Patients clinical information
The RNA sequencing data and detailed clinical prognostic information resources of 478 COAD samples in the TCGA database were included in the study. All patients were divided into FUNDC1 high expression group 239 cases and low expression group 239 cases. And summarized the patient's age, gender, histological grade, pathological stage (T, N and M), OS time and survival results and other clinical information (Table 1).
Table 1
The corresponding clinical information of patients with different expression levels of FUNDC1 obtained from the TCGA database
|
Characteristic
|
Low expression of FUNDC1
|
High expression of FUNDC1
|
p
|
n
|
239
|
239
|
|
Gender, n (%)
|
|
|
< 0.001
|
Female
|
93 (19.5%)
|
133 (27.8%)
|
|
Male
|
146 (30.5%)
|
106 (22.2%)
|
|
Age, n (%)
|
|
|
0.050
|
<=65
|
86 (18%)
|
108 (22.6%)
|
|
>65
|
153 (32%)
|
131 (27.4%)
|
|
OS event, n (%)
|
|
|
< 0.001
|
Alive
|
189 (39.5%)
|
146 (30.5%)
|
|
Dead
|
50 (10.5%)
|
93 (19.5%)
|
|
Pathologic stage, n (%)
|
|
|
0.023
|
Stage I
|
43 (9.2%)
|
38 (8.1%)
|
|
Stage II
|
96 (20.6%)
|
81 (16.9%)
|
|
Stage III
|
69 (14.8%)
|
74 (15.5%)
|
|
Stage IV
|
29 (6.2%)
|
37 (7.9%)
|
|
T stage, n (%)
|
|
|
0.446
|
T1
|
3 (0.6%)
|
8 (1.7%)
|
|
T2
|
44 (9.2%)
|
39 (8.2%)
|
|
T3
|
160 (33.5%)
|
163 (34.2%)
|
|
T4
|
31 (6.5%)
|
29 (6.1%)
|
|
N stage, n (%)
|
|
|
0.008
|
N0
|
146 (30.5%)
|
128 (26.8%)
|
|
N1
|
48 (10%)
|
60 (12.6%)
|
|
N2
|
45 (9.4%)
|
51 (10.6%)
|
|
M stage, n (%)
|
|
|
0.381
|
M0
|
177 (42.7%)
|
172 (41.4%)
|
|
M1
|
29 (7%)
|
37 (8.9%)
|
|
Expression of FUNDC1 in tumor tissues
Compared with normal tissues in TCGA, the expression of FUNDC1 was significantly increased in COAD samples (Fig 1B, p=1.1e−13). Two data sets (GSE37364, GSE110224) from the GEO database were selected for verification, which also showed that the expression of FUNDC1 was significantly up-regulated in tumor tissues (Fig 2A, B; p=7.5e-05, 5.9e-05). Searching in the HPA database revealed that the FUNDC1 encoded protein FUN14 domain-containing protein 1 was up-regulated in tumor tissues (Fig 2C, D).
The relationship between FUNDC1 high expression and clinical information of COAD patients
When analyzing the correlation between FUNDC1 expression level and clinicopathological parameters in COAD samples (Fig 1), the results showed that FUNDC1 mRNA level was not significantly different with age (p=0.578), gender (p=0.731), and weight (p=0.324). The expression level of FUNDC1 is positively correlated with tumor stage and N stage (p = 1.324e-03, 3.278e-04).
Survival analysis
The KM curve was constructed according to the method mentioned above, and the results showed that the high expression of FUNDC1 in COAD showed a shorter overall survival (OS, p=8.58e-3, Fig3B). The Time ROC curve shows that FUNDC1 has a certain predictive power for the prognosis of COAD patients (5-Years, AUC=0.76, 95%CI (0.695-0.813), Fig3C). In the univariate Cox model, high FUNDC1 expression and high pathological grade and staging (TNM) are both negative predictors of OS in COAD patients. In the multivariate regression analysis, the expression level of FUNDC1 was shown to be related to OS as an independent factor (Fig4).
PPI network construction
The String database was used to construct the PPI network of FUNDC1, and the top 10 proteins that interact with FUNDC1 and their corresponding gene names, annotations and scores were listed (Fig 5A). These proteins include: MAP1LC3B, PGAM5, MAP1LC3A, ULK1, ATG5, GABARAPL1, GABARAPL2, GABARAP, ATG12. MAP1LC3B, Microtubule-associated proteins 1A/1B light chain 3B, is ubiquitin-like modifier that involved in formation of autophagosomal vacuoles. Plays a role in mitophagy which contributes to regulate mitochondrial quantity and quality by eliminating the mitochondria to a basal level to fulfill cellular energy requirements and preventing excess ROS production[24,25]. Figure 5B shows the proteins predicted to interact with FUNDC1 in the GENEMANIA database and the enrichment pathways of related genes.
Screen for co-expressed genes
In order to further study the mechanism of FUNDC1 in COAD, we used the Oncomine database to mine the genes co-expressed with FUNDC1 in COAD samples. FUNDC1 is co-expressed with 104 genes, of which the highest correlation coefficient is KDM6A. The GEPIA database was selected to further analyze the relationship between FUNDC1 and KDM6A (Fig 6). KDM6A encodes lysine-specific demethylase 6A protein, which mainly plays a role in the regulation of gene expression[26].
Immune Infiltration and Correlation Analysis with Immune
We used the TIMER database to analyze the correlation between FUNDC1 expression and six types of infiltrating immune cells (CD8+ T cells, CD4+ T cells, B cells, dendritic cells, macrophages and neutrophils) and tumor purity (Fig 7). The results showed that the level of FUNDC1 was significantly correlated with CD8+ T cells (r=0.191, p=1.04e-04) and macrophages (r=0.155, p=1.82e-03). There was no significant correlation with tumor purity (r=0.007, p=8.38e-01), CD4+ T cells (r=0.011, p=8.29e-01), neutrophils (r=0.028, p=5.71e-01), and dendritic cells (r=0.012, p=8.03e-01). P <0.05 is considered statistically significant. The results of immune correlation analysis using R software showed that FUNDC1 levels were correlated with B cells (p=0.012, Spearman=-0.12), CD4+ T cells (p=0.028, Spearman=-0.10), endothelial cell (p=9.7e-05, Spearman=-0.18), and NK cells (p=0.009, Spearman=-0.12), CD8+ T cells (p=1.9e-04, Spearman=-0.17), macrophages (p=2.36e-05, Spearman=-0.20) are negatively correlated. Among them, it has the strongest correlation with macrophages. P <0.05 is considered statistically significant (Fig 7).