NAV3 Is a Novel Prognostic Biomarker Affecting the Immune Status of the Tumor Microenvironment in Colorectal Cancer

Abstract

Colorectal cancer (CRC) is one of the most common malignant tumors in the world. Tumor microenvironment (TME) plays a crucial role in the development of CRC. With the deep understanding of TME function, growing studies have demonstrated that alteration in tumor-infiltrating immune cells (TICs) and gene expressions are associated with clinical outcomes of various tumors. In this study, we aimed to recognize critical prognostic genes involved in immune states in TME of CRC. Hence, the proportion of TICs and the number of immune and stromal components in CRC samples from TCGA datasets were calculated by the use of CIBERSORT and ESTIMATE calculation methods. Different assays were applied to collect differential expression genes (DEGs) shared by the ImmuneScore and StromalScore. DEGs were further analyzed by the use of univariate Cox regression. Our attention focused on neuron navigator 3 (NAV3) which was highly expressed in CRC specimens and associated with an advanced clinical stage and poor prognosis of CRC patients. KEGG assays revealed that NAV3 may be involved in Alzheimer disease, amyotrophic lateral sclerosis, Huntington disease, FoxO signaling pathway, and human papillomavirus infection. Correlation assays showed that macrophage M0 and B cells memory, NK cells activated, dendritic cells resting, T cells CD4 memory activated, and T cells CD8 were correlated with NAV3 expression, indicating that NAV3 may represent the immune status of TME. Finally, RT-PCR confirmed that NAV3 expression was distinctly increased in CRC cells, and its knockdown suppressed the proliferation of CRC cells. Overall, NAV3 could be used as a novel predictor for TME of CRC and might be a novel prognostic biomarker. In the future, drugs targeting NAV3 might be developed as a potential immunotherapy for CRC patients.

Figure 1

Relationships of ESTIMATEScore, StromalScore, and ImmuneScore with survival in CRC patients. Kaplan-Meier survival investigations of CRC patients suffering from low and high (a) ImmuneScores, (b) StromalScores, and (c) ESTIMATEScores.

Figure 2

Relationship of scores showing clinicopathological staging features: (a–c) ImmuneScore in gender, age, and stage; (d–f) StromalScores in gender, age, and stage; (g–i) ESTIMATEScores in gender, age, and stage.

Figure 3

DEGs shared by the StromalScore and ImmuneScore and functional enrichment investigation. (a) The heat map of DEGs obtained in accordance with the ImmuneScore. (b) The heat map of DEGs obtained in accordance with the StromalScore. (c, d) Venn plots showing common increased and reduced DEGs shared by the ImmuneScore and StromalScore. (e, f) GO and KEGG assays with the use of the above DEGs.

Figure 4

Univariate Cox regression investigation of DEGs.

Figure 5

The expression of NAV3 and its clinical significance in CRC. (a) The expression of NAV3 was distinctly downregulated in CRC specimens. (b, c) The expressions of NAV3 in groups which were divided by age and gender. (d) Higher levels of NAV3 were observed in CRC specimens at different stages. (e) Kaplan-Meier curves estimating the overall survival according to the expression of NAV3 in patients with CRC.

Figure 6

Pathway enrichment investigations of NAV3-related DEGs. (a) The numbers of genes enriched in each GO category. (b) KEGG pathways are analyzed, and the top 30 pathways are mapped.

Figure 7

TIC profile within tumor samples and relationship investigation. (a) The landscape of immune infiltration in CRC tissues. (b) Relationship matrix of all 22 TIIC proportions.

Figure 8

Relationship of TIC proportion with NAV3 expression. (a) The levels of 21 types of immunization cells in the two groups (high and low). (b, c) The relationship investigation between the levels of 21 types of immunization cells and the levels of NAV3 in CRC.

Figure 9

NAV3 promoted CRC cell proliferation in vitro. (a) NAV3 expressions in five CRC cells examined via RT-qPCR. (b) The efficiency of NAV3 silencing was examined through RT-qPCR in the HCT116 and SW480 after the transfection with siRNAs. (c) CCK-8 experiments confirming that silencing of NAV3 inhibited the proliferation of HCT116 and SW480 cells. ^∗∗p < 0.01.