Down-Regulation of ESYT3 mRNA and protein Expression in LUAD tissue Based on TCGA Database
In this study, the expression of ESYT3 mRNA in various cancer types from TCGA was analyzed using TIMER 2.0 database. We found that the expression of ESYT3 mRNA was low in various cancers (Figure 1A), including LUAD. Compared with unmatched adjacent normal tissues, ESYT3 was expressed remarkably lower in LUAD tissues (Figure 1B). These results were confirmed in tumor tissues and matched normal tissue (Figure 1C). Further investigation of the protein expression level of ESYT3 was performed using UALCAN database, and we observed a significant reduction in ESYT3 protein levels in LUAD tissues compared with adjacent normal tissues (Figure 1D).
Correlation analysis between ESYT3 expression and clinical pathological characteristics of LUAD patients via TCGA cohort
As the function of ESYT3 in LUAD is still unclear, we further analyzed ESYT3 expression on the basis of different clinical characteristics in LUAD samples from TCGA. In subgroup analyses based on gender, age, smoking habits, stage, nodal metastasis and TP53 mutation status, ESYT3 expression was significantly lower in LUAD patients than the normal control (Figure 2). Specifically, patients with nodal metastasis had lower ESYT3 mRNA expression than those with non-nodal metastasis (Figure 2E). Furthermore, patients with TP53 mutations have a lower expression of ESYT3 (Figure 2F).
A total of 340 patients with LUAD were divided into low and high ESYT3 groups based on cut-off value. The correlations between ESYT3 expression and clinical characteristics in LUAD were presented in Table 1. Results showed that ESYT3 expression was related to gender (p = 0.009), N classification (P < 0.001), and stage (P = 0.002) (Table 1).
TABLE 1 Correlation of ESYT3 mRNA expression with the clinicopathological characteristics of LUAD cases from TCGA.
Down-Regulation of ESYT3 Expression Was Associated with Poor Prognosis in LUAD
The prognostic value of ESYT3 expression was assessed in LUAD patients using GEPIA, Kaplan-Meier Plotter and UALCAN databases. These results showed that low levels of ESYT3 mRNA expression were significantly related to poor OS among LUAD patients in GEPIA (Log rank p = 1.3E-05, HR = 0.51; Fig. 3A), Kaplan-Meier Plotter (Log rank p = 0.00013, HR = 0.56; Fig. 3B) and UALCAN (p= 0.0095; Fig. 3C).
Going further, we conducted univariate and multivariate Cox regression analyses. According to univariate analysis, T, N, M, stage and low ESYT3 mRNA level were associated with a worse overall survival (Table 2). In multivariate cox analysis, only T and N were associated with poor OS (Table 2).
Table 2 Univariate Analysis and Multivariate Analysis of OS in 340 patients with LUAD
Functional enrichment analysis of co-expressed genes of ESYT3.
In order to further investigate the molecular mechanism of ESYT3 in LUAD, we applied the c-BioPortal web server to identify top 300 co-expressed genes with ESYT3 in three different studies from TCGA (TCGA, Firehose Legacy; TCGA, Nature 2014; TCGA, PanCancer Atlas). A total of 236 ESYT3 co-expressed genes were identified in three TCGA studies, as shown in Figure 4A. To further explore enrichment function of co-expressed genes of ESYT3, 236 ESYT3 co-expressed genes were selected through Metascape to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. In Figure 4B, co-expressed genes of ESYT3 were highly enriched in cell division, spindle elongation, FOXM1 pathway and G2/M Transition. As shown in Figures 4C and 4D, ESYT3 enrichment terms were displayed by cluster ID and p-value. Furthermore, the top six co-expressed genes of ESYT3 arranged by the values of Spearman’s rank correlation coefficient were identified. In a correlation analysis, ESYT3 exhibited a strong positive correlation with Surfactant Associated 3 (SFTA3), BTB Domain Containing 9 (BTBD9), Hepatic Leukemia Factor (HLF), NK2 Homeobox 1 (NKX2-1), Cyclin Dependent Kinase Like 2 (CDKL2), and Adhesion G Protein-Coupled Receptor F5 (ADGRF5) (Figure 5A-F). Overall survival analysis further confirmed that deregulation of expression levels of SFTA3, BTBD9, HLF, NKX2-1, CDKL2, and ADGRF5 were associated with favorable prognosis of LUAD (Figure 5G-L).
ESYT3 Expression Was Associated with Tumor-Infiltrating Immune Cells in LUAD
In LUAD, TIMER2 database was used to assess the relationship between ESYT3 expression and immune infiltration. There was a positive correlation between ESYT3 expression and CD4+ T cell (r=0.134, P=72.81e-03), B cell (r=0.214, P=1.66e-06), NK cell (r=0.316, P=7.41e-13), neutrophil (r=0.257, P=17.38e-09), and monocyte (r=0.242, P=5.65e-08) (Figure 6). Additionally, a number of immune marker genes corresponding to different tumor-infiltrating immune cells (CD8+ T cells, T cells, B cells, monocytes, mast cells, neutrophils, NK cells, Th1, Th2, Tfh, Th17) were also investigated by TIMER and ESYT3 was found to be associated with most of these marker genes (Table 3). Generally, ESYT3 expression were positively correlated with the majority of gene markers of different functional Tumor-Infiltrating Immune cells, which suggested that ESYT3 plays critical roles in immune microenvironment in LUAD. Then, we analyzed the correlation between ESYT3 expression and immune checkpoint-related genes and the results showed ESYT3 expression was significantly negatively correlated with PDCD1 and LAG3. These results suggested that patients with high levels of ESYT3 expression may benefit from immunotherapy
Analysis of ESYT3 methylation in LUAD
A hypermethylated DNA promoter inhibits gene expression while a hypomethylated promoter activates gene expression [10]. To examine whether ESYT3 expression might be influenced by DNA methylation states in LUAD, we used MethSurv tool to visualize the correlation between gene expression and DNA methylation. Using MethSurv tool, ESYT3 DNA methylation sites and the prognostic values of each CpG obtained from TCGA database were analyzed. Differentially methylated CpG sites of ESYT3 gene were identified as shown in Figure 7A. In survival analysis, we found that hypermethylation of cg15579376, cg16802792, cg04810656 and cg01905210 in the ESYT3 gene promoter was associated with poor survival (Figure 7B). Figure 7C shows that the methylation level of ESYT3 was higher in tumor tissues compared with paired normal tissues.
Table 3 Correlations Between ESYT3 and Biomakers of Immune Cells