A Preliminary Exploration Of The Effects Of RASAL3 And GBE1 On The Tumor Microenvironment In Lung Adenocarcinoma

Objective:The tumor immune microenvironment(TME)plays a crucial role in the development and progression of cancer,and is closely associated with clinical outcomes in cancer patients.Tumor immunotherapy mainly relies on the recognition and attack of tumor cells by immune cells in the TME,and its immunological mechanisms are related to CD8+T lymphocytes and tumor-associated macrophages in the immune microenvironment.CD8+ T lymphocytes and tumor-associated macrophages(TAMs)are the main effector cell components in the TME,and they affect the occurrence and development of lung adenocarcinoma(LUAD).The aim of this study is to explore key genes associated with the infiltration of CD8+T lymphocytes and TAMs in LUAD.Methods:In this study,we analyzed lung adenocarcinoma patients’ data from The Cancer Genome Atlas(TCGA)database to screen genes related to clinical prognosis and tumor microenvironment.Kaplan-Meier survival curves and multivariate Cox regression were used to identify genes associated with clinical prognosis.The CIBERSORT algorithm was used to analyze the correlation between the target genes and CD8-positive T lymphocytes and CD 163-positive tumor-associated macrophages infiltration.External validation was performed using lung adenocarcinoma tissue microarrays,the PrognoScan database,and the TIMER database.Furthermore,enrichment analysis was conducted on differentially expressed target genes.Finally,a prediction model was constructed based on the target genes,and the model’s performance was evaluated using ROC and calibration curves.Results:According to the data of 515 LUAD patients from the TCGA database,we extracted expression data of 20,530 genes and analyzed their relationship with prognosis,as well as their correlation with T-stage,N-stage,M-stage,CD8-positive T lymphocytes,and CD 163-positive tumor-associated macrophages.We found that RAS protein activator like 3(RASAL3)was expressed at lower levels in LUAD tissues compared to normal tissues.Patients with high RASAL3 expression had better prognosis,while low RASAL3 expression was associated with later T-stage,M-stage,TNM stage,and worse pathological grade.The mRNA expression levels of CD8 in TCGA-LUAD patients were positively correlated with RASAL3 mRNA expression.CIBERSORT analysis showed that RASAL3 expression was positively correlated with CD8-positive T lymphocyte infiltration.In the TCGA database cohort,the mRNA expression levels of CD8,a marker for CD8-positive T lymphocytes,were positively correlated with RASAL3 mRNA expression.Furthermore,we validated our findings using tissue microarrays,two LUAD datasets(GSE13213 and GSE31210)from the PrognoScan database,and the TIMER database,and the results were consistent with those from the TCGA database and the CIBERSORT algorithm.GSEA analysis of RASAL3 expression in LUAD patients from the TCGA database suggested that abnormal RASAL3 expression may affect lipid metabolism reprogramming.In addition,we found that the expression level of 1,4-alpha-glucan branching enzyme 1(GBE1)in LUAD tissue was significantly higher than that in adjacent normal tissue in the TCGA database.The high expression of GBE1 was associated with poor overall survival(OS)in LUAD.Furthermore,high expression of GBE1 was correlated with later T stage,N stage,M stage,TNM stage,and worse pathological grade in LUAD patients.GBE1 was positively correlated with the infiltration level of CD 163-positive tumor-associated macrophages in LUAD.CIBERSORT analysis showed that the expression of GBE1 was positively correlated with the infiltration of M2 macrophages,CD4-positive memory activated T cells,and resting dendritic cells in LUAD.We validated our findings using the PrognoScan database,TIMER database,and tissue microarray.Analysis of three LUAD cohorts(HARVARD-LC,GSE31210,jacob-00182UM)in the PrognoScan database showed that patients with high expression of GBE1 had worse prognosis.Analysis of the correlation between GBE1 expression and tumor immune cell infiltration levels in TIMER database showed that GBE1 expression was positively correlated with macrophage infiltration in LUAD.GBE1 was also found to be positively correlated with immune marker genes CD163,VSIG4,and MS4A4A of M2 macrophages in LUAD through TIMER database analysis.The expression of CD 163 mRNA in TCGA cohort was also positively correlated with GBE1 mRNA expression.Furthermore,we used tissue microarray-based IHC to confirm the relationship between GBE1 expression and the infiltration of CD 163-positive tumor-associated macrophages.Finally,GSEA analysis of differential expression of GBE1 in lung adenocarcinoma patients in TCGA revealed that abnormal expression of GBE1 may be involved in fatty acid metabolism through the mTORC1 signaling pathway.We further constructed an "improved TNM staging" prediction model by combining the expression of RASAL3 and GBE1 with the traditional TNM staging.The calibration curve showed a good fit between the improved TNM staging and the diagonal line,and the model provided a more accurate prediction of the five-year survival rate.Although the AUC value of the improved TNM staging was higher than that of the traditional TNM staging,the difference was not statistically significant.Conclusion:This study reveals a positive correlation between RASAL3 expression and CD8+T cell infiltration in lung adenocarcinoma,as well as a positive correlation between GBE1 expression and CD163+tumor-associated macrophage infiltration.The expression levels of RASAL3 and GBE1 can be used to predict the prognosis of lung adenocarcinoma,providing new avenues for exploring the mechanisms of tumor immune microenvironment related to CD8+T lymphocytes and CD 163+tumor-associated macrophages in lung adenocarcinoma.Moreover,RASAL3 and GBE1 may serve as prognostic and immune biomarkers for LUAD.