mRNA Expression level of Eph/Ephrins in LUAD based on different databases
First, the Oncomine database was used to analyze mRNA expression levels of Eph/Ephrins in LUAD (Fig. 1A). The following threshold were used to analyze the data: 2-fold change, P value < 0.0001, and a gene grade of 10%. In most datasets, EPHA1, EPHA10, EFNA3, EFNA4, EPHB1, EPHB2, EPHB3, and EFNB3 were up-regulated in LUAD tissue compared with normal lung tissue. EPHA2, EPHB4, EPHB6, and EFNB1 were down-regulated in LUAD tissue. In order to further evaluate Eph/Ephrins expression, The Cancer Genome Atlas (TCGA) was used.
Since there were fewer normal samples in the TCGA dataset, the GTEx dataset based on GEPIA website was included for further analysis of the differential expression of Eph/Ephrins between normal and LUAD tissue (Fig. 1B). As showed in Fig. 1C, the results were consistent with data from the Oncomine database and TCGA database. The data is as follows: EPHA10, EFNA3, EFNA4, EFNA5, EPHB1, and EPHB2 expression levels were significantly upregulated in LUAD. In addition, EFNB1, EFNB2, and EPHB6 expression levels were significantly downregulated in LUAD. Based on these results, EPHA10, EFNA3, EFNA4, EFNA5, EPHB1, EPHB2, EFNB1, EFNB2, EPHB6 were used for the next analysis.
EFNA3 acts as the most valuable prognostic biomarker in LUAD patients
To determine the prognostic value of the selected Eph/Ephrins in LUAD patients, the Kaplan–Meier plotter database was used to analyze the relationship between expression levels and OS or PFS (Fig. 2 and Fig. 3). First, the relationship between OS was analyzed. In Figure.2A-I, upregulation of EFNA3, EFNB2, EFNB1, and EPHB6 expression showed significant correlation with poor OS in LUAD patients. In contrast, EFNA5 and EFNB2 upregulation signified a better prognosis. Conversely, EPHA10 and EFNA4 expression did not show significant correlation with OS, so were exclude from this study.
Next, EFNA3, EFNB2, EFNB1, EPHB6, EFNA5, and EFNB2 were selected for correlation of the relationship between expression levels and PFS. As shown in Fig. 3A-H, upregulation of EFNA3, EFNB1, and EPHB2 expression showed significant correlation with poor PFS in LUAD patients. In contrast, EFNB2 upregulation signified a better prognosis. Similarly, EPHA10, EFNA5, and EPHB6 expression did not show a significant correlation with PFS.
EFNA3 was selected next since it showed the higher HR value both in prognostic analysis for OS and PFS in LUAD patients. Patients with different stage cancers require different therapeutic strategies, and have different prognoses, therefore subgroup analysis was applied[19]. As shown in Fig. 3A-C, a higher EFNA3 expression was associated with significantly worse OS, regardless if the patient was diagnosed at stage 1 (Fig,3A), stage 2 (Fig,3B) or stage 3 (Fig,3C). Smoking was also an important risk factor for LUAD. EFNA3 expression showed prognostic value in smoking LUAD patients (Fig. 3D), but not in patients with no smoking history (Fig. 3E).
Genetic variations of EFNA3
To determine if upregulation of EFNA3 in LUAD tissues were caused by genetic variations, genetic variations of EFNA3 were assessed using the cBioPortal database. This database contains information of 1272 samples from five studies (Broad, Cell 2012; MSKCC, Science 2015; OncoSG, Nat Genet 2020; TCGA, Firehose Legacy; TSP, Nature 2008). Genetic variations of EFNA3 showed incidence rates of 12.98% in TCGA, 7.65% in Broad, and 7.28% in OncoSG (Fig. 5). Amplification was the most common type (67/67 in TCGA; 1/14 in Broad; and 7/7 in OncoSG). Based on these results, amplification might be one of the main mechanisms by which EFNA3 is highly expressed in LUAD.
The expression of the EFNA3 protein was increased in LUAD tissues and related to prognosis of LUAD patients
To explore the clinical significance of the EFNA3 protein in LUAD patients, IHC to investigate the expression of EFNA3 in a tissue microarray (TMA) containing 74 LUAD tissues and adjacent normal lung tissues. Compared with normal lung tissue, the expression of the EFNA3 protein was significantly increased in LUAD tissue samples (Fig. 6A). Further analysis revealed that the IHC score for EFNA3 was significantly upregulated in cases of larger tumor sizes (Fig. 6B), lymph node metastasis (Fig. 6C), and advanced TNM stage (Fig. 6D).
LUAD patients were divided into two groups: EFNA3-high group and EFNA3-low group according to IHC scores. Chi-square test showed that EFNA3 protein expression were significantly correlated with larger tumor size (P = 0.004), lymph node metastasis (P = 0.035), and a higher TNM stage (P = 0.002) (Table 1). Moreover, EFNA3-negative patients presented with a shorter overall survival time than EFNA3-positive patients (P = 0.039) (Fig. 6E). Finally, cox regression analysis of overall survival showed that higher EFNA3 expression is the independent risk prognosis factor (HR = 3.108; 95% CI = 1.077–8.963; P = 0.036) (Table.2). Based on these results, EFNA3 could represent a new prognostic biomarker for LUAD.
Table 1
Correlation between the expression of EFNA3 and clinical characteristics in LUAD patients (n = 74).
Clinical Pathological | Number | EFNA3 expression | P value |
Parameters | | High(n = 38) | Low(n = 36) | |
Age | | | | 0.367 |
< 60 | 29 | 13 | 16 | |
≥ 60 | 45 | 25 | 20 | |
Sex | | | | 0.902 |
Male | 48 | 27 | 39 | |
Female | 26 | 11 | 15 | |
Tumor size | | | | 0.004* |
T1 | 15 | 5 | 10 | |
T2 | 43 | 19 | 24 | |
T3 + T4 | 16 | 14 | 2 | |
LN metastasis | | | | 0.035* |
No | 51 | 22 | 29 | |
Yes | 23 | 16 | 7 | |
TNM stage | | | | 0.002* |
Ⅰ | 31 | 10 | 21 | |
Ⅱ | 35 | 20 | 15 | |
Ⅲ+Ⅳ | 8 | 8 | 0 | |
*Significant correlation. |
Table 2
Cox regression analysis of overall survival in LUAD patients.
Variables | Univariate analysis | Multivariate analysis |
HR | 95% CI | P value | HR | 95% CI | P value |
Age (years) (≤ 60 vs > 60) | 1.180 | 0.529–2.629 | 0.686 | | | |
Gender (male vs female) | 0.949 | 0.419–2.150 | 0.900 | | | |
pT stage | 1.489 | 0.898–2.469 | 0.123 | | | |
pN stage | 3.162 | 1.435–6.965 | 0.004* | 1.336 | 0.479–3.725 | 0.580 |
pTNM stage | 2.583 | 1.505–4.432 | < 0.001* | 1.530 | 0.738–3.173 | 0.253 |
EFNA3 expression | 4.272 | 1.597–11.423 | 0.004* | 3.108 | 1.077–8.963 | 0.036* |
Factors for which P < 0.05 in univariate analysis were subsequently used for multivariate analysis. |
In the purple module genes, nuclear division, synaptic function, and ion channel activity-related pathways were the most frequently noted pathways in the GO analysis. The most enriched GO terms in the Biological Process (BP) category was “meiotic cell cycle.” In the Cellular Component (CC) category, “chromatoid body” was most enriched and in the Molecular Function (MF) category “ion gated channel activity” was most abundant. Furthermore, KEGG pathway enrichment analysis results contained "neuroactive ligand-receptor interaction," and "calcium signaling pathway" enrichments (Fig. 8B). Finally, GSEA was used to identify the mechanism and functional differences between the EFNA3-high expression group and the EFNA3-low expression group. As showed in Fig. 8C, "cholesterol homeostasis," "DNA repair," "glycolysis," and “oxidative phosphorylation" was enriched in the EFNA3-high expression group, whereas, "IL2-STAT5 signaling" was enriched in the EFNA3-low expression group.