Prognostic significance of high npc2 expression in gastric cancer

ABSTRACT Gastric cancer is one of the most common malignancies worldwide, and the third leading cause of cancer-related death. The identification of novel biomarkers and therapeutic targets

is critical to improve the prognosis. A total of 380 patients with primary gastric cancer from the TCGA database were analyzed. The receiver operating characteristic curves were plotted. We

further evaluated the independent prognostic ability of NPC2 expression for overall survival (OS) and relapse-free survival (RFS) through the Kaplan–Meier curve and Cox analysis. The NPC2

expression was significantly higher (_P_ < 0.001) in gastric cancer. High NPC2 expression was significantly (_P_ < 0.0001) associated with poor OS and poor RFS. The age, stage,

radiation therapy, residual tumor, and NPC2 expression showed independent prognostic value for OS. The gender and NPC2 expression showed independent prognostic value for RFS. The higher NPC2

expression was observed in gastric cancer, compared with adjacent normal tissue (_P_ < 0.001), confirmed by the IHC staining. The CCK-8 assay showed that NPC2 knockdown inhibits cell

proliferation while NPC2 overexpression promotes cell proliferation (_P_ < 0.05). NPC2 expression may serve as a promising prognostic biomarker for patients with gastric cancer. SIMILAR

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PRPC EXPRESSION AND PATIENT PROGNOSIS IN PRIMARY ESOPHAGEAL SQUAMOUS CELL CARCINOMA AND PRIMARY HEPATOCELLULAR CARCINOMA Article Open access 28 December 2024 INTRODUCTION Gastric cancer,

originated from gastric mucosal epithelium, is one of the most common malignancies worldwide threatening human health1. Gastric cancer is the third leading cause of cancer-related death with

more than 1 million new cases each year2. Due to the limited understanding of molecular mechanisms and poor prognosis, the mortality rate of gastric cancer is still high3. Gastric cancer

has an insidious onset and atypical early symptoms are often not appreciated by patients4. The majority of patients are found to have advanced gastric cancer, and their 5-year survival rates

tend to be less than 10%5. Therefore, identification of novel biomarkers and therapeutic targets is critical to improve the diagnosis, treatment and prognosis of gastric cancer6, 7. NPC2

(NPC intracellular cholesterol transporter 2) is an intracellular cholesterol transporting protein that affects the cholesterol homeostasis regulatory system8, 9. In recent years, increasing

evidence has revealed the important role of NPC2 in the development and progression of various human cancers, including gastric cancer10. There is a very strong association between NPC2

expression and tumors, with a signature of overexpression in breast, colon, as well as lung cancers11. However, the clinical significance and prognostic value of NPC2 expression in gastric

cancer remain largely unknown. The Cancer Genome Atlas (TCGA) is a comprehensive and integrated database that provides a wealth of genomic, transcriptomic, and clinical information on

multiple types of cancer, including gastric cancer12, 13. In this study, we aimed to assess the correlation between NPC2 expression in gastric cancer and clinicopathologic characteristics

through analyzing data from the TCGA database. The receiver operating characteristic (ROC) curves were plotted to analyze the diagnostic value of NPC2. We further evaluated the independent

prognostic ability of NPC2 expression for overall survival (OS) and relapse-free survival (RFS) through the Kaplan–Meier curve and Cox analysis. The ability of NPC2 to predict OS and RFS was

reflected by nomogram. Finally, the NPC2 expression was validated in the samples collected from patients and gastric cancer cell. The findings could provide new insights into the clinical

significance of NPC2 expression in gastric cancer and its potential as a diagnostic and prognostic biomarker. METHODS DATA COLLECTION Data was obtained from TCGA database

(https://www.cancergenome.nih.gov). The raw data was preprocessed to remove outliers and normalize the expression levels of NPC2. A total of 380 patients with primary gastric cancer were

involved (Supplementary Table 1). The clinicopathologic characteristics and expression levels of NPC2 in gastric cancer were analyzed by Fisher’s exact test and chi-square test, and

non-parametric rank sum test, respectively14. The study was approved by the Ethics Committee of the First Hospital of Shanxi Medical University, and all methods were performed in accordance

with the relevant guidelines and regulations. The 30 patient samples were obtained with appropriate informed consent and processed with approval. DIAGNOSTIC VALUE ANALYSIS The diagnostic

value of NPC2 expression was analyzed by plotting the ROC curve and calculating the area under the curve (AUC). The AUC was used to determine the accuracy of NPC2 in diagnosing gastric

cancer. SURVIVAL ANALYSIS The Kaplan–Meier curve was used to evaluate the prognostic value of NPC2 expression for OS and RFS15. The log-rank test was used to compare the survival curves

between different subgroups. UNIVARIATE AND MULTIVARIATE COX ANALYSIS Univariate Cox analysis was performed to assess the univariate association between NPC2 expression and survival.

Multivariate Cox analysis was performed to evaluate the independent prognostic ability of NPC2 expression after adjusting for other potential confounding factors. NOMOGRAM ANALYSIS The

nomogram was used to reflect the ability of NPC2 to predict OS and RFS. The concordance index (C-index) was calculated to assess the predictive accuracy of the nomogram16. GSEA ANALYSIS The

online GSEA analysis was performed after searching the TCGA for evaluation of NPC2 expression and enriched pathways17. REAL-TIME QUANTITATIVE PCR The RT-qPCR was performed using the kit

(Invitrogen, Thermo Fisher, USA) according to the instruction. 2−ΔΔCt method was used for quantification. The NPC2 primers (forward primer: 5′-TGT GGC TCA GTG GCT TAG G-3′; reverse primer:

5′-CCA GGA AGG GAT TTC ACA CA-3′) and β-actin primers (forward primer: 5′-ACC CCA AAG CCA ACA GA-3′; reverse primer: 5′-CCA GAG TCC ATC ACA ATA CC-3′) were used. IMMUNOHISTOCHEMISTRY The

immunohistochemistry (IHC) staining was performed as reported18. Anti-NPC2 primary antibody (MA5-29450, Thermo Fisher, USA) and corresponding rabbit secondary antibody were used. CELL

CULTURE AND TRANSFECTION The cells were purchased from American Tissue Culture Collection and cultured in the necessary environment as reported19. There were four groups including si-NPC2

transfected group, NPC2 overexpressed group, si-NC transfected group as negative control, and un-transfected group as control. The validation of transfection efficiency was performed using

qRT-PCR and western blot. WESTERN BLOT To confirm NPC2 knockdown, Western blot analysis was performed as previously reported20. Primary antibody NPC2 (sc-30346, Santa Cruz Biotechnology,

USA) and goat secondary antibody (sc-2354, Santa Cruz Biotechnology, USA) were used. CELL PROLIFERATION ASSAY The cell proliferation assay was performed by CCK-8 in AGS and SGC-7901 cell as

reported20. After plasmid transfection, the cells were cultured for 24 h. Then, the CCK-8 reagent (10 μL) was added and kept for 20 min. The absorbance at 490 nm was measured. STATISTICAL

ANALYSIS All statistical analyses were performed using the R software (version 3.5.2). The Wilcoxon rank sum and the Kruskal–Wallis test was used for comparing two and multiple groups,

respectively. A two-sided P value of < 0.05 was considered statistically significant. RESULTS 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. 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). 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). 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). 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. 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 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 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).

DISCUSSION Clinical stage at diagnosis directly determines the prognosis of patients with gastric cancer. Patients with localized, early-stage gastric cancer usually have a high 5-year

overall survival rate (OS > 60%), whereas patients with local and distant metastases of gastric cancer have a significantly decreased 5-year OS of 30% and 5%, respectively21.

Unfortunately, due to the occult nature and atypical nature of early clinical symptoms of gastric cancer, more than 60% of patients present with regional or distant metastasis at

diagnosis22. Surgical resection is the best treatment option for patients with early-stage gastric cancer, and chemotherapy is the most important treatment for patients with unresectable or

advanced metastasis. However, gastric cancer patients often respond poorly or even do not respond to chemotherapy due to their inherent or acquired resistance, becoming the most common cause

of treatment failure. Therefore, the low rate of early diagnosis and chemoresistance are the main reasons for the poor prognosis of gastric cancer. In the present study, we assessed the

correlation between NPC2 expression and clinicopathologic characteristics in gastric cancer patients using data from The Cancer Genome Atlas (TCGA) database. Our findings indicated that NPC2

expression is significantly associated with certain clinicopathological features and can serve as a promising prognostic biomarker for gastric cancer patients. Abnormal expression of NPC2

is closely associated with the progression of multiple malignant tumors. NPC2 expression is increased in patients with lung adenocarcinoma and their pleural effusions, but it is not clear

whether NPC2 plays a role in lung adenocarcinoma23. The presence of NPC2 protein in pleural effusions of lung adenocarcinoma suggests that it may be used as a potential diagnostic marker for

lung cancer. NPC2 overexpression correlates with the HER-2 subtype, suggesting that NPC2 upregulation may be a favorable prognostic predictor in breast cancer24. Notably, reduced expression

of NPC2 was particularly observed in liver tumor tissues compared with normal counterpart tissues25. NPC2 is associated with lipid metabolism and the innate immune system, and it influences

the formation and progression of various types of cancer cells, playing critical regulatory functions. Wei et al. found a relatively higher expression of NPC2 in glioblastoma and NPC2

overexpression inhibited cell proliferation26. Adachi et al. found silkworm NPC2 protein inhibits the growth of FM3A murine breast cancer cells27. Wang et al. reported that NPC2

overexpression decreased PDGF-BB-induced cell proliferation by inhibiting p38, JNK, and AKT phosphorylation28. Yet, the mechanism underlying the involvements of NPC2 in proliferation has

reached no consensus. The TCGA database involving huge data of gene related expression profile is keeping updated, and the number of patients may vary slightly at different time points.

Further studies with larger sample sizes and longer follow-up periods are needed to validate our results and to determine the optimal cutoff value for NPC2 expression in gastric cancer

patients. Additionally, it is essential to evaluate the feasibility and accuracy of NPC2 expression as a diagnostic and therapeutic target in gastric cancer. CONCLUSION In conclusion, our

findings provide evidence that NPC2 expression may serve as a promising prognostic biomarker for patients with gastric cancer. This information could have important implications for the

early diagnosis, therapeutic strategies and personalized management of gastric cancer. The findings of this study can improve the therapy of GC patients by providing a novel therapeutic

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Thank for the TCGA database for providing data. FUNDING This study was funded by the First Hospital of Shanxi Medical University (No. SYYYRC-2022002). AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Department of Gastroenterology, First Hospital of Shanxi Medical University, Taiyuan, 030000, China Xing Chen * Faculty of Graduate Studies, Shanxi Medical University,

Taiyuan, 030000, China Yunzhuang Yao, Jinnan Ren, Junhui Lu, Yue Sui & Jingwen Gong * Heping Hospital Affiliated to Changzhi Medical College, Changzhi, China Yunzhuang Yao Authors *

Yunzhuang Yao View author publications You can also search for this author inPubMed Google Scholar * Jinnan Ren View author publications You can also search for this author inPubMed Google

Scholar * Junhui Lu View author publications You can also search for this author inPubMed Google Scholar * Yue Sui View author publications You can also search for this author inPubMed 

Google Scholar * Jingwen Gong View author publications You can also search for this author inPubMed Google Scholar * Xing Chen View author publications You can also search for this author

inPubMed Google Scholar CONTRIBUTIONS X.C., and Y.Y. designed this study and had full access to all of data in the study; J.R. extracted the data; J.L. analyzed and interpreted the data;

S.Y., and J.G. wrote the paper. All authors reviewed the manuscript. CORRESPONDING AUTHOR Correspondence to Xing Chen. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no

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in gastric cancer. _Sci Rep_ 13, 20710 (2023). https://doi.org/10.1038/s41598-023-47882-3 Download citation * Received: 19 April 2023 * Accepted: 20 November 2023 * Published: 24 November

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