cell experiments were conducted to verify the effect of PBK gene knockout on the proliferation and migration of LUAD cell line H1299.Results:1.Based on the differentially expressed genes related to prognosis,the LUAD patients in the TCGA dataset were divided into high and low risk groups according to the best median value using LASSO regression analysis.The survival analysis showed that patients in high-risk group had poor prognosis(P = 0.007).2.Clinical correlation analysis showed that clinical T stage(P < 0.01),N stage(P <0.05)and Stage stage(P < 0.01)were significantly different in different risk groups.Single-factor and multi-factor Cox analysis showed that the risk score of the prognosis model could predict the prognosis of LUAD patients(P < 0.001).3.Three GEO data sets GSE30219,GSE31210 and GSE50081 were used to verify the accuracy of the model.The survival analysis results showed that the survival time of patients in the high-risk group was shorter,which was consistent with the results of the training group.4.GO analysis showed that the biological processes(BP),cellular components(CC)and molecular functions(MF)of differential genes between high and low risk groups were mainly enriched by organelle division,mitosis,chromosome region,spindle,microtubule and tubulin binding.The pathways enriched by KEGG analysis mainly include Cell cycle,Progesterone-mediated oocyte maturation,Oocyte meiosis,Linoleic acid metabolism,Arachidonic acid metabolism,IL-17 signal pathway,p53 signal pathway,etc.5.Tumor mutation burden(TMB)analysis showed that patients in high-risk group had higher TMB and poor prognosis(P = 0.007).6.The analysis of immunocyte infiltration showed that the infiltration of neutrophils and macrophages increased in the high-risk group,and the analysis of immune microenvironment showed that the tumor purity in the high-risk group increased significantly(P=8.8e-05),and was associated with poor prognosis(P=0.005).7.Low-risk people were more likely to receive anti-CTLA4,CD28,CD48 and IDO2 treatment.8.PBK was significantly expressed in tumor tissues of LUAD patients in TCGA dataset,and the expression of high PBK was associated with poor prognosis of LUAD patients(P = 0.003),which was verified in three validation sets GSE13213(P = 0.003),GSE31210(P < 0.001)and GSE72094(P < 0.001).PCR and Western blotting experiments showed that PBK expression increased in LUAD cell line.9.The expression of PBK can affect the proliferation activity,migration and scratch healing ability of H1299 cells.Conclusions:1.The constructed prognosis model can better distinguish high-risk and low-risk populations and predict the prognosis,immune,TMB and immune treatment response of LUAD patients.2.PCR and Western blotting experiments show that PBK expression was increased in LUAD cell line.Cell experiments have shown that PBK can significantly affect the proliferation and migration of H1299 cells.