The expression of TC2N is elevated in gastric cancer
Firstly, we analyzed TC2N transcription levels in multiple GC studies from TCGA. Data in the UALCAN database revealed that the mRNA expression of TC2N was significantly higher in GC tissues than in normal tissues (P ˂0.001) (Fig. 1A). Further sub-group analysis of multiple clinic pathological features of GC samples in the TCGA consistently showed that the transcription levels of TC2N were significantly higher in GC tissue than normal tissue in subgroup analyses based on gender, age, ethnicity, disease stages, tumor grade, TP53 mutation and H.pylori infection (Fig. 1B-H). The mRNA level of TC2N in GC tissues and normal gastric tissues from the GEPIA database also showed an increase in gastric cancers compared with normal gastric tissues (Fig. 2A). Next, RT-qPCR was conducted on RNA samples derived from several gastric cancer cell lines and normal gastric epithelium cell line GES1. When compared with GES1, it was found that TC2N mRNA expressions were higher in AGS, MKN28 and HGC27 (Fig. 2B) and the tendency of protein level tested by western blot was in accordance with the RNA level (Fig. 2C). To further confirm the TC2N expression in gastric cancer samples, TC2N was detected by RT-qPCR and WB in 12 pairs of gastric cancer tissues and adjacent normal gastric mucosa. We found that both the mRNA and protein levels of TC2N were significantly elevated in gastric cancer tissues in comparison with adjacent normal gastric tissues (Fig. 2D, E). Thus, TC2N expression may serve as a potential diagnostic indicator in GC.
Relationships between the expression of TC2N and clinicopathological features in gastric cancer
To explore the associations between TC2N expression and GC clinicopathologic characteristics, firstly, we performed IHC to detect TC2N expression in the GC tissue array, which contained 55 cases of GC tissue and paired adjacent normal tissue from GC patients. As shown in Fig. 3, TC2N protein was distributed both in the nucleus and cytoplasm (Fig. 3A, B). Meanwhile, we found that the protein expression of TC2N was significantly higher in primary GC compared with adjacent normal tissues (Fig. 3C). Next, the expression of TC2N protein in 232 primary gastric cancer tissue samples was also detected by IHC, 55 of which was collected from the same cases with tissue array mentioned above. The patients included 159 males and 73 females, ranging in age from 27 to 83 years (mean 58.6 years). Patients were followed up for 1 to 84 months, with a median follow-up time of 51.2 months. As shown in Fig. 3D, there were 71 cases (30.6%) with IHC score 1, 78 cases (33.6%) with IHC score 2, 44 cases (19.0%) with IHC score 3, and 39 cases (16.8%) with negative staining (IHC score 0). Negative and weak staining were defined as low TC2N expression (47.4%, 110/117), whereas moderate and strong staining were defined as high TC2N expression (52.6%,122/117). The association between TC2N expression and clinicopathological features of this cohort was further evaluated. As shown in Table 1, High TC2N expression was associated with poorly differentiated histological classification(P < 0.001), large tumor size(P < 0.05), advanced distant metastasis(P < 0.05) and tumor-node-metastasis (TNM) stage(P < 0.05).
Table 1
Relationships between TC2N expression and clinical pathological parameters in gastric cancer
Parameters | Cases number | TC2N expression | P value |
Low | High |
Gender | | | | 0.862 |
Male | 159 (68.5%) | 76 (69.1%) | 83 (68.0%) | |
Female | 73 (31.5%) | 34 (30.9%) | 39 (32.0%) | |
Age | | | | 0.926 |
< 50 | 47 (20.3%) | 22 (20.0%) | 25 (20.5%) | |
≥ 50 | 185 (79.7%) | 88 (80.0%) | 97 (79.5%) | |
Histological classification | | | | < 0.001 |
Well and moderately differentiated | 79 (34.1%) | 54 (49.1%) | 25 (20.5%) | |
Poorly differentiated | 153 (65.9%) | 56 (50.9%) | 97 (79.5%) | |
Tumor size | | | | 0.014 |
≤ 4 cm | 109 (47.0%) | 61 (55.5%) | 48 (39.3%) | |
> 4 cm | 123 (53.0%) | 49 (44.5%) | 74 (60.7%) | |
Tumor location | | | | 0.944 |
Upper stomach | 55 (23.7%) | 27 (24.5%) | 28 (22.9%) | |
Middle stomach | 34 (14.7%) | 17 (15.5%) | 17 (13.9%) | |
Lower stomach | 89 (38.4%) | 40 (36.4%) | 49 (40.2%) | |
More than two parts | 54 (23.3%) | 26 (23.6%) | 28 (23.0%) | |
Depth of invasion | | | | 0.516 |
T1 | 19 (8.2%) | 8 (7.2%) | 11 (9.0%) | |
T2 | 30 (12.9%) | 15 (13.6%) | 15 (12.2%) | |
T3 | 137 (59.1%) | 61 (55.5%) | 76 (62.3%) | |
T4 | 46 (19.8%) | 26 (23.6%) | 20 (16.4%) | |
Lymph node metastasis | | | | 0.184 |
N0 | 78 (33.6%) | 42 (38.1%) | 36 (29.5%) | |
N1 | 80 (34.5%) | 45 (40.9%) | 35 (28.7%) | |
N2 | 41 (17.7%) | 15 (13.6%) | 26 (21.3%) | |
N3 | 33 (14.2%) | 8 (7.3%) | 25 (20.5%) | |
Distant metastasis | | | | 0.032 |
M0 | 182 (78.4%) | 93 (84.5%) | 89 (73.0%) | |
M1 | 50 (21.6%) | 17 (15.5%) | 33 (27.0%) | |
TNM stage | | | | 0.020 |
IA | 14 (6.0%) | 8 (7.3%) | 6 (4.9%) | |
IB | 21 (9.1%) | 14 (12.7%) | 7 (5.7%) | |
II | 52 (22.4%) | 29 (26.4%) | 23 (18.9%) | |
IIIA | 47 (20.3%) | 25 (22.7%) | 22 (18.0%) | |
IIIB | 21 (9.1%) | 5 (4.5%) | 16 (13.1%) | |
IV | 77 (33.2%) | 29 (26.4%) | 48 (39.3%) | |
a: The pathological diagnoses and classifications were made according to American Joint Committee on Cancer (AJCC) cancer staging manual, 7th ed |
High TC2N expression indicated worse prognosis in patients with gastric cancer
For all subjects in the present study, the follow-up period ranged from 1 to 84 months, with a median follow-up time of 51.2 months. And 123 (53.0%) patients were confirmed dead at the follow-up end point. The median overall survival time was 64.4 ± 11.6 months and the 5-year overall survival (OS) rate was 50.7%. In GC patients with high TC2N expression, the overall 5-year survival rate was significantly poorer than that in GC patients with low expression of TC2N (39.0% vs. 59.4%; P < 0.001, Fig. 4A). By comparing the Kaplan-Meier curves of the relapse-free survival (RFS) stratified by TC2N expression (Fig. 4B), it showed the consistent results that high level of TC2N is a signal of poor prognosis in GC patients. Furthermore, we also detected the prognostic indicator effect of TC2N expression in early (TNM stage I and II) (Fig. 4C, D) and advanced (TNM stage III and IV) (Fig. 4E,4F) gastric cancer, respectively. In early gastric cancer, the 5-year overall survival was 54.3% and 31.6% in the groups with TC2N low and high expression, respectively. And in advanced gastric cancer, the 5-year overall survival was 35.0% and 25.8% in the TC2N low and high expression groups, respectively. Similar results have been observed when RFS was used to be the comparative index. These results suggested that patients with high TC2N expression in gastric cancer tissues showed poorer prognosis than those with low TC2N expression regardless of TNM stage.
We also performed univariate Cox regression to estimate the value of prognostic evaluation of TC2N expression and the clinicopathological features for patients with gastric cancer. Univariate analysis showed that high TC2N level together with female, younger, advanced differentiation degree, large tumor size, more than two parts of stomach involved and advanced T, N, and M stage were significantly associated with poor overall survival of gastric cancer patients (Table 2). To correct the effects of confounding factors, multivariate Cox regression analysis was performed by using the method “Forward Stepwise (Conditional LR)”. It was revealed that high TC2N expression was also associated with poor survival of gastric cancer patients after adjusting for tumor size, depth of tumor invasion, lymph node metastasis and distant metastasis (HR = 1.616; 95%CI: 1.121–2.332, p = 0.010) (Table 2). These results indicated that the protein expression of TC2N could be an independent prognostic predictor for patients with gastric cancer.
Table 2
Association of various factors with overall survival determined by Cox regression.
Variables | Univariate Cox analysis | Multivariate Cox analysis |
HR | 95%CI | P value | HR | 95%CI | P value |
Lower | Upper | | | Lower | Upper | |
Gender | | | | | | | | |
Male | 1 | | | | | | | |
Female | 1.414 | 1.167 | 2.016 | 0.022 | | | | |
Age | | | | | | | | |
< 50 | 1 | | | | | | | |
≥ 50 | 0.624 | 0.428 | 0.975 | 0.043 | | | | |
Histological classification | | | | | | | | |
Well and moderately differentiated | 1 | | | | | | | |
Poorly differentiated | 2.016 | 1.679 | 3.183 | 0.001 | | | | |
Tumor size | | | | | | | | |
≤ 4 cm | 1 | | | | 1 | | | |
> 4 cm | 2.138 | 1.152 | 4.126 | < 0.001 | 1.573 | 1.315 | 2.422 | 0.029 |
Tumor location | | | | 0.007 | | | | |
Upper stomach | 1 | | | | | | | |
Middle stomach | 0.945 | 0.527 | 1.893 | 0.981 | | | | |
Lower stomach | 0.695 | 0.621 | 1.190 | 0.238 | | | | |
More than two parts | 1.239 | 1.311 | 2.678 | 0.039 | | | | |
Depth of invasiona | | | | < 0.001 | | | | 0.006 |
T1 | 1 | | | | 1 | | | |
T2 | 1.489 | 0.318 | 8.812 | 0.622 | 2.012 | 0.413 | 9.879 | 0.398 |
T3 | 8.786 | 2.056 | 28.745 | 0.003 | 5.622 | 1.445 | 22.423 | 0.018 |
T4 | 22.773 | 5.023 | 92.101 | < 0.001 | 7.691 | 1.698 | 36.012 | 0.007 |
Lymph node metastasisa | | | | < 0.001 | | | | 0.003 |
N0 | 1 | | | | 1 | | | |
N1 | 2.123 | 1.129 | 3.147 | 0.004 | 1.891 | 0.801 | 2.234 | 0.901 |
N2 | 6.012 | 3.761 | 9.012 | < 0.001 | 2.712 | 1.654 | 5.812 | 0.001 |
N3 | 7.071 | 3.613 | 10.972 | < 0.001 | 2.231 | 1.414 | 3.914 | 0.012 |
Distant metastasisa | | | | | | | | |
M0 | 1 | | | | 1 | | | |
M1 | 3.418 | 3.011 | 6.746 | < 0.001 | 2.087 | 1.294 | 3.294 | 0.003 |
TC2N level | | | | | | | | |
Low | 1 | | | | 1 | | | |
High | 2.003 | 1.354 | 2.691 | < 0.001 | 1.451 | 1.312 | 2.241 | 0.013 |
a: The pathological diagnoses and classifications were made according to American Joint Committee on Cancer (AJCC) cancer staging manual, 7th ed. |
TC2N promotes gastric cancer cell proliferation, migration and invasion in vitro
To explore the potential role of TC2N in the development of GC, TC2N was knocked-down in AGS cells or overexpressed in MKN28 cells, and the expression of TC2N was verified by WB (Fig. 5A). We then assessed the role of TC2N in cell proliferation by CCK8 assay (Fig. 5B). The data showed that TC2N knockdown markedly impeded the proliferation of AGS cells, while TC2N overexpression promoted the growth of MKN28 cells. Next, transwell assays were performed to examine the effect of TC2N on migratory and invasive capacity of GC cells. The results showed that the downregulation of TC2N inhibited the migration and invasion of AGS cells, whereas TC2N overexpression promoted the migration and invasion of MKN28 cells (Fig. 5C, D). These above results suggested that TC2N might act as a potential oncogene in gastric cancer.
Analysis of genes differentially expressed in correlation with TC2N in GC
The Function module of LinkedOmics was used to analyze mRNA sequencing data from 415 GC patients in the TCGA. As shown in the volcano plot (Fig. 6A), 3,733 genes (dark red dots) showed significant positive correlations with TC2N, whereas 5,420 genes (dark green dots) showed significant negative correlations (false discovery rate [FDR] < 0.01). The 50 significant gene sets positively and negatively correlated with TC2N as shown in the heat map (Fig. 6B, 6C). This result suggests a widespread impact of TC2N on the transcriptome. We noticed that TC2N expression showed a strong positive association with expression of CATSPERB (positive rank #1, Pearson correlation = 0.75, P = 4.694e-76) and some other genes which have been proved to be connected with cancer, such as GALNT3 (positive rank #2, Pearson correlation = 0.58, P = 5.76E-38), RBM47 (positive rank #4, Pearson correlation = 0.60, P = 1.86E-42). The scatter plot shows Pearson correlation of TC2N expression with expression of CATSPERB(Fig. 6D). In addition, we analyzed the PPI networks of TC2N by using the GeneMANIA and STRING program (Fig. 6E,6F), and predicted a strong interaction between the proteins of TC2N and CATSPERB, as well as the proteins of TC2N and GALNT3.