Open Access

CCNA2 and NEK2 regulate glioblastoma progression by targeting the cell cycle

  • Authors:
    • Hao-Yu Zhou
    • Yi-Chang Wang
    • Tuo Wang
    • Wei Wu
    • Yi-Yang Cao
    • Bei-Chen Zhang
    • Mao-De Wang
    • Ping Mao
  • View Affiliations

  • Published online on: March 13, 2024
  • Article Number: 206
  • Copyright: © Zhou et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Glioblastoma (GBM) is characterized by significant heterogeneity, leading to poor survival outcomes for patients, despite the implementation of comprehensive treatment strategies. The roles of cyclin A2 (CCNA2) and NIMA related kinase 2 (NEK2) have been extensively studied in numerous cancers, but their specific functions in GBM remain to be elucidated. The present study aimed to investigate the potential molecular mechanisms of CCNA2 and NEK2 in GBM. CCNA2 and NEK2 expression and prognosis in glioma were evaluated by bioinformatics methods. In addition, the distribution of CCNA2 and NEK2 expression in GBM subsets was determined using pseudo‑time analysis and tricycle position of single‑cell sequencing. Gene Expression Omnibus and Kyoto Encyclopedia of Genes and Genome databases were employed and enrichment analyses were conducted to investigate potential signaling pathways in GBM subsets and a nomogram was established to predict 1‑, 2‑ and 3‑year overall survival probability in GBM. CCNA2 and NEK2 expression levels were further validated by western blot analysis and immunohistochemical staining in GBM samples. High expression of CCNA2 and NEK2 in glioma indicates poor clinical outcomes. Single‑cell sequencing of GBM revealed that these genes were upregulated in a subset of positive neural progenitor cells (P‑NPCs), which showed significant proliferation and progression properties and may activate G2M checkpoint pathways. A comprehensive nomogram predicts 1‑, 2‑ and 3‑year overall survival probability in GBM by considering P‑NPCs, age, chemotherapy and radiotherapy scores. CCNA2 and NEK2 regulate glioblastoma progression by targeting the cell cycle, thus indicating the potential of novel therapy directed to CCNA2 and NEK2 in GBM.


Glioblastoma (GBM) is the most common type of malignant tumor in the central nervous system among adults and is associated with a poor survival rate (1). Despite significant advancements in surgical techniques, radiotherapy and chemotherapy, the prognosis for GBM remains dismal (2). The malignant behavior of cancer cells is regulated by oncogenes and tumor suppressor genes through various signaling pathways, which significantly affect the efficacy of clinical treatments and patient outcomes (3,4). The development of GBM requires specific molecular aberrations, including mutations in the P53 and retinoblastoma signaling pathways, as well as alterations in the receptor tyrosine kinase/Ras/phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways and the epithelial growth factor receptor (5,6). Despite efforts to target these abnormal changes, GBM treatment has not yielded favorable results. Hence, there is an urgent need to identify novel biomarkers and therapeutic targets in GBM.

A previous study revealed that NIMA related kinase 2 (NEK2) overexpression was significantly correlated with the grade, proliferation and prognosis of GBM (7). In addition, it enhances malignancy through the NIK/NF-κB pathway (8). The NEK family is a group of protein kinases that share similarities with NIMA kinase, found in higher eukaryotes (9). This family comprises 11 members, designated as NEK 1 to 11, with NEK2 exhibiting the highest similarity to NIMA (9). NEK2 is primarily located in cell centrosomes and is involved in various cellular processes, including centrosomal circulation during cell mitosis, formation of bipolar mitotic spindles (10) and stabilization of microtubules (11). Abnormal overexpression of NEK2 has been observed in several types of human cancers, including non-small cell lung cancer (12), myeloma (13), pancreatic (14) and breast cancer (15). This overexpression has been associated with various aspects of malignant transformation, such as tumorigenesis, therapy resistance and tumor progression (16).

Cyclin A2 (CCNA2) is a gene related to the cell cycle that has been suggested as a possible molecular marker in low-grade gliomas (17). This gene is in the Q27 region of human chromosome 4 and plays a crucial role in promoting the transition through the G1/S and G2/M phases of the cell cycle by binding and interacting with cyclin-dependent kinases CDK1 and CDK2 (18). Furthermore, the complete absence of CCNA2 leads to embryonic lethality in mice (19). CCNA2 is differentially expressed in several types of tumors in the digestive, urinary and central nervous systems (20). It has been found to play a regulatory role in the development of kidney cancer (20), breast cancer (21), lung cancer (22), colorectal cancer (23) and other types of tumors. Moreover, CCNA2 has been demonstrated to regulate epithelial-mesenchymal transition in distinct types of cancer, including oral squamous cell carcinoma (24), colorectal cancer (25) and bladder cancer (26). CCNA2 and NEK2 have been also identified as crucial genes in cancer transformation induced by hepatitis B (27) and the progression and prognosis of pancreatic cancer (28). However, the specific roles and correlation of CCNA2 and NEK2 with GBM have not been yet elucidated.

In the present study, it was observed that high expression of CCNA2 and NEK2 in glioma indicates poor clinical outcomes. Furthermore, CCNA2 and NEK2 were significantly co-expressed in neural progenitor cells (NPCs) at the single-cell level. Moreover, CCNA2 and NEK2, along with NPCs, were closely linked to the cell cycle in GBM and controlled the malignant advancement of tumor cells. Based on these findings, a comprehensive nomogram that supports a clinical prognosis analysis has been developed, which may prompt the development of new treatment strategies.

Materials and methods

Data collection

A total of 693 expression profiles of bulk sequencing and relevant clinical data for glioma were extracted from the Chinese Glioma Genome Atlas (CGGA; (29) and The Cancer Genome Atlas Program (TCGA; (30). The single cell RNA sequencing (scRNA-seq) expression profile and relevant clinical data of nine isocitrate dehydrogenase (IDH) wildtype GBM samples were collected from the Gene Expression Omnibus (GEO) database, with accession number GSE131928 (31). Data were obtained using 10X scRNA-seq.

Cellular clustering, gene analysis and cell type annotation of scRNA-seq

The gene expression matrix and corresponding clinical information for the nine IDH wildtype GBM samples were imported into RStudio (v.4.3.0; and analyzed using the Seurat package (v.4.0; R; (32). Downstream analysis was performed on the primary expression data, removing low-quality single cells. Cells with <300 expressed genes, >10% mitochondrial transcripts, >0.5% red blood cell transcripts, or genes that were expressed in fewer than three individual cells were also excluded. After excluding 2,841 low-quality cells, the expression data from the remaining 13,360 single cells were normalized using the ‘LogNormalize’ method. The top 5,000 highly variable features were then selected using the ‘FindVariableFeatures’ method, with variance stabilizing transformation. Next, the gene expression data were transformed using the z-score method and scaled by the ‘ScaleData’. The linear method was used to scale and center the top 5,000 highly variable features in this dataset, after which the principal component analysis was performed to reduce the dimensionality of the data. The top 5,000 most variable genes in the dataset were used for this analysis. The first 20 principal components (PCs) were analyzed using the ‘JackStrawPlot’ and ‘ElbowPlot’. These 20 PCs were then used for downstream calculations based on ‘FindNeighbors’ with default parameters. The resolution parameter applied to identify clusters by ‘FindClusters’ was 0.5, which was determined based on the range of 0.1 to 1 for the single-cell datasets. The t-distributed stochastic neighbor embedding (t-SNE) was employed to perform non-linear dimensionality reduction and visualize different single-cell clusters. Marker genes for each cluster were identified using ‘FindAllMarkers’. Only the top five significantly upregulated genes were selected for presentation in the heatmap, based on the following criteria: Adjusted P-value <0.05, minimal percentage >0.25 and log2-fold change (log2FC) >0.25. Subsequently, SingleR (Bioconductor-SingleR) and Cellmarker 2.0 ( were used together to annotate cell types for the different single-cell clusters (33,34).

Evaluation of stemness in single-cell clusters using scRNA-seq

Cellular trajectory reconstruction analysis using gene counts and expression (CytoTRACE) is an emerging computational method for evaluating the transcriptional diversity of each single-cell cluster in terms of differential or stemness status based on scRNA-seq. This method has been validated in large-scale datasets and has exceeded pre-existing computational strategies for evaluating stemness (35). The CytoTRACE package (v.0.3.3; CytoTRACE) was used to calculate the CytoTRACE score for each single-cell cluster. This score ranges from 0 to 1, with a higher score indicating greater stemness or fewer differentiation characteristics.

Pseudo-time trajectory analysis of scRNA-seq

The Monocle 2 package (v.2.28.0; Monocle; was utilized to perform trajectory analysis, assuming that the one-dimensional variable ‘time’ captures the high-dimensional expression characteristics and reveals the transformation of cell status based on scRNA-seq data (36). The cell types identified as astrocytes and NPC clusters were further analyzed for trajectory features, using Monocle 2. The ‘newCellDataSet’ function was applied to establish an analysis purpose with the parameter ‘expressionFamily=negbinomial.size’. Subsequently, the highly variable genes generated from the ‘VariableFeatures’ were utilized to reduce dimensions and sort cells in pseudo-time, using the ‘reduceDimension’ function with the ‘DDRTree’ algorithm. To identify candidate genes that separate cells into branches, a filtering criterion of ‘mean expression ≥0.5’ and ‘dispersion empirical ≥1× dispersion_fit’ was used. The branch expression analysis modeling (BEAM) was employed to analyze expression data and identify significant genes with a Q-value <0.0001. These genes were then grouped into different subgroups based on their expression patterns.

Estimating the cell cycle phase of scRNA-seq data

The tricycle package (v.1.8.0; was used to evaluate cell cycle phases by leveraging critical characteristics of cell cycle biology. This method has been positively compared with gold-standard experimental assays and has demonstrated significant predictive potential with multiple cell types or tissues in single-cell datasets (37). The preprocessed scRNA-seq dataset was evaluated for cell cycle phase, using the default parameters of the tricycle package. This package calculated a cell cycle position, represented by polar coordinates ranging from 0 to 2π, indicating distinct phases.

Estimating the proportion of positive NPCs (P-NPCs) in bulk sequencing samples

P-NPCs were defined as NPCs with CCNA2 and NEK2 expression levels >0. The marker genes were identified using ‘FindMarkers’, with a threshold of log2FC >1, an adjusted P-value <0.05 and a minimum percentage >0.70. The proportion of P-NPCs in bulk sequencing samples was evaluated through single-sample gene set enrichment analysis (ssGSEA), using marker genes.

Analysis of differentially expressed genes (DEGs) in bulk sequencing

Preprocessing of the expression profile from bulk sequencing included background correction, gene symbol transformation and normalization using RStudio programming. Significant DEGs in these datasets were found using the limma package (version 3.48.3; Bioconductor). Genes were considered as upregulation with adjusted P-value <0.05 and log2-fold change (log2FC) >1.5 and the downregulated genes had an adjusted P-value of <0.05 and a log2-fold change (log2FC) <-1.5.

Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA)

Candidate genes resulting from bulk sequencing analysis or scRNA-seq analysis were used to conduct GO and KEGG analyses, using the clusterprofiler package (v.4.8.1; Bioconductor). Gene sets were evaluated according to the hallmark gene sets in the MSigDB database ( (38). The results met the requirements, with an adjusted P-value of <0.05 (39). Furthermore, the GSEA was utilized to identify enriched pathways from the previous analyses of bulk sequencing or scRNA-seq. The significantly enriched results were subsequently validated using GSVA.

Establishing and assessing the predictive nomogram

To construct a nomogram that predicts the prognosis of patients with GBM, a multivariate Cox regression analysis to identify significant independent risk and protective factors was conducted. Based on these factors, a comprehensive nomogram was developed. The predictive potential of the nomogram was also demonstrated by the calibration curve. The suitability of the current predictors used in the nomogram was tested by evaluating the Schoenfeld residuals and deviance residuals to assess the proportional hazards assumption and identify outliers. Decision curve analysis (DCA) was also performed to assess the clinical applicability of the nomogram and the net benefit of diverse prediction models at different threshold probabilities. This was achieved by adding the benefits and minimizing the harms. The time-dependent receiver operating characteristic (ROC) curve was used to detect the discrimination of the nomogram and the Kaplan-Meier curve was used to estimate the prognostic value of the nomogram score.

Acquisition of glioma tissue

A total of 18 samples included World Health Organisation (WHO) II, III and IV grade gliomas were obtained from patient samples with a pathological diagnosis, who underwent craniotomy at the First Affiliated Hospital of Xi'an Jiao Tong University from April 2022 to December 2022. Of 18 patients, 8 were female and 10 were male. The mean age was 56 years (range, 42–69 years). The grades of the 18 glioma samples were confirmed by two independent pathologists. Inclusion criteria: i) Glioma patients meeting the 5th edition of the World Health Organization classification of tumors of the central nervous system in 2021; ii) All patients received conventional imaging examination within 1 week before surgery and were diagnosed with glioma; iii) the first diagnosed case without any invasive or non-invasive treatment; iv) Age above 18 years old, regardless of gender; v) The tumor was supratentorial. Exclusion criteria: i) Patients with other types of brain tumors; ii) patients who could not receive surgical treatment due to physical, financial or other reasons. The research involving the utilization of these biological specimens received ethical approval from the Ethics Committee of the hospital located in Xi'an, Shaanxi, China (approval no. 2020-G13). Informed consents for the utilization of clinical samples were approved and signed by patients.

Immunohistochemistry staining

For the immunohistochemical study, tissue blocks were fixed in 4% paraformaldehyde (PFA) at 4°C for 48 h, embedded in paraffin and sectioned at a thickness of 4 µm. The processed sections were blocked with 5% bovine serum albumin (BSA, EZ2811C238; BioFroxx) for 1 h at room temperature. Then the tissue sections were incubated with primary antibodies (1:100) to NEK2 and CCNA2 overnight at 4°C, after which they were incubated with biotinylated secondary antibodies (1:2,000) at room temperature for 1 h. Next, sections were incubated with horseradish peroxidase-conjugated avidin (PK4002; NEOBIOSCIENCE) at room temperature for 1 h, washed with PBS and stained with 3,3-diaminobenzidine (30 mg dissolved in 100 ml of Tris buffer containing 0.03% H2O2) at room temperature for 5 min. The sections were then rinsed in water and counterstained with hematoxylin at room temperature for 3 min. For the evaluation of NEK2 and CCNA2 expression, 10 visual fields per section were randomly selected and examined by light microscopy. The present study utilized the following antibodies: Mouse anti-NEK2 primary antibodies (1:100, sc-55601; Santa Cruz Biotechnology, Inc.) and rabbit anti-CCNA2 primary antibodies (1:100, ab181591; Abcam). Goat anti-rabbit IgG (1:2,000, ab97051; Abcam) and Goat anti-mouse IgG (1:2,000, PK4002; NEOBIOSCIENCE) was used as the secondary antibody. The results were analyzed using the ImageJ (v.1.8.0) software (National Institutes of Health).

Western blot analysis

The samples were prepared in RIPA buffer; Servicebio Biological) containing a protease inhibitor cocktail (MilliporeSigma) were used for western blot analysis. And the protein concentration was measured using a BCA protein assay kit (Beyotime Biotechnology). Equal amounts of protein lysates were loaded onto the wells (20 µg/lane) of a 10% precast SDS-PAGE gel and transferred to a PVDF membrane (Thermo Fisher Scientific, Inc.; Invitrogen). The membrane was then incubated with 5% skimmed milk for 1 h at 25°C, followed by overnight treatment with the target antibodies (anti-NEK2, 1:500; anti-CCNA2 primary antibody, 1:1,000) at 4°C. Following three washes with TBS-Tween 20 (TBST; 0.1% Tween) for 10 min each, the membrane was incubated with horseradish peroxide-conjugated secondary antibodies (1:5,000) for 1 h at room temperature. The Amersham ECL Western Blot System (Cytiva) was applied to visualize the protein expression levels of each sample, using GAPDH (1:5,000) as the loading control. The following antibodies were used: Mouse anti-NEK2 primary antibody, rabbit anti-CCNA2 primary antibody, mouse GAPDH antibody (1:5,000, sc-47724; Santa Cruz Biotechnology, Inc.), Goat Anti-Mouse secondary antibody (1:5,000, DY60203; DIYIBio) and mouse anti-rabbit secondary antibody (1:5,000, sc-2357; Santa Cruz Biotechnology). Western blot analysis results were analyzed using the ImageJ (v.1.8.0) software.

Statistical analysis

Statistical analyses were performed using RStudio. Data were presented as the mean ± standard deviation of triplicate determinations. The normality of the data distribution was evaluated by the Shapiro-Wilk test. Unpaired two-tailed Student's t-tests were performed to evaluate statistical differences in two groups and one-way ANOVA analyses following Tukey's multiple comparisons test was applied for comparisons between multiple groups and other statistical analysis methods were consistent with R packages listed in the manuscript. The log-rank test was utilized to conduct the Kaplan-Meier survival analysis. Multivariate Cox stepwise regression was selected for further analysis of survival. The P-value <0.05 indicated statistically significant differences.


Highly expression of CCNA2 and NEK2 in glioma

The TCGA and CGGA databases were utilized to investigate the functions of CCNA2 and NEK2 in glioma. Results indicated a significant increase in the expression levels of CCNA2 and NEK2 with the progression of WHO grade in GBM (Fig. 1A, B, D and E). A significant positive correlation between the expression of CCNA2 and NEK2 in GBM was also observed (Fig. 1C and F). The Kaplan-Meier survival analysis revealed that patients with high expression of CCNA2 and NEK2 had a shorter overall survival time in GBM and all grades (Fig. 1G-K). To investigate the consistency of CCNA2 and NEK2 expression levels in clinical glioma samples, IHC staining was performed on glioma samples. Results indicated that CCNA2 and NEK2 gene expression levels significantly increased with the progression of WHO grade in glioma (Fig. 1L). The western blot analysis of clinical samples yielded comparable results (Fig. 1M). Original figures of western blot were presented in Fig. S1.

Figure 1.

Highly expressed CCNA2 and NEK2 in glioma. (A) Analysis of NEK2 expression in different WHO grades of glioma, using TCGA database (*P<0.05 and ****P<0.0001, with independent t-test). (B) Analysis of NEK2 expression in different WHO grades of glioma, using TCGA database. (C) Co-expression analysis of CCNA2 and NEK2 expression in GBM, using TCGA database (P<0.0001, with Pearson correlation coefficient). (D) Analysis of NEK2 expression in different WHO grades of glioma, using the CGGA database. (E) Analysis of NEK2 expression in different WHO grades of glioma, using the CGGA database (****P<0.0001, with one-way ANOVA followed by Tukey's multiple comparison test). (F) Co-expression analysis of CCNA2 and NEK2 expression in GBM, using the CGGA database (P<0.0001, with Pearson correlation coefficient). (G) The Kaplan-Meier analysis of overall survival in patients with glioma based on CCNA2 expression, using the CGGA database (P<0.0001, with log-rank test). (H) The Kaplan-Meier analysis of overall survival in patients with GBM based on CCNA2 expression, using the CGGA database (P=0.012, with log-rank test). (I) Kaplan-Meier analysis of overall survival in patients with glioma based on NEK2 expression, using the CGGA database (P<0.0001, with log-rank test). (J) The Kaplan-Meier analysis of overall survival in patients with GBM based on NEK2 expression, using the CGGA database (P=0.026, with log-rank test). (K) The Kaplan-Meier analysis of overall survival in patients with glioma and GBM based on CCNA2 and NEK2 expression, using the CGGA database (P<0.0001, for all glioma and P=0.013, for GBM, respectively, with log-rank test). (L) Immunohistochemical staining of CCNA2 and NEK2 in GBM samples, compared with adjacent normal tissue. (M) Protein expression of CCNA2 and NEK2 in GBM samples, compared with adjacent normal tissue. T, tumor tissue; N, adjacent normal tissue. Original blots are presented in Fig. S1. Western blot and immunohistochemical analyses were conducted to detect CCNA2 and NEK2 protein levels in GBM samples (*P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001, with independent t-test). Data represent the mean ± SD of triplicate determinations from three independent experiments. CCNA2, cyclin A2; NEK2, NIMA related kinase 2; WHO, World Health Organization; TCGA, The Cancer Genome Atlas; GBM, glioblastoma; CGGA, Chinese Glioma Genome Atlas.

CCNA2 and NEK2 are co-expressed in NPC subtypes

Although a consistent expression of CCNA2 and NEK2 was demonstrated, the heterogeneity of GBM contributed to ambiguity regarding the reciprocal regulation within the same cells (40). Therefore, the expression pattern of CCNA2 and NEK2 was explored using scRNA-seq information collected from the GEO database (dataset no. GSE131928) to identify whether they were expressed in the same cell types. After conducting quality control, low-quality cells were eliminated and 13,360 cells were retained for further analysis (Fig. S2). Cells from nine patients were clustered into 16 distinct groups, using t-SNE dimensionality reduction (Fig. 2A). To differentiate between cell types in the GBM microenvironment, immune and non-immune clusters were classified based on marker genes identified in previous studies (4158). Genes such as C1QB (41), CD68 (42), CSF1R (43), PTPRC (44), C1QC (45), P2RY12 (46), CX3CR1 (47), CD163 (48), PTGS2 (49) and CD86 (50) were used to identify immune clusters; whereas genes AQP4 (51), PTPRZ1 (52), CLDN5 (53), CD34 (54), GFAP (55), FDGFRA (56), OLIG2 (57) and PLP1 (58) were used to identify non-immune clusters. Clusters 0, 2, 6, 14 and 15 were identified as immune cells, whereas clusters 1, 3, 4, 5, 7, 8, 9, 10, 11, 12 and 13 were identified as non-immune cells based on the average expression of PTPRC, as demonstrated in Fig. 2B and C. The top five significantly DEGs (marker genes) among these 16 clusters are presented in Fig. 2D. Based on the information provided by the singleR and CellMarker databases, nine distinct cell types out of the 16 clusters that were identified through the use of ‘FindAllMarkers’ were accurately annotated (Fig. 2E). According to Neftel et al (31), the following cell types were considered malignant: Astrocytes, cancer stem cells, mesenchymal, NPCs, oligodendrocytic precursor cells and radial glia. Notably, the results showed significant heterogeneity in the proportions of various cell types present among different patients (Fig. 2F). Consequently, gene expression levels were analyzed in various cell types, revealing that CCNA2 and NEK2 were expressed in NPCs, belonging to cluster 11, which were derived from patient ‘MGH143’ (Fig. 2G-I). Although clusters 3, 11 and 13 were defined as NPCs, only cluster 11 expressed CCNA2 and NEK2, indicating that different subtypes of NPCs may possess distinct functions. CCNA2 is a ubiquitously expressed member of the cyclin family (59,60), it is commonly associated with cell proliferation and it is expressed at high levels in many cancers (61). NEK2 is a core component of the human centrosome (62), where it regulates a key step in the centrosome cycle, namely centrosome disjunction (63), which has been associated with the progression of a variety of cancers (64). Therefore, it was hypothesized that cluster 11, which co-expressed CCNA2 and NEK2, has the potential to stimulate cell proliferation and differentiation.

NPCs exhibit significant stemness in GBM

As aforementioned, it was revealed that cluster 11, which exhibited high expression of CCNA2 and NEK2, was upregulated in the ‘MHG143’ patient. GBM cells obtained from this patient were then divided into 10 clusters, using t-SNE dimensionality reduction (Fig. 3A). Marker genes were identified as the top three differential genes in each cluster (Fig. 3B). According to the SingleR and CellMarker databases, four cell types including NPC, astrocytes, neurons and tumor-associated macrophages (TAMs) were annotated. Clusters 3 and 4 were identified as NPCs (Fig. 3C. Since NPCs may function as the initiating cells of GBM (65,66), these four cell types were evaluated using the CytoTRACE package. The results demonstrated that NPCs had a higher potential to proliferate and differentiate (Fig. 3D and E). Moreover, when projected into these four cell types, CCNA2 and NEK2 were co-expressed in cluster 3 (Fig. 3F-H).

The trajectory of GBM cell states reveals branched progression

Based on the aforementioned results, it was found that NPCs showed a higher level of stemness, indicating that these cell types may contribute to the initiation and progression of GBM. In addition, CCNA2 and NEK2 expression levels significantly increased in NPCs. To comprehend the function of CCNA2 and NEK2 in GBM, the pseudo-time analysis was utilized to delineate the differentiation pathways of astrocytes and NPCs from the ‘MHG143’ patient, employing the Monoce 2 package. As depicted in Fig. 4A and B, it was observed that astrocytes and NPCs followed a trajectory of differentiation with two branched progressions originating from NPCs. It was observed that NPCs were mainly in the first half of pseudo-time trajectory, whereas astrocytes were mainly in the second half. Furthermore, NPCs highly expressed CCNA2 and NEK2, which were related to cell cycle. It was hypothesized that NPCs may differentiate into astrocytes, but the specific differentiation mechanism remains unclear. Furthermore, when examining the different clusters, it was observed that clusters 3 and 4 (NPCs) were primarily located in the starting position, whereas the remaining clusters were dispersed throughout various time periods (Fig. S3). This supported the hypothesis that NPC contributes to the onset of GBM. Moreover, as demonstrated in Fig. 4C, it can be observed that CCNA2 and NEK2 expression levels were higher at the beginning and end stages of GBM and exhibited a ‘U’-shaped pattern of distribution (Fig. 4D). It was hypothesized that CCNA2 and NEK2-highly expressing NPCs may have an advantage to grow in the middle and later development of GBM, which has polyclonal sources (67), but it requires further research to investigate it.

As aforementioned, astrocytes and NPCs follow two different paths at a distinct bifurcation point. According to earlier studies, cells on different trajectories exhibit distinct cellular functions or procedural changes. Therefore, analyzing bifurcation points in cells will help understand the underlying genes responsible for these changes (68,69). The BEAM analysis was performed on this bifurcation point, resulting in the clear division of 1,542 genes into three clusters with distinct expression patterns. Cluster 3 comprises 190 genes, including CCNA2 and NEK2, which were found to be overexpressed in the primary stage, based on a branched expression pattern in pseudo-time dimension. This suggested that these genes may play a role in the progression of GBM (Fig. 4E). Furthermore, the GSEA analysis revealed that these clusters exhibited distinct functions. Specifically, cluster 3 was associated with chromosome segregation, mitotic nuclear division, sister chromatid segregation, nuclear chromosome segregation and nuclear division (Fig. 4F), which are related to the cell cycle.

GBM prognosis may be associated with P-NPCs

As aforementioned (Figs. 2 and 4), CCNA2 and NEK2 were co-expressed in a group of early active cells known as NPCs. To investigate the clinical significance of these cells in patients with GBM, marker genes that were differentially expressed between P-NPCs and negative cells in the patient ‘MGH143’ were identified (Fig. 5A). The ‘AddModileScore’ was utilized to assess all single-cell datasets and validate the suitability of marker genes (Fig. S4). The results were consistent with those shown in Fig. 2I, thus indicating that this set of genes accurately represents P-NPCs. Subsequently, ssGSEA was employed to investigate the expression of marker genes in gliomas of varying grades in the CGGA database. Results indicated a significant difference in expression levels with increasing WHO grades, as demonstrated in Fig. 5B. Comparable results were observed in TCGA database (Fig. 5C). As revealed in Fig. 5D, the goodness-of-fit was investigated, and the results indicated that the proportion of P-NPCs was more appropriate in GBM, suggesting that P-NPCs may be more representative in GBM. The Kaplan-Meier analysis demonstrated that patients with high expression of marker genes exhibited a shorter overall survival in GBM and gliomas of all grades (Fig. 5E). These results indicated that marker genes, which may represent P-NPCs, may be utilized to differentiate the WHO grade of GBM patients and assess their prognosis.

P-NPCs are associated with the co-expression of CCNA2 and NEK2 in the G2M checkpoint pathway

Based on the aforementioned survival analysis for patients with GBM in the CGGA database (Fig. 5), a total of 27 patients with poor survival and 106 patients with improved survival rates were observed. An analysis of DEGs in patients with varying prognoses was then conducted. Results indicated that 455 genes were significantly upregulated, whereas 29 genes were significantly downregulated (Fig. 6A). Enrichment analysis of GEO and KEGG pathways for the DEGs indicated that the low survival rate of patients with GBM was associated with increased activity in mitosis and cell cycles (Fig. 6B and C). Subsequently, the GSEA analysis revealed that five pathways, namely DNA repair, epithelial-mesenchymal transition, G2M checkpoint, hypoxia and MTORC1 signaling, were significantly activated in patients with poor survival (Fig. 6D). To validate the aforementioned results, ssGSEA analysis was utilized to reevaluate them. As a result, a total of 23 pathways, which included the five aforementioned pathways, were found to be significantly enriched (Fig. S5). Furthermore, a correlation between the ssGSEA score of P-NPCs and the expression of CCNA2 and NEK2 in the G2M checkpoint pathway was observed (Fig. 6E). These results suggested that the differential prognosis of these patients may be related to the role of P-NPCs in driving the cell cycle and that the G2M checkpoint may be a key factor in these differences.

G2M checkpoint pathway is activated in P-NPCs

NPCs from patient ‘MGH143’ exhibited high activity levels, but the specific functions of the other cells remain unclear. To further examine the underlying pathways in scRNA-seq, TAMs and neurons were removed. Next, the marker genes of the remaining eight clusters were used to conduct significant differential pathway analysis through GSEA. As a result, hallmark pathways were significantly enriched, including mTORC1, hypoxia, Myc-targets-v1, TNFα-signaling-via-NF-κB and G2M checkpoint (Fig. 7A). The top four pathways (mtorc1, cell cycle, oxidative, and NF-κB) were selected to generate butterfly plots, based on the enrichment score of each cluster (Fig. 7B). Clusters 3 and 4 belonged to NPCs, as demonstrated in Fig. 3C. These clusters revealed a strong proliferation of E2F targets and G2M checkpoints, as depicted in Fig. 7A. Furthermore, they were assigned to cell cycle quadrants, as illustrated in Fig. 7B. However, they were not equally distributed among the four quadrants. Cluster 3, as compared with cluster 4, exhibited significantly enriched pathways, including the mitotic spindle and the G2M checkpoint (Fig. 7C). This indicated a stronger association with the cell cycle. The tricycle analysis (37) was used to evaluate the cell cycle position of single-cell data from patients. The position of cells in the cell cycle was denoted by an angle θ and cells in the same stage always appeared at a similar θ. To accurately represent the position of cells in the cell cycle, a circular color scale that considers the circular nature of the cell cycle was utilized, with positions ‘wrap around’ from 0 to 2π (Fig. 7D). For instance, cells at the G2/M stage are represented by colors centered at 1.75π (Fig. 7D). It was revealed that cluster 11 (NPCs) accounted for a high proportion of cells in the G2/M phase (Fig. 7D). In general, NPCs or a subset of them (cluster 3) were strongly enriched at the G2M checkpoint. These cells may contribute to GBM cell population proliferation and disease progression.

Establishment and evaluation of a nomogram with the TCGA dataset

The forest plot presented P-NPCs, age and chemotherapy score as independent risk factors (Fig. S6). As radiotherapy has been widely used in clinical practice and its effectiveness has been previously reported in the literature (70,71), it was included in the present study. The Schoenfeld residual test indicated that all variables equally satisfied the assumption of proportional hazards (Fig. S6). Outliers were not observed, based on the deviance residual test (Fig. S6). After considering all the aforementioned significant predictive factors, a comprehensive nomogram that includes P-NPCs, age, chemotherapy and radiotherapy score was developed (Fig. 8A). The calibration curves for one, two and three years revealed a satisfactory calibration efficiency. A closer alignment with the dashed line indicated an improved prediction performance (Fig. 8B). The DCA was used to evaluate the clinical application of the nomogram and the net benefit of various prediction models at different threshold probabilities. This was achieved by weighing the benefits against the harms and minimizing the latter. The comprehensive nomogram demonstrated a more favorable probability and an improved net benefit compared with P-NPCs and clinical index, as shown in Fig. 8C-E. Subsequently, the nomogram score model and clinical index score model were evaluated using the concordance index. Results demonstrated that the nomogram score had a higher prediction accuracy than the clinical index score (Fig. 8F). Furthermore, the time-dependent ROC curve analysis demonstrated that the predictive performance of the nomogram gradually improved over time (Fig. 8G). The Kaplan-Meier analysis indicated that a higher nomogram score was associated with a poorer prognosis for patients with GBM (Fig. 8H). Therefore, a comprehensive nomogram was established based on multiple prognostic factors that exceeded the predictive power of each factor alone. The nomogram may assist clinicians in making more precise assessments of patient prognosis.


The cell cycle, a complex process tightly regulated by various proteins (67), is closely linked to the development and advancement of cancer (72,73). NEK2 is a member of the NIMA-related family of serine/threonine protein kinases, which are considered to play a role in regulating the cell cycle (74). Upregulation of NEK2 in human cells leads to premature splitting of the centrosome, whereas overexpression of a NEK2 kinase-dead mutant results in centrosome abnormalities and aneuploidy (10,75). These factors are significant contributors to tumorigenesis (76). NEK2 has two splice variants, namely NEK2A and NEK2B. NEK2A is necessary for centrosome separation during the G2/M transition (77). The abnormal expression of NEK2A may play a role in regulating genetic stability and tumorigenesis. In the present study, a positive correlation was observed between the expression of CCNA2 and NEK2 and the progression of glioma. Furthermore, CCNA2 and NEK2 were upregulated in a subset of P-NPCs in GBM, which exhibited significant proliferation and progression properties. CCNA2 is located on chromosome 4 and is encoded by the human CCNA2 gene. Belonging to the highly conserved cyclin family, this protein promotes progression through the S-phase and transition from G2 to M phase by binding to CDK in the mitotic cell cycle (78). Jiang et al (79) proposed that CCNA2 promotes proliferation, migration, invasion and regulates macrophage polarization in glioma. It is recognized that changes in cell cycle proteins may trigger cancer. CCNA2 and NEK2 are classified as cell cycle regulatory genes, which are crucial in the advancement and prognosis of pancreatic cancer (28). However, the correlation and underlying mechanism between CCNA2 and NEK2 has not been fully explored in GBM. The present study presented a novel finding on the co-expression of CCNA2 and NEK2 in GBM and their association with NPCs, thus suggesting their involvement in regulating the cell cycle.

GBM are the most common malignant tumors in the nervous system, which are known for their rapid progression and short survival rates (80,81). Therapeutic approaches for GBM have slowly progressed, unlike other multimodal therapies for tumors (82). Ongoing efforts are being made to understand the highly heterogeneous nature of GBM. Differences have been observed among various tumor types, individuals with identical diagnoses, non-tumor cell types and states and individual tumor cell clones (83). NPCs are the precursor cells of the central nervous system (CNS) that generate many, if not all, of the glial and neuronal cell types that populate the CNS (84). It has been proposed that NPCs are the preferred cell of origin for GBM (85). In addition, NPCs have a strong potential to migrate (86), renew (87) and maintain the population (88,89). In the present study, patients with GBM were analyzed at the single-cell level and it was discovered that CCNA2 and NEK2 were co-expressed in NPCs. These findings suggested that they may have a significant role in the development and progression of GBM. Furthermore, the abundance of NPCs in patient ‘MGH143’ was higher during the initial stages of tumorigenesis and was similar to the peak expression of CCNA2 and NEK2. Therefore, it was hypothesized that changes in CCNA2 and NEK2 may drive the progression of NPCs and ultimately contribute to the development of GBM. CCNA2 and NEK2 were found to be differentially expressed in NPC subtypes, indicating cellular heterogeneity in the glioma-associated microenvironment. This finding agrees with previous research on NPCs heterogeneity (90).

In the present study, NPCs exhibiting high expression levels of CCNA2 and NEK2 as P-NPCs were defined. Moreover, the marker genes based on P-NPCs significantly distinguish the prognosis of patients with GBM. In GBM, NPCs highly expressed stemness-associated cell membrane antigens such as CD133, CD15/SSEA, CD44, or A2B5 and intracellular markers such as Sox2 and Nestin (91,92) and have demonstrated the potential for cellular transitions (31). Similar to normal neurogenesis processes, NPCs may generate more differentiated phenotypes with astrocytic features in GBM (91). As aforementioned, CCNA2 and NEK2 are closely related to the cell cycle, which is responsible for cell differentiation and fate (93). The origin of glioma cells is polyclonal (40) and in the development of cancer, the population with competitive advantages will be selected to develop (94). Therefore, it was hypothesized by the authors that CCNA2 and NEK2 are only expressed in some NPC subtypes, which may have a competitive advantage, but the specific process requires further discussion. In the analysis of different pathways among these patients with varying prognoses, it was found that the G2M checkpoint was highly enriched. The G2/M checkpoint prevents cells with damaged DNA from entering mitosis, allowing repair of DNA that was damaged during the late S or G2 phases before mitosis. A weakened G2/M checkpoint under therapeutic conditions may trigger cell death through mitotic catastrophe in cells with irreparable DNA damage and faulty mitotic machinery (95). It was revealed that the G2M checkpoint is significantly increased in patients with poor prognosis. This increase may inhibit mitotic catastrophe, which in turn promotes rapid tumor growth. Currently, there are numerous drugs that target the G2M checkpoint (96). The present study may offer a novel approach to the pharmacological mechanism. By recognizing the significance of P-NPCs, a comprehensive nomogram that incorporates clinical characteristics to predict patient prognosis more accurately has been developed.

However, the data on the regulatory relationship between NEK2 and CCNA2 expression in GBM are limited, which cannot accurately reflect the heterogeneity of glioma. At the same time, single-cell sequencing loses the information of spatial location, thus it is impossible to characterize their co-localization relationship in spatial location. Mapping research through multiple samples will be conducted. Moreover, the authors have marginal information on the regulatory association between CCNA2 and NEK2 in GBM and the role they play in different stages of the cell cycle is unclear, thus more experiments are needed to determine their regulatory role in GBM. To date, it is not feasible to precisely determine the composition of these cells before surgery. This information is only inferred through postoperative sequencing. To address this practical issue, a non-invasive preoperative assessment method should be developed for these cells.

Supplementary Material

Supporting Data


Not applicable.


The present study was supported by the National Natural Science Foundation of China (grant nos. 82072781 and 81602207) and Medical Foundation-Clinical Integration Program of Xi'an Jiaotong University (grant no. YXJLRH2022040).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

MDW and PM conceptualized and designed the study. HYZ, TW and WW developed methodology. HYZ, WW, YYC and YCW acquired data. HYZ, BCZ and WW analysed and interpreted the data. HYZ, WW and YYC wrote the manuscript. YYC performed experiments. HYZ and YCW confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

The WHO grade II, III and IV gliomas were obtained from patient samples with a pathological diagnosis who underwent craniotomy at the First Affiliated Hospital of Xi'an Jiao Tong University (Shaanxi, China). The research involving the utilization of these biological specimens received ethical approval from the Ethics Committee of the hospital located in Xi'an, Shaanxi, China (approval no. XJTU1AF2022LSK-428). Informed consent was obtained through the signing of consent forms for the utilization of clinical samples, all experimental protocols used in the present study were in accordance with the guidelines of the Declaration of Helsinki.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.



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Volume 27 Issue 5

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Zhou H, Wang Y, Wang T, Wu W, Cao Y, Zhang B, Wang M and Mao P: CCNA2 and NEK2 regulate glioblastoma progression by targeting the cell cycle. Oncol Lett 27: 206, 2024
Zhou, H., Wang, Y., Wang, T., Wu, W., Cao, Y., Zhang, B. ... Mao, P. (2024). CCNA2 and NEK2 regulate glioblastoma progression by targeting the cell cycle. Oncology Letters, 27, 206.
Zhou, H., Wang, Y., Wang, T., Wu, W., Cao, Y., Zhang, B., Wang, M., Mao, P."CCNA2 and NEK2 regulate glioblastoma progression by targeting the cell cycle". Oncology Letters 27.5 (2024): 206.
Zhou, H., Wang, Y., Wang, T., Wu, W., Cao, Y., Zhang, B., Wang, M., Mao, P."CCNA2 and NEK2 regulate glioblastoma progression by targeting the cell cycle". Oncology Letters 27, no. 5 (2024): 206.