Transcriptional expression data of NMB and corresponding clinical information were downloaded from TCGA official website [6]. The 18 enrolled cancer types and the RNA-Seq gene expression data were converted to log2 conversion for further studies. Because all the data were obtained from TCGA, this study was not required to provide the approval from the Ethics Committee.

The RNA-Seq expression data of NMB in GBM was obtained from TCGA. Then 166 GBM and 5 normal brain tissue data were captured for analysis. The mRNA expression data were presented as the mean values with standard deviation (mean $\pm$ SD).

UALCAN (http://ualcan.path.uab.edu/) [7], a publicly available web resource for analyzing cancer data, was used to perform the analysis of NMB protein expression from Clinical Proteomic Tumor Analysis Consortium (CPTAC) in our study.

The Human Protein Atlas (HPA) includes tumor and normal tissues information regarding the acquisition and expression profiles for protein level of human genes [8]. We performed HPA to compare the protein expression of NMB between normal brain and GBM samples in our study.

The STRING database can retrieve the protein networks online [9] (version 11.0, http://string-db.org/). In this study, we performed STRING to search for co-expression genes and to build the protein-protein interaction (PPI) networks with an interaction score $>$0.4. Gene ontology (GO) enrichment analyses and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of these co-expression of genes were conducted.

TIMER database is a user-friendly online resource for systematic analysis of immune infiltrates about amount of cancer types [10]. We conducted TIMER to verify the relationship between NMB expression in GBM and six immune cells (CD4${}^{+}$ T cells, CD8${}^{+}$ T cells, B cells, neutrophils, macrophages, and dendritic cells) infiltration.

TISIDB is an interactive online web resource integrated repository portal for tumor-immune system interaction [11]. We performed TISIDB to verify the expression of NMB and tumor-infiltrating lymphocytes (TILs) among human cancers. According to the gene expression profile, the relative abundance of TILs was speculated through gene set variation analysis. The correlations of NMB and TILs were calculated by Spearman’s test.

The PrognoScan is a very powerful online database to assess the correlation between gene expression and survival rates for all types of cancers [12]. We used PrognoScan to analyze the correlation between NMB gene expression and overall survival in two different datasets (GSE4412_GPL96 and GSE4271_GPL96).

The U-87 MG GBM cells were cultured in DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin, and 1% streptomycin in 5% CO${}_2$ at 37 °C in a humidified incubator. Cells were seeded into 96-well tissue culture plates at a density of 1 $\times$ 10${}^4$ cells per well in 100 $\mu$L of medium. After overnight incubation, cells were treated with various concentrations of NMB (10 μM, 50 μM, 100 μM) for 24 hours. CCK8 solution was added to the plate and the optical density (OD) was measured at 450 nm using an ELISA plate reader (EL340 Microplate Reader; Winooske, VT, USA). Untreated cells at different times (24 h, 48 h, 72 h) were used as a negative control (100% viability). Each experiment was performed at least three times.

All statistical analyses were applied with R (V 4.1.0, R Foundation for Statistical Computing, Vienna, Austria) and R package was performed to visualize the expression differences. Paired t-test and Mann-Whitney U-test were performed to examine the differences between GBM and normal brain tissues. ROC curve was applied to verify the cutoff value of NMB using the pROC package [13]. Kaplan-Meier and log-rank tests were performed to assess the effect of NMB on survival rates.

To assess the mRNA expression of NMB in different cancer types, we obtained 19 cancer types from the analysis datasets that contained at least five samples in the control group. As shown in Fig. 1, NMB mRNA expression was significantly increased in 14 of the 19 cancer types compared with the control group. Compared with the control group, NMB mRNA expression was significantly decreased in breast invasive carcinoma (BRCA), kidney renal clear cell carcinoma (KICH) and lung squamous cell carcinoma (PRAD) cancers. The result showed that NMB mRNA expression was abnormal in all cancer types.

Fig. 1.

Pan-cancer expression pattern of NMB. The mRNA expression of NMB was increased in 14 of 19 cancer types compared with normal tissues. *p 0.05, **p 0.01, ***p 0.001. ns, not significance; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; ESCA, esophageal carcinoma; GBM, glioblastoma; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; STAD, stomach adenocarcinoma; THCA, thyroid carcinoma; UCEC, uterine corpus endometrial carcinoma.

To verify NMB mRNA expression in GBM, we analyzed NMB expression in TCGA database. The characteristics of GBM patients were analyzed from TCGA and Table 1 lists the baseline characteristics. Unpaired data analyses demonstrated that NMB mRNA expression in GBM (n = 169) significantly increased compared with control tissues (n = 5) (as shown in Fig. 2A, 2.565 $\pm$ 0.993 vs. 6.534 $\pm$ 1.293, Mann-Whitney U-test, p 0.001).

Fig. 2.

The mRNA and protein expression of NMB in GBM. (A) The mRNA expression levels of NMB in 169 GBM samples and five normal samples. ***p 0.001, compared with normal tissues. (B) The NMB protein expressed in normal brain tissue and GBM. The NMB expression sourced from the Human Protein Atlas. (C) The NMB protein is expressed and located in U-251 MG cells based on the Human Protein Atlas. The subcellular location of NMB protein expression was shown in green, the nucleus in blue, and the microtubules in red.

Next, we examined NMB protein expression in GBM using the HPA database. As shown in Fig. 2B,C, NMB expression, evaluated with immunohistochemical staining, was enhanced in GBM (Fig. 2C) compared with normal brain (Fig. 2B). NMB protein expression located in the cytoplasm and membrane of U-251 MG cells (Fig. 2D) using immunofluorescence staining. These results showed that both NMB mRNA and protein expression were increased in GBM patients.

To obtain a NMB value for distinguishing GBM from normal brain samples, we conducted ROC curve analysis. As shown in Fig. 3A, by analyzing the ROC curve was determined that the AUC value of NMB was 0.984 (95% CI: 0.971–0.997). At the 4.490 cutoff, the specificity and sensitivity of NMB expression were 96.2% and 96.4%, respectively. The positive and negative predictive values were 78.4% and 99.5%, respectively. The 4.490 cutoff was beneficial in distinguishing high NMB expression from low NMB expression in GBM patients. These results demonstrated that NMB expression could be a prospective biomarker for differentiating GBM patients from healthy individuals.

Fig. 3.

ROC and Kaplan-Meier curves for NMB. (A) ROC curve showed that NMB had an AUC value of 0.984 to discriminate GBM from healthy control tissues. With a cutoff of 4.490, the sensitivity and specificity were 96.4% and 96.2%, respectively. (B) Kaplan-Meier survival curves indicated that GBM patients with a high level of NMB mRNA expression had a long OS compared with those with low-level of NMB expression (16.3 vs. 12.7 months, p = 0.002). Low expression of NMB was associated with poor overall survival in datasets GSE4412_GPL96 (C) and GSE4271_GPL96 (D) analyzed using PrognoScan. HR, Hazard Ratio.

To determine the relationship between NMB expression and relative OS in GBM patients, Kaplan-Meier curves were constructed, and the PrognoScan database was applied to the analysis OS. As shown in Fig. 3B, the OS in GBM patients with higher expression levels of NMB was significantly longer than the OS in those with lower expression levels (12.7 vs. 16.3 months, p = 0.002). These results shown in Fig. 3C,D, also demonstrated that high NMB expression was associated with longer OS in GBM patients using two different datasets (GSE4412 and GSE4271). These results indicated that low NMB expression is one of the biomarkers of poor prognosis in GBM patients.

To construct the PPI networks of NMB and the relative functional annotations, we used the STRING database to construct the PPI network, and performed GO analysis and KEGG pathway analysis. As shown in Fig. 4A, the NMB PPI network was constructed including its 10 co-expression genes. Fig. 4B shows that these biological processes of NMB were associated with neuroactive ligand-receptor interactions, receptor ligand activity, and neuropeptide signaling pathways. Functional annotations showed that these genes participated in the axon initial segment and neuronal cell body, as well as in hormone activity. As shown in Fig. 4C,D, the correlation coefficients were significant between NMB expression and co-expressed genes in GBM patients.

Fig. 4.

PPI networks and functional enrichment analyses of NMB. (A) A network of NMB and its co-expression genes. (B) Functional enrichment analyses of 11 involved genes. NMB was associated with actin filament organization, regulation of actin filament-based process, and actin cytoskeleton organization. These genes were involved in purine ribonucleoside binding, GTP Binding, and GTPase Activity. (C,D) The correlation analyses between the expression of NMB and co-expressed genes in GBM. BRS3, bombesin receptor subtype 3; GRP, gastrin releasing peptide.

We performed a correlation analysis between NMB expression and TILs using the TIMER database. Fig. 5A shows that NMB expression was correlated with tumor purity (r = 0.105, p = 3.1 $\times$ 10${}^{-2}$), B cells (r = 0.143, p = 2.89 $\times$ 10${}^{-5}$), CD8${}^{+}$ T cells (r = 0.16, p = 1.02 $\times$ 10${}^{-3}$), macrophages (r = 0.136, p = 5.50 $\times$ 10${}^{-3}$), neutrophils (r = 0.135, p = 5.62 $\times$ 10${}^{-3}$), and dendritic cells (r = –0.112, p = 2.24 $\times$ 10${}^{-2}$). Next we determined the correlation between NMB expression and TILs in the TISIDB. Fig. 5B shows the relation between NMB expression and TILs in 28 human cancer types. Fig. 5C shows that NMB expression was associated with abundance of CD4${}^{+}$ T cells (r = –0.286, p = 2.03 $\times$ 10${}^{-4}$), CD8${}^{+}$ T cells (r = –0.194, p = 1.25 $\times$ 10${}^{-2}$), Th1 cells (r = –0.168, p = 3.09 $\times$ 10${}^{-2}$), Treg cells (r = –0.256, p = 8.93 $\times$ 10${}^{-4}$), memory B cells (r = –0.278, p = 2.97 $\times$ 10${}^{-4}$), NK cells (r = –0.191, p = 1.38 $\times$ 10${}^{-2}$), CD56${}^{\text{bright}}$ cells (r = 0.185, p = 1.71 $\times$ 10${}^{-2}$), and mast cells (r = –0.165, p = 3.41 $\times$ 10${}^{-2}$). These data demonstrated that NMB expression may play an important role in immune cell infiltration in GBM patients.

Fig. 5.

Correlation of NMB expression with TILs. (A) NMB expression is negatively related to tumor purity and is correlated with B cells, CD8${}^{+}$ T cells, neutrophils, macrophages, and dendritic cells in GBM. (B) Relationships between the NMB expression and TILs across 28 types of human cancers. (C) NMB was associated with an abundance of CD8${}^{+}$ T cells, CD4${}^{+}$ T cells, Th1 cells, Treg cells, B cells, NK cells, CD56${}^{\text{bright}}$ cells, and mast cells.

We measured the proliferation effect of NMB on glioblastoma using the CCK8 assay. U-87 MG cells are treated with 0-, 10-, 50-, and 100 $\mu$M NMB for 24-, 48-, and 72-h. The morphology of the glioblastoma cells becomes large and shows cell stretch out due to the increasing concentrations of NMB. After treatments with 10- and 50-$\mu$M NMB for 48 h to U-87 MG cells, the proliferation rates are 1.28 $\pm$ 0.27 and 1.59 $\pm$ 0.34. After treatments with 10- and 50-$\mu$M NMB for 72 h to U-87 MG cells, the proliferations are 1.40 $\pm$ 0.37 and 1.67 $\pm$ 0.45. Compared to the 24 h group, there was a significant increase in U-87 MG cell proliferations in the 48 h group and 72 h group (Fig. 6). These results show that NMB increased glioblastoma proliferation in a time-dependent manner.

Fig. 6.

The proliferation effects of NMB on glioblastoma cells. The proliferative effects of varying concentrations of NMB (10 uM, 50 uM, 100 uM) in U-87 MG cell lines were performed by the CCK8 assay. Untreated cells at 24-, 48-, and 72-h were used as a negative control (100% viability). The relative ratios of cell viability are given as mean $\pm$ SD. of three independent experiments. *p 0.05 and **p 0.01, compared to 24 h group.