Characteristics of patients with gastric cancer
The characteristics of patients with gastric cancer were evaluated in both groups according to high and low NPC2 expression (Supplementary Table 2). The histologic grade (P = 0.048), T classification (P = 0.0081), and vital status (P = 0.018) were significantly different in two groups. At the same time, there were no statistical differences in age (P = 0.8347), gender (P = 0.4134), histological type (P = 0.0594), stage (P = 0.2087), N classification (P = 0.198), M classification (P = 0.7266), radiation therapy (P = 0.0581), and residual tumor (P = 0.62).
NPC2 expression in gastric cancer
Compared with normal gastric tissue, the NPC2 expression was significantly higher (P < 0.001) in gastric cancer (Fig. 1A). Besides, the subgroup analysis was performed (Fig. 1B–L), including vital status, radiation therapy, residual tumor, TNM classification, stage, histological type, histologic grade, gender, and age. However, no statistical differences were found.
NPC2 expression in gastric cancer. (A) NPC2 expression in tumor versus paired normal tissue. NPC2 expression grouped by (B) vital status, (C) radiation therapy, (D) residual tumor, (E) M classification, (F) N classification, (G) T classification, (H) stage, (I) histologic grade, (J) histological type, (K) gender, and (L) age.
Diagnostic value of NPC2 expression
The ROC curve analysis showed that NPC2 expression had a high diagnostic value for gastric cancer with AUC of 0.696 (Supplementary Figure 1A). Moreover, the AUC for stage I-IV was 0.660 (Supplementary Figure 1B), 0.721 (Supplementary Figure 1C), 0.678 (Supplementary Figure 1D), and 0.716 (Supplementary Figure 1E).
High NPC2 expression is associated with poor OS and RFS
The Kaplan–Meier curves showed high NPC2 expression was significantly (P < 0.0001) associated with poor OS (Fig. 2A) and poor RFS (Fig. 2B).
High NPC2 expression is associated with poor survival. (A) Overall survival. (B) Relapse free survival.
OS grouped NPC2 expression
The OS grouped NPC2 expression was significantly different in stage I/II, stage III/IV, stage G1/G2, stage G3/G4, stage I, stage II, stage III, stage G2, stage G3, T3, T4, N0, M0, R0, male, and older patients (Fig. 3A–P). Others were not significant (Supplementary Figure 2).
Overall survival grouped NPC2 expression. Overall survival group by NPC2 in (A) stage I/II, (B) stage III/IV, (C) G1/G2, (D) G3/G4, (E) stage I, (F) stage II, (G) stage III, (H) stage G2, (I) stage G3, (J) T3, (K) T4, (L) N0, (M) M0, (N) R0, (O) male, and (P) older.
By univariate and multivariate analysis (Supplementary Figure 3A–B), the age [hazard ratio (HR): 1.825, 95% confidence interval (CI): 1.122–2.967, P = 0.015], stage (HR: 1.320, 95% CI: 1.049–1.661, P = 0.018), radiation therapy (HR: 0.471, 95% CI: 0.318–0.699, P < 0.001), residual tumor (HR: 1.695, 95% CI: 1.290–2.227, P < 0.001), and NPC2 expression (HR: 1.664, 95% CI: 1.190–2.327, P = 0.003) showed independent prognostic value for OS.
RFS grouped NPC2 expression
The RFS grouped NPC2 expression was significantly different in stage I/II, stage III/IV, stage G3/G4, stage II, stage III, stage G3, T3, T4, N0, M0, R0, R1/R2, male, female, and older patients (Fig. 4A–O). Others were not significant (Supplementary Figure 4).
Relapse free survival grouped NPC2 expression. Relapse free survival group by NPC2 in (A) stage I/II, (B) stage III/IV, (C) G1/G2, (D) stage II, (E) stage III, (F) stage G3, (G) T3, (H) T4, (I) N0, (J) M0, (K) R0, (L) R1/R2/Rx, (M) male, (N) female, and (O) older.
By univariate and multivariate analysis (Supplementary Figure 5A–B), the gender (HR: 1.960, 95% CI: 1.132–3.394, P = 0.016) and NPC2 expression (HR: 2.097, 95% CI: 1.289–3.412, P = 0.003) showed independent prognostic value for RFS.
Predictive value of NPC2 expression in OS and RFS
The nomogram was used to further evaluate the ability of NPC2 to predict OS (Fig. 5A,B) and RFS (Fig. 6A,B). The results showed that NPC2 expression was a significant predictor of OS and RFS, with high predictive accuracy (Figs. 5C–H, 6C–H). Specifically, high NPC2 expression could predict shorter OS and RFS.
Predictive value of NPC2 expression in overall survival. (A, B) ROC curves evaluating the NPC2 expression for predicting overall survival. (C–E) Nomogram predicted 1-year, 3-year, and 5-year overall survival versus actual overall survival. (F–H) Decision curve analysis reflects the feasibility of NPC2 expression in predicting 1-year, 3-year, and 5-year overall survival.
Predictive value of NPC2 expression in relapse free survival. (A, B) ROC curves evaluating the NPC2 expression for predicting relapse free survival. (C–E) Nomogram predicted 1-year, 3-year, and 5-year relapse free survival versus actual relapse free survival. (F–H) Decision curve analysis reflects the feasibility of NPC2 expression in predicting 1-year, 3-year, and 5-year relapse free survival.
High NPC2 expression-enriched pathways
The GSEA analysis (Supplementary Table 3) showed that high NPC2 expression was significant associated (P < 0.05) with asthma (Fig. 7A), cytokine-cytokine receptor interaction (Fig. 7B), drug metabolism other enzymes (Fig. 7C), natural killer cell mediated cytotoxicity (Fig. 7D), primary bile acid biosynthesis (Fig. 7E), and systemic lupus erythematosus (Fig. 7F).
High NPC2 expression-enriched pathways. (A) Asthma. (B) Cytokine-cytokine receptor interaction. (C) Drug metabolism other enzymes. (D) Natural killer cell mediated cytotoxicity. (E) Primary bile acid biosynthesis. (F) Systemic lupus erythematosus.
High NPC2 expression in tissue
The higher NPC2 expression was observed in gastric cancer, compared with adjacent normal tissue (P < 0.001, Fig. 8A). The IHC staining further confirmed the high NPC2 expression in gastric cancer (Fig. 8B). Moreover, the prognostic significance of NPC2 expression was validated by RT-PCR with another cohort consisting of 58 living patients and 32 deceased patients (Supplementary Figure 6). Higher NPC2 expression was associated with poor survival (P < 0.05).
High NPC2 expression in tissue and cell. (A) NPC2 expression in 30 gastric cancer and adjacent normal tissues by qRT-PCR. (B) Representative IHC image of gastric cancer and normal gastric tissue. Scale bar = 100 μm. (C) Relative NPC2 expression in HaCaT, RGM-1, MGC-803, HGC-27, SGC-7901, and AGS by qRT-PCR. (D) Relative NPC2 expression in AGS cells transfected with control, si-NC, si-NPC2, and O-NPC2 by qRT-PCR. (E) Relative cell proliferation of AGS cell by CCK-8 assay. NS, no significance; *P < 0.05; **P < 0.01; ***P < 0.001.
High NPC2 expression in cell
Besides, the NPC2 expression was evaluated in normal cells including HaCaT and RGM-1 (Human gastric epithelial cell), and gastric cancer cells including MGC-803 (Human low differentiated gastric cancer), HGC-27 (Human undifferentiated gastric cancer), SGC-7901 (Human gastric cancer with lymph node metastasis), AGS (Untreated human gastric adenocarcinoma cells). The NPC2 expression was higher in gastric cancer than normal cells (P < 0.05, Fig. 8C). Particularly, the NPC2 expression was highest in AGS, which was used for subsequent cell proliferation assay.
NPC2 knockdown inhibits cell proliferation
First, the knockdown and overexpression efficiency were examined (Supplementary Figure 7 and Fig. 8D). The si-NPC2 treated AGS cell showed significant decreased NPC2 expression (P < 0.05), and O-NPC2 treated AGS cell showed significant increased NPC2 expression (P < 0.01). Then, the CCK-8 assay showed that NPC2 knockdown inhibits AGS cell proliferation while NPC2 overexpression promotes cell proliferation (P < 0.05, Fig. 8E). The involvements of NPC2 in proliferation was validated in SGC-7901 cell as well (Supplementary Figure 8).