High expression of NXPH4 correlates with poor prognosis, metabolic reprogramming, and immune infiltration in colon adenocarcinoma

Abstract

Background: Colon adenocarcinoma (COAD) is a prevalent gastrointestinal malignant disease with high mortality rate, and identification of novel prognostic biomarkers and therapeutic targets is urgently needed. Although neurexophilin 4 (NXPH4) has been investigated in several tumors, its role in COAD remains unclear. The aim of this study was to explore the prognostic value and potential functions of NXPH4 in COAD.

Methods: The expression of NXPH4 in COAD were analyzed using The Cancer Genome Atlas (TCGA) and datasets from the Gene Expression Omnibus (GEO) database. The prognostic value of NXPH4 was determined using Kaplan-Meier analysis and Cox regression analysis. To investigate the possible mechanism underlying the role of NXPH4 in COAD, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) were employed. The correlation between NXPH4 expression and immune cell infiltration levels was examined thorough single-sample gene set enrichment analysis (ssGSEA). Furthermore, the competing endogenous RNA (ceRNA) regulatory network that may be involved in NXPH4 in COAD was predicted and constructed through a variety of databases.

Results: NXPH4 expression was significantly higher in COAD tissue compared with normal colon tissues. Meanwhile, high expression of NXPH4 was associated with poor prognosis in COAD patients. GO-KEGG and GSEA analyses indicated that NXPH4 was associated with glycolysis and hypoxia pathway, and may promote COAD progression and metastasis by modulating metabolic reprogramming. ssGSEA analysis demonstrated that NXPH4 expression also associated with immune infiltration. Furthermore, we identified various microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) as upstream regulators of NXPH4 in COAD.

Conclusions: The present study revealed that high expression of NXPH4 is associated with tumor progression, metabolic reprogramming, and immune infiltration. These findings suggest that NXPH4 could serve as a reliable prognostic biomarker and a promising therapeutic target in COAD.

Keywords: Neurexophilin 4 (NXPH4); colon adenocarcinoma (COAD); immune infiltration; metabolic reprogramming; prognosis.

Figure 1

Expression status and prognostic value of NXPH4 in pan-cancer. (A,B) Expression of NXPH4 in different tumor tissues and normal tissues according to the TCGA database. (C) Prognostic analysis of NXPH4 expression levels in different human cancers. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significance. NXPH4, neurexophilin 4; TPM, transcripts per million; ACC, adrenocortical carcinoma; HR, hazard ratio; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; COAD, colon adenocarcinoma; KIRP, kidney renal papillary cell carcinoma; LIHC, liver hepatocellular carcinoma; READ, rectum adenocarcinoma; UCEC, uterine corpus endometrial carcinoma; UVM, uveal melanoma; TCGA, The Cancer Genome Atlas.

Figure 2

NXPH4 expression in COAD patients. (A,B) Expression of NXPH4 in COAD tissues and adjacent normal colon tissues according to the TCGA database. (C,D) Expression of NXPH4 in COAD tissues and normal colon tissues according to the GEO database. (E-G) Correlation between NXPH4 expression and clinical parameters in COAD. **, P<0.01; ***, P<0.001. NXPH4, neurexophilin 4; TPM, transcripts per million; COAD, colon adenocarcinoma; TCGA, The Cancer Genome Atlas; GEO, Gene Expression Omnibus.

Figure 3

Prognostic and diagnostic value of NXPH4 in COAD. (A-C) Kaplan-Meier survival curve analysis of OS, DSS and PFI in COAD cohort. (D) ROC curve of NXPH4 expression in COAD cohort. NXPH4, neurexophilin 4; HR, hazard ratio; FPR, false positive rate; TPR, true positive rate; AUC, area under the curve; CI, confidence interval; COAD, colon adenocarcinoma; OS, overall survival; DSS, disease-specific survival; PFI, progression-free interval; ROC, receiver operating characteristic.

Figure 4

Prognostic prediction model of NXPH4 in COAD patients. (A) Nomogram for 1-, 3- and 5-year OS of COAD patients. (B) Calibration plots for 1-, 3- and 5-year OS prediction. (C) Time-dependent ROC curves and AUC values for 1-, 3- and 5-year OS prediction. CEA, carcinoembryonic antigen; NXPH4, neurexophilin 4; FPR, false positive rate; TPR, true positive rate; AUC, area under the curve; COAD, colon adenocarcinoma; OS, overall survival; ROC, receiver operating characteristic; AUC, area under the curve.

Figure 5

Functional enrichment analysis of NXPH4 in COAD. (A) Volcano plot of DEGs between the high and low NXPH4 expression groups. (B,C) Heat map showing the top 20 positively and negatively co-expressed genes with NXPH4 in COAD. (D) GO-KEGG enrichment analysis of NXPH4 expression-correlated DEGs. ***, P<0.001. NXPH4, neurexophilin 4; TPM, transcripts per million; BP, biological process; CC, cellular component; MF, molecular function; KEGG, Kyoto Encyclopedia of Genes and Genomes; COAD, colon adenocarcinoma; DEGs, differentially expressed genes; GO, Gene Ontology.

Figure 6

PPI analysis of NXPH4. The PPI network constructed on (A) GeneMANIA database and (B) STRING database. NXPH4, neurexophilin 4; PPI, protein-protein interaction; GeneMANIA, Gene Multiple Association Network Integration Algorithm; STRING, Search Tool for the Retrieval of Interacting Genes.

Figure 7

Enrichment plots from the GSEA. Genes related to NXPH4 in COAD were enriched in (A) interferon gamma response, (B) allograft rejection, (C) inflammatory response, (D) IL6 Jak STAT3 signaling, (E) complement, (F) glycolysis, (G) KRAS signaling Dn, (H) hypoxia, and (I) coagulation. NES, normalized enrichment score; FDR, false discovery rate; GSEA, gene set enrichment analysis; NXPH4, neurexophilin 4; COAD, colon adenocarcinoma.

Figure 8

Analysis of genes co-expressed with NXPH4 and related to glycolysis and hypoxia in COAD. (A,B) Chord diagrams of the relationships between the expression of NXPH4 and 10 glycolysis-related genes and 10 hypoxia-related genes in TCGA-COAD. (C) Scatter plots of the correlation between the expression of NXPH4 and B3GALT6, ENO2, and P4HA1. (D) Analysis of B3GALT6, ENO2, and P4HA1 expression in tumor tissues and adjacent normal tissues in TCGA-COAD. (E) Association between B3GALT6, ENO2, and P4HA1 expression and OS in COAD patients. **, P<0.01; ***, P<0.001. NXPH4, neurexophilin 4; TPM, transcripts per million; B3GALT6, β-1,3galactosyltransferase 6; ENO2, enolase 2; P4HA1, prolyl 4-hydroxylase subunit α1; HR, hazard ratio; COAD, colon adenocarcinoma; TCGA, The Cancer Genome Atlas; OS, overall survival.

Figure 9

Correlation analysis of NXPH4 expression and immune infiltration in COAD. (A) The infiltrating levels of 24 subtypes of immune cells in high and low NXPH4 expression groups in COAD. (B) The correlation between NXPH4 expression and 24 tumor-infiltrating immune cells in COAD. (C-K) The correlation of NXPH4 expression with immune infiltration levels of NK cells, NK CD56bright cells, cytotoxic cells, macrophages, aDC cells, Th1 cells, neutrophils, Treg cells, and Tcm cells. *, P<0.05; **, P<0.01; ***, P<0.001. NXPH4, neurexophilin 4; aDC, activated dendritic cells; DC, dendritic cells; iDC, immature dendritic cells; NK, natural killer; pDC, plasmacytoid dendritic cells; Tem, effector memory T; Tcm, central memory T; TFH, follicular helper T; Tgd, gamma-delta T; Th1, T helper 1; Treg, regulatory T; TPM, transcripts per million; COAD, colon adenocarcinoma.

Figure 10

Correlation analysis of NXPH4 expression with immunomodulators, chemokines, and chemokine receptors. (A-D) Heatmap analysis of the correlation between NXPH4 and immunoinhibitors (A), immunostimulators (B), chemokines (C), and chemokine receptors (D) in COAD. NXPH4, neurexophilin 4; COAD, colon adenocarcinoma.

Figure 11

The ceRNA network of NXPH4 was predicted and constructed in COAD. (A) The Venn diagram displays the target miRNAs of NXPH4 predicted by ENCORI and TargetScan databases. (B) Correlation analysis between NXPH4 expression and hsa-miR-204-5p, hsa-miR-485-5p, hsa-miR-497-5p, and hsa-miR-320c. (C) Analysis of hsa-miR-204-5p, hsa-miR-485-5p, hsa-miR-497-5p, and hsa-miR-320c in tumor tissues and adjacent normal tissues in TCGA-COAD. (D) Correlation analysis between miRNAs (hsa-miR-485-5p, hsa-miR-497-5p, and hsa-miR-320c) and lncRNAs (MELTF-AS1, RPARP-AS1, SNHG7, SNHG10, AC129492.1, AGAP2-AS1, and TYMSOS) in COAD. (E) Correlation analysis between NXPH4 expression and MELTF-AS1, RPARP-AS1, SNHG7, SNHG10, AC129492.1, AGAP2-AS1, and TYMSOS in COAD. (F) Analysis of MELTF-AS1, RPARP-AS1, SNHG7, SNHG10, AC129492.1, AGAP2-AS1, and TYMSOS expression in tumor tissues and adjacent normal tissues in TCGA-COAD. (G) A ceRNA network based on NXPH4. **, P<0.01; ***, P<0.001. NXPH4, neurexophilin 4; COAD, colon adenocarcinoma; RPM, reads per million; FPKM, fragments per kilobase per million; TPM, transcripts per million; ceRNA, competing endogenous RNA; TCGA, The Cancer Genome Atlas; miRNAs, microRNAs; lncRNAs, long noncoding RNAs.