Fig 1.
The clinical relevance of CCNB1 in GBM.
(A-D) The expression level of CCNB1 mRNA in normal brain tissues, LGG and GBM tissues in CGGA-693 (A), CGGA-325 (B), CGGA-301 (C), and TCGA + GTEx (D) database. *p < 0.05. (E) The expression level of CCNB1 mRNA in LGG and GBM tissues in TCGA database. (F) CCNB1 protein level in UALCAN database. (G) Immunohistochemistry analysis of CCNB1 protein in normal brain tissues and GBM tissues obtained from HPA database. Scale bar = 200μm. (H-I) The relationship between CCNB1 mRNA expression and OS in GBM patients in CGGA (H), and TCGA (I) clinical cohorts. (J-K) ROC curve analysis of CCNB1 expression for 1-, 3-, and 5-year OS in CGGA (J), and TCGA (K) GBM clinical cohorts.
Fig 2.
scRNA-seq data analysis of CCNB1.
(A) T-SNE plot displaying the annotated 12 cell types in the scRNA-seq data from GBM tissue in CGGA database. Clusters are color-coded and labeled to highlight cellular heterogeneity. (B) Dot plot showing the expression patterns of selected marker genes across different cell types. Dot size indicates the percentage of cells expressing each gene, while color intensity represents the average expression level. (C) T-SNE of MKI67, CDK1, CCNB1 and PLK1 expression by proliferative cells. The KEGG (D) and GO (E) enrichment analysis of upregulated genes in proliferative cells subset.
Fig 3.
Spatial transcriptomics analysis of CCNB1.
(A) H&E staining of four tumor samples (269-T, 243-T, 265-T, 275-T) with zoomed-in regions. (B-C) Spatial expression patterns of key cell cycle-related genes (MKI67, CDK1, CCNB1, PLK1) across the tumor samples. Deeper blue color indicates higher expression of the gene at the corresponding spatial location. The differences in color intensity across different regions present the difference in gene expression levels among various regions in the spatial transcriptome. (D) Spatial functional spatial enrichment maps showing biological processes, including chromosome segregation, RNA splicing, regulation of cell cycle phase transition, DNA replication, and mitotic nuclear division. Color intensity indicates relative expression or activity levels, with darker shades representing higher expression/activity.
Fig 4.
Prognosis model of CCNB1 and its interacting genes in GBM cohorts.
(A) PPI network of CCNB1 and its interacting genes (CDK1, ESPL1, CDK2, CDC20, CKS2, PLK1, CKS1B, CDC27, ANAPC10, ANAPC4). (B) Forest plot of univariate Cox regression analysis of CCNB1 interacting genes in GBM patients. (C) RiskScore is obtained based on LASSO regression model (RiskScore = (−0.1498 × ESPL1) + (0.3847 × CDC20) + (0.2241 × CKS2) + (0.0842 × PLK1) + (0.6745 × ANAPC4) + (−0.2054 × CCNB1). The Kaplan-Meier curve showed that GBM patients in the high riskScore group had a worse prognosis (cutoff = the median riskScore). (D-E) The risk curve(D), and scatter plot (E) showed the relationship between risk of death and the riskScore. (F) Heat maps showed the relationship between CCNB1 and its interacting genes expression and prognosis in GBM patients. (G) Forest plot showing the hazard ratios and p-values for various clinical factors and molecular markers. Factors include gender, age, radiotherapy (Radio), chemotherapy (Chemo), IDH mutation, 1p/19q co-deletion, MGMT methylation, and risk score. (H) Nomogram predicting 1-, 3-, and 5-year survival probabilities, based on risk score, PRS type, gender, age, Radio, Chemo, IDH mutation, 1p/19q co-deletion, and MGMT methylation status. Each variable contributes points to calculate overall survival probability.
Fig 5.
The effects of CCNB1 to proliferation and cell cycle of U251 and U87 cells.
(A) WB assay was used to evaluate the efficiency and specificity of three siRNAs targeting the CCNB1. (B) EdU assay was used to assess the changes in DNA synthesis capacity of U251 and U87 cells following CCNB1 knockdown. (C) The quantification of the EDU positive rate (n = 3). (D) CCK8 assay detected the proliferation of GBM cells after CCNB1 knockdown (n = 3). Flow cytometric analysis of cell cycle distribution in U251 (E) and U87 (F) cells after CCNB1 knockdown. Left: representative DNA content histograms. Right: quantification of the percentages of cells in G1, S, and G2/M phases (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Fig 6.
Resveratrol limits the proliferation of GBM cells and inhibits CCNB1 expression.
(A) CCK8 assay detected the proliferation of U251 and U87 cells after resveratrol treatment (n = 3). (B) EdU assay was used to assess the changes in DNA synthesis capacity of glioma cells after resveratrol treatment. (C) The quantification of the EDU positive rate (n = 3). (D) Volcano plot showed the DEGs in resveratrol and PBS treated U251 cells. (E) The GO enrichment analysis of downregulated genes in U251 cells treated with resveratrol. (F) RT-qPCR detected the expression of CCNB1 and its interacting genes after resveratrol treatment in U251 and U87 cells (n = 3). (G) CCK-8 assay showing cell proliferation of U251 and U87 cells under siNC, siCCNB1, Res, and siCCNB1 combined with resveratrol treatments over time (n = 3). (H) Representative bioluminescent images (at 10, 17, 24, and 31 days) of the NPG mice were captured after U87 cells were intracranially injected into NPG mice. CON = control, RES = resveratrol. Ten-day post-tumor injection, resveratrol (50 mg/kg, 1 time/day) was administered orally for 28 days. (I) Upper panel: bioluminescent intensity of glioma-bearing mice on day 31 (n = 5), Lower panel: Kaplan‒Meier survival curves of glioma-bearing mice (resveratrol treatment group or PBS treatment group, n = 5). RES: resveratrol. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.