| Objective:Lung cancer is a global health problem,which poses serious challenges to human life safety and public health systems of all countries.Lung adenocarcinoma(LUAD)is one of the most common subtypes of non-small cell lung cancer.In recent years,the rapid development of molecular targeted therapy and immunotherapy has brought hope for the clinical treatment of a series of malignant tumors,but the prognosis of lung adenocarcinoma is still not optimistic.It has been found that Liquid-liquid phase separation(LLPS)is involved in many biological functions,including gene transcription,gene damage repair and gene expression.In addition,many cancer-related proteins are regulated by phase separation,such as RAS,cyclic GMP-AMP synthase(c GAS),and m TOR.Since phase separation closely regulates the function of cancer-related proteins,poorly regulated phase separation may become a novel strategy for cancer treatment.Lactate dedease A(LDHA)in phase separation related genes is responsible for converting pyruvate into lactic acid and NAD +,promoting oxidative metabolism.Studies have found that LDHA is highly expressed in a variety of tumors,such as gastric cancer and breast cancer,and its expression is positively correlated with poor prognosis of patients.However,the relationship between LDHA expression and lung adenocarcinoma as well as the potential mechanism affecting the prognosis of lung adenocarcinoma need further study.Therefore,this study established a prognostic model of lung adenocarcinoma from the perspective of phase separation,mined new prognostic biomarkers of lung adenocarcinoma,explored the potential of LDHA as a prognostic marker of lung adenocarcinoma,and provided a new idea for improving the prognosis of lung adenocarcinoma patients.Methods : Part Ⅰ: Construction of phase separation related prognostic model of lung adenocarcinoma.(1)The Cancer Genome Atlas(TCGA)data set of lung adenocarcinoma was downloaded as a training set to construct a prognostic model.The transcripts of two datasets GSE30219 and GSE50081 in the Gene Expression Omnibus(GEO)database were used as validation sets to test the model,and phase separation-related genes were downloaded from the Pha Sep DB database.(2)"limma" R package was used to select differentially expressed genes by unpaired T-test,and prognostic genes were obtained by univariate Cox regression analysis in "survival" package.Genes retained after intersection were selected as candidate genes.(3)A prognostic model for lung adenocarcinoma phase separation was established by LASSO regression analysis.(4)Lung adenocarcinoma patients were divided into high and low risk groups according to the median risk score.Kaplan-Meier curve and ROC curve were plotted by the "survminer","survival" and "time ROC" R packages to evaluate the effect of this model.(5)Enrichment analysis of differentially expressed genes in two groups of high and low risk was performed using the "cluster Profiler" package to analyze the pathways and functions involved in this model.Part Ⅱ: Analysis of expression,prognosis and potential biological function of LDHA.(1)The expression of LDHA in pancarcinoma and lung adenocarcinoma was analyzed by TIMER2.0 and "limma" R package.(2)The tumor tissues of lung adenocarcinoma patients were collected,and the expressions of LDHA in lung adenocarcinoma tissues and lung adenocarcinoma cell line A549 were detected by quantitative PCR(Polymerase Chain Reaction).(3)"limma" and "ggpubr" R packages were used to analyze the correlation between LDHA expression and clinical features of lung adenocarcinoma patients.(4)Pearson test was used to screen genes related to LDHA expression,and "circlize" and "Corrplot" R packages were used to construct co-expression networks.(5)LUAD patients were divided into high and low expression groups according to the median LDHA expression value,and then enrichment analysis was performed using "Cluster Profiler" and "gsva" R package to explore the potential biological function of LDHA.(6)CIBERSORT was used to calculate the proportion of immune cells in lung adenocarcinoma samples,and "maftools" R package was used to calculate TMB based on genomic mutation data,and then the association between LDHA expression and immune cell infiltration,immune checkpoint and tumor mutation load was analyzed.Results: Part Ⅰ: Construction of phase separation related prognostic model of lung adenocarcinoma.(1)After obtaining 539 lung adenocarcinoma samples and 59 adjacent normal samples as the training set,202 lung adenocarcinoma samples as the verification set,and 5541 phase separation-related genes,35 candidate genes were obtained by extracting the expression matrix of phase separation-related genes,differential expression gene screening and univariate Cox regression analysis.(2)A 13-gene phase separation related prognostic model was constructed by LASSO regression analysis.(3)Kaplan-Meier curve showed that patients with low risk score had longer survival than those with high risk score,and the difference was statistically significant(P = 1.261e-10).The AUC for 1-year,2-year,and 3-year overall survival predicted by phase separation-related prognostic models were 0.730,0.721,and 0.718,respectively.(4)Univariate and multivariate Cox regression analysis demonstrated that the model could be used as an independent prognostic factor to evaluate the overall survival of patients with lung adenocarcinoma(training set: HR = 3.013,95%CI = 2.155-4.212,P < 0.001;Verification set: HR = 4.559,95%CI = 1.811-11.477,P < 0.001).(5)The GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)analyses showed that the model was involved in some key functions and pathways,their genomes being analyzed jointly.Such as ciliary movement,microbundle formation,neuroactive ligand-receptor interaction,etc.Part Ⅱ: Analysis of expression,prognosis and potential biological function of LDHA.(1)LDHA was highly expressed in lung adenocarcinoma,and this finding was confirmed by quantitative PCR in cell lines and lung adenocarcinoma tissues(cell line:P=0.0058;Lung adenocarcinoma tissue: P=0.0047),and the prognosis of lung adenocarcinoma patients with low LDHA expression was better(overall survival:P<0.001;Progression-free survival: P=0.002).(2)There was no correlation between LDHA expression and age and gender,but the expression level of LDHA in stage Ⅰpatients was significantly different from that in other stages(P<0.05).(3)The expression of LDHA was strongly correlated with the expression of ERO1 A,TPI1,PGAM1,PGK1,PKM and GAPDH,and the coexpression cycle of LDHA was plotted.(4)GSEA analysis showed that LDHA was involved in several pathways related to cancer development,such as cell cycle,gene replication,Wnt signaling pathway,interleukin-17 signaling pathway,etc.(5)By analyzing the relationship between LDHA expression and immune cell infiltration,it was found that the expression level of LDHA was negatively correlated with the immune infiltration level of plasma cells and resting mast cells,but positively correlated with the immune infiltration level of activated CD4 memory T cells,resting NK cells,M0 macrophages,activated mast cells and neutrophils.The correlation between the expression of LDHA and the expression of immune checkpoint characteristic genes was evaluated.A positive correlation was found between the expression of LDHA and the expression of CD276(R=0.35,P<0.05).In addition,the association between LDHA expression level and Tumor Mutation Burden(TMB)was very weak(R=0.093,P=0.037).Conclusion: 1.Based on bioinformatics,we constructed a prognosis signature of lung adenocarcinoma composed of 13 genes.Including 7 risk factors(IGF2BP1,LDHA,CDC25 C,DLGAP5,FSCN1,PKP2,PTPRH)and 6 protective factors(ZC3H12D,GPRIN2,KHDRBS2,STK32 A,TTLL2,RPS6KL1),This may provide an accurate prognosis prediction for LUAD patients with good predictive performance.2.LDHA is significantly highly expressed in lung adenocarcinoma and patients with high expression have a poorer prognosis.LDHA expression varies between different stages.LDHA was associated with infiltration of seven immune cell types,and they are more likely to influence patient prognosis through the overall shaping of the tumor immune microenvironment.In the immune checkpoint analysis,there was a positive correlation between the expression of the immune checkpoint CD276 gene and LDHA,and the results of GSEA enrichment analysis indicated that high LDHA expression was associated with multiple pathways affecting tumorigenesis,progression,metastasis,and invasion. |
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