Neurexophilin 4 is a prognostic biomarker correlated with immune infiltration in bladder cancer

Abstract

Recent studies have shown that NXPH family member 4 (NXPH4) plays an important role in the progression of cancer. However, the potential role of NXPH4 in bladder cancer (BCa) remains to be explored. The purpose of the present study was to identify whether NXPH4 could be used as a biomarker to predict the prognosis of BCa. We first examined the expression of NXPH4 in pan-cancer, and then focused on BCa. Univariate and multivariate Cox regression analysis were used to investigate whether NXPH4 could be used as an independent prognostic indicator. Gene set enrichment analysis (GSEA) was used for functional analysis of NXPH4-related genes. CIBERSORT algorithm was used to calculate immune cell infiltration levels with different NXPH4 expression. Finally, the expression of NXPH4 was validated in clinical tissue specimens and bladder cancer cell lines by immunohistochemistry and qRT-PCR. The tumor-promoting effects of NXPH4 were further investigated using counting kit-8 (CCK-8), colony formation, EdU assays, and tumor xenograft model. Our results showed that NXPH4 was highly expressed in BCa tissues. Patients in the high NXPH4 expression group had shorter overall survival (OS) and progression-free survival (PFS). We found that immune-related pathways were enriched in NXPH4-related genes. Immune cell infiltrations in BCa were also associated with different NXPH4 expression. NXPH4 was further found to be highly expressed in our validation specimens. The proliferative effect of NXPH4 was confirmed in BCa in vivo and in vitro. Overall, NXPH4 is a biomarker for predicting BCa prognosis and associated with immune infiltration.Abbreviations: NXPH4: Neurexophilin 4; BCa: Bladder cancer; TCGA-BLCA: The Cancer Genome Atlas Urothelial Bladder Carcinoma; shRNA: short hairpin RNA; NC: Negative control; OS: Overall survival; PFS: Progression-free survival; TME: Tumor microenvironment; IPS: immunophenoscore; ICIs: Immune checkpoint inhibitors; DEGs: Differential expression genes.

Keywords: Bladder cancer; NXPH4; immune infiltration; prognosis; progression.

Graphical abstract

Figure 1.

NXPH4 is overexpressed in BCa. Unpaired (a) and paired (b) expression data analyses indicated that NXPH4 expression was markedly higher in BCa. ***p < 0.001.

Figure 2.

NXPH4 expression affects the prognosis of BCa patients. Patients in the high NXPH4 expression group had shorter OS (a) and PFS (b) compared with those in the low NXPH4 expression group. Correlation analysis between NXPH4 and different pathological features in BCa (c). *p < 0.05, **p < 0.01

Figure 3.

NXPH4 is an independent risk factor for BCa. Univariate (a) and multivariate (b) Cox regression analysis showed that NXPH4 was an independent risk factor correlated with the survival in BCa. A prognostic nomogram with NXPH4 and clinical variables was constructed (c). The 1-, 3- and 5-year calibration plots demonstrated the performance of the nomogram (d).

Figure 4.

Biological function of NXPH4 in BCa. Co-expressed genes significantly associated with NXPH4 were shown as correlation circle plots (a). Red, positive correlation; green, negative correlation. The top 50 upregulated DEGs and top 50 downregulated DEGs were displayed in the heatmap (b). Red, upregulated genes; blue, downregulated genes. Enrichment analysis is shown as a circle graph (c). GSEA was used to identify signaling pathways that were NXPH4-related differentially expression genes activated in BCa (d).

Figure 5.

Association of NXPH4 with immune cell infiltration in the TME of BCa. The proportion of 22 immune cells infiltrating in low and high NXPH4 expression groups (a). (b) Relationships between the expression of NXPH4 and 22 types of immune infiltration cells. (c-f) The relationships between the expression of NXPH4 and memory B cells, naive B cells, M0 macrophages, and plasma cells. (g) ESTIMATE algorithm was used to investigate the correlation between the two groups in Immune scores and Stromal scores. *p < 0.05, **p < 0.01,***p < 0.001.

Figure 6.

Role of NXPH4 in immunotherapeutic responses. (a-b) The association between ICIs and NXPH4. (c-f) The correlation between immunophenoscore and different NXPH4 expression groups. (g-j) Low NXPH4 expression group was positively associated with a higher IC50 of Doxorubicin, Gemcitabine, Tipifarnib, and Methotrexate.

Figure 7.

Clinical and in vitro validation of NXPH4 expression. (a-b) Immunohistochemistry results showed that NXPH4 were significantly highly expressed in cancer tissues. Scale bar: 100 and 200 μm. (c) The mRNA expression levels of NXPH4 were higher in cancer tissues. (d) NXPH4 was significantly increased in BCa cell lines (UMUC3, T24, and 5637) compared with SV-HUC-1. (e-f) CCK8, (g-h) colony formation, and (i-j) EdU assays showed that NXPH4 silencing inhibited the proliferation and colony formation of T24 and UMUC3 cells. Scale bar: 50 μm. *p < 0.05, **p < 0.01,***p < 0.001.

Figure 8.

NXPH4 knockdown inhibited BCa cells tumorigenesis in vivo. (a) T24 cells transfected with sh-NXPH4 or sh-NC were injected subcutaneously into nude mice. (b-c) The tumor volume and tumor weight were lower in sh-NXPH4 group. (d-e) Immunohistochemical staining of Ki-67 indicated that tumor proliferation was remarkably reduced in the sh-NXPH4 group. Scale bar: 100 and 200 μm. NC: negative group; sh: short hairpin. *p < 0.05, **p < 0.01,***p < 0.001.