RNA-seq and microarray data were retrieved from the GEO database (http://www.ncbi.nlm.nih.gov/geo/). Microarray data comprised GSE35493 (based on Affymetrix Human Genome U133 Plus 2.0 Array, GPL570) (11) and GSE39182 (based on Agilent-014850 Whole Human Genome Microarray 4×44K G4112F, GPL6480) (12). GSE35493 included 21 MB and 9 normal brain samples. GSE39182 included 20 MB and 5 normal samples. In addition, RNA-seq data GSE148389 (based on NextSeq 550, GPL21697) contained 14 normal and 26 tumor tissues (13). All probes were annotated by annotation files. Data processing was performed using R 3.6.0 software (https://www.r-project.org/). DEGs between MB and normal samples in the GSE35493 and GSE39182 microarray datasets were screened using the Limma package (14), and GSE148389 RNA-seq data were analyzed using the edgeR package (15). An adjusted P<0.05 and |log2 fold change (FC)|≥1 were set as thresholds to identify the DEGs. Venn diagrams (http://bioinformatics.psb.ugent.be/webtools/Venn/) were utilized to detect and present the common DEGs among the three datasets.

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID, http://davidd.ncifcrf.gov/), which is a database which can be used for processing functional annotation with gene lists (16). The DEG results were entered to obtain the enrichment of the biological process, molecular function and cellular component terms of GO analysis and KEGG pathway terms. A P<0.05 was considered to indicate a statistically significant difference.

To assess the functional associations among the upregulated DEGs, the Search Tool for the Retrieval of Interacting Genes (STRING, http://string-db.org/) database was used to construct the PPI network of DEGs (17). Interactions with a combined score >0.4 were considered significant. The PPI network was then visualized using Cytoscape (version 3.8.0; http://cytoscape.org/) (18). Subsequently, hub genes among the upregulated DEGs were identified using the Cytoscape plugin cytoHubba. The top 10 hub genes were calculated according to the maximal clique centrality (MCC) algorithm in cytoHubba (19).

At the beginning of the present study, a number of datasets comprising MB and normal brain samples were downloaded following a search of the GEO database. GSE35493, GSE39182 and GSE148389 were selected to identify DEGs between MB and normal samples as these three datasets had relatively larger sample sizes and were based on different platforms, which was considered beneficial for obtaining more DEGs. The remaining datasets were then used as validation sets to verify the high expression level of PBK in MB, including GSE74195 (20), GSE50161 (21), GSE42656 (22), GSE19360 (23), GSE109401 (24), GSE86574 (25) and GSE62600 (26). PBK expression in the four MB subgroups was also examined in datasets GSE85217 (27), GSE37418 (28) and GSE21140 (29). To date, only a few MB datasets contain survival information, of which GSE85217 is the one with the largest cohort, and the sample size of other datasets is too small to conduct the survival analysis for four subgroups. Therefore, the prognostic values of PBK were tested in GSE85217 with the clinical data of a large cohort of patients with MB (27). Survival curves were drawn using Graphpad Prism 9 (GraphPad Software, Inc.). Survival analysis was completed using the Kaplan-Meier method and overall survival was analyzed using the log-rank test.

The Daoy (HTB-186) and D341 (HTB-187) MB cell lines were obtained from the American Type Culture Collection (ATCC). Daoy cells were cultured in Dulbecco's modified Eagle's medium (DMEM; cat. no. C11995, Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; cat. no. 10270-106, Gibco; Thermo Fisher Scientific, Inc.), 1% penicillin/streptomycin (P/S; cat. no. 15140-122, Gibco; Thermo Fisher Scientific, Inc.) and 1% non-essential amino acid (NEAA; cat. no. 11140-035, Gibco; Thermo Fisher Scientific, Inc.). D341 cells were cultured in DMEM (cat. no. C11995; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 20% FBS, 1% P/S and 1% NEAA. Both cell lines were maintained under a 95% O₂ and 5% CO₂ humidified atmosphere in an incubator at 37°C. The PBK inhibitor, HI-TOPK-032, was purchased from MedChemExpress (cat. no. HY-101550).

MB cells were plated at a density of 8.5×10⁵ cells in 100-mm cell culture dishes and harvested following treatment with HI-TOPK-032 for 48 h. Daoy cells were treated at 0 (vehicle, DMSO), 1, 2 or 4 µM and D341 cells were treated at 0, 1 or 2 µM. Cells were lysed using RIPA lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology), and the protein concentrations were determined using the BCA kit (cat. no. P0012; Beyotime Institute of Biotechnology). Proteins were then separated using SDS-PAGE (10% gel for PBK, ERK1/2, Akt and β-tubulin; 12% gel for cleaved caspase-3 and GAPDH) and transferred to PVDF membranes (Merck Millipore). The membranes were blocked in 5% bovine serum albumin (cat. no. CCS30014.01, MRC, EN MOASAI Biological Technology Co., Ltd.) for 1 h at room temperature, and incubated overnight at 4°C with the following primary antibodies: Anti-PBK (1:1,000; cat. no. 4942S, Cell Signaling Technology, Inc.), anti-phosphorylated (p-)p44/42 MAPK (ERK1/2) (Thr202/Tyr204, 1:1,000; cat. no. AF1015, Affinity Biosciences), anti-p44/42 MAPK (ERK1/2) (1:1,000, cat. no. BF8004, Affinity Biosciences), anti-p-Akt (Ser473, 1:1,000, cat. no. T56569, Abmart Pharmaceutical Technology Co., Ltd.), anti-Akt (1:1,000, cat. no. T55561, Abmart Pharmaceutical Technology Co., Ltd.), anti-cleaved caspase-3 (1:500, cat. no. ab32042, Abcam), anti-β-tubulin (1:5,000, cat. no. AP0064, Bioworld Technology, Inc.) and anti-GAPDH (1:2,000, cat. no. TA-08, OriGene Technologies, Inc.). Subsequently, the membranes were incubated with secondary antibodies (peroxidase-conjugated goat anti-rabbit IgG, 1:5,000; cat. no. ZB-2301, or peroxidase-conjugated goat anti-mouse IgG, 1:5,000, cat. no. ZB-2305; both from OriGene Technologies, Inc.) for 1 h at room temperature. After washing, the membranes were visualized using an enhanced chemiluminescence reagent (Merck Millipore) and a Tanon 5200 Chemiluminescent Imaging System. Gel densities were measured using ImageJ software (Version 1.53m, National Institutes of Health).

Total RNA was extracted using TRIzol^® reagent (cat. no. 15596026, Thermo Fisher Scientific, Inc.) and the RNA concentrations were assayed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). cDNA was synthesized using reverse transcription kits (FastKing gDNA Dispelling RT SuperMix; cat. no. KR118, Tiangen Biotech, Co., Ltd.) according to the manufacturer's thermocycler guidelines (the reaction setup comprised of 4 µl of 5X FastKing-RT SuperMix and 1 µg of total RNA, and the final volume was made up to 20 µl with RNase-Free ddH₂O; temperature protocol: 15 min at 42°C for gDNA removing and reverse transcription followed by 3 min at 95°C for enzyme inactivation). The sequences of the PCR primer pairs were as follows: PBK forward, CCAAACATTGTTGGTTATCGTGC and reverse, GGCTGGCTTTATATCGTTCTTCT; and actin beta (ACTB) forward, CATGTACGTTGCTATCCAGGC and reverse, CTCCTTAATGTCACGCACGAT. qPCR was then performed according to the manufacturer's instructions [one cycle of initial denaturation at 95°C for 3 min, 40 cycles of PCR (5 sec at 95°C for denaturation and 15 sec at 60°C for annealing/extension), and ended with a melting/dissociation curve stage (15 sec at 95°C, 1 min at 60°C and 1 sec at 95°C)] at a final volume of 20 µl/well using SYBR-Green Talent qPCR Premix (10 µl; cat. no. FP209, Tiangen Biotech, Co., Ltd.), forward and reverse primers (0.6 µl, Sangon Biotech, Co., Ltd.), cDNA (1 µl/50 ng), ROX Reference Dye (0.4 µl, Tiangen Biotech, Co., Ltd.) and RNase-Free ddH₂O (7.4 µl, Tiangen Biotech, Co., Ltd.). The reaction was performed on the Applied Biosystems QuantStudio 3 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). In each run, the expression levels of PBK were normalized to those of ACTB as a housekeeping gene [i.e., the expression level of ACTB was set as 1; the ΔCq value was calculated as follows: ΔCq=Cq(PBK)-Cq(ACTB); the difference in the expression level between PBK and ACTB was 2^ΔCq-fold and the expression level of PBK was then normalized as 1/2^ΔCq].

The Cell Counting Kit-8 (CCK-8; cat. no. GK10001, Glpbio Technology Inc.) assay was used to detect the viability of the control and HI-TOPK-032-treated cells. The Daoy and D341 cells were seeded in 96-well plates at a density of 2,000 and 10,000 cells per well in 100 µl of culture medium, respectively. For the cytotoxicity assays, the cells were treated with HI-TOPK-032 at 0 (vehicle, DMSO), 0.5, 1, 1.5, 2, 2.5 and 3 µM. Following treatment for 24 h (Daoy cells) or 72 h (D341 cells), 10 µl of CCK-8 solution was added to each well followed by incubation for 3 h. The absorbance values were measured using an ELX808 microplate reader (BioTek Instruments, Inc.) at a wavelength of 450 nm. Cell viability was calculated using the absorbance value of the treated groups divided by the absorbance value of the control groups and multiplied by 100%. Sigmoidal dose-response curves were fitted using non-linear regression in Graphpad Prism 9 (GraphPad Software, Inc.) to determine the IC₅₀ values. For the cell proliferation assay, the absorbance values were measured following treatment with HI-TOPK-032 at 0, 1 or 2 µM for 0, 24, 48, or 72 h.

The cells were plated in 96-well plates at a density of 5,000 (Daoy cells) and 10,000 (D341 cells) cells per well and treated with HI-TOPK-032 at 0, 1 or 2 µM. Following incubation at 37°C for 24 h, EdU assay was performed using a Click EdU cell proliferation kit with Alexa Fluor 594 (cat. no. C0078, Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Images were obtained using an ECLIPSE Ti2-E inverted microscope (Nikon Corporation) and the percentage of EdU-positive cells was quantified using ImageJ software (Version 1.53m; National Institutes of Health).

The MB cells were seeded at a density of 1×10⁵ cells in 60-mm cell culture dishes and treated with HI-TOPK-032 at 0, 2 (D341 cells) or 4 µM (Daoy cells) for 24 h. The cells were then collected and washed twice with PBS followed by staining with Annexin V-APC/7-AAD (Apoptosis Detection kit; cat. no. KGA1017, Nanjing KeyGen Biotech Co., Ltd.) and Hoechst 33342 (cat. no. C1027, Beyotime Institute of Biotechnology) at room temperature for 10 min protected from light. After staining, the cells were transferred to a 24-well plate and fluorescence microscopy images were obtained using an ECLIPSE Ti2-E inverted microscope (Nikon Corporation). Positive cells were quantified using ImageJ software (Version 1.53m; National Institutes of Health).

Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software Inc.). All experiments were repeated three times. Quantitative results are presented as the mean ± standard deviation (SD). Statistically significant differences were assessed using an unpaired Student's t-test, one-way or two-way ANOVA with Tukey's HSD post hoc test. A value of P<0.05 was considered to indicate a statistically significant difference.

The present study first screened for genes differentially expressed between MB and normal brain samples to identify potential therapeutic targets in MB. Following RNA-seq and microarray data analyses, differential mRNA expression between MB and normal samples in three datasets was found (Fig. 2A). In GSE148389, 6,906 DEGs were identified (3,216 upregulated and 3,690 downregulated genes). In GSE35493, 5,568 DEGs were determined (2,793 upregulated and 2,775 downregulated genes). In GSE39182, 2,405 DEGs were detected (818 upregulated and 1,587 downregulated genes). Venn diagrams presented the overlapping DEGs in the three datasets. A total of 282 upregulated and 436 downregulated genes were identified as common between the three datasets (Fig. 2B).

Subsequently, the upregulated DEGs were submitted to DAVID to examine their functions and potential roles in the molecular tumorigenesis of MB. Following the enrichment analysis, the top 10 significant terms of GO analysis in three categories (biological process, molecular function and cellular component) and the significant KEGG enrichment terms were screened. In biological process, the upregulated genes were associated predominately with cell division, mitotic nuclear division, DNA replication and the G2/M transition of mitosis (Fig. 2C). In molecular function, the upregulated genes were involved primarily in protein binding, DNA binding and ATP binding (Fig. 2D). In addition, the cytosol, cytoplasm, nucleus and nucleoplasm were significantly associated with upregulated genes in the cellular component (Fig. 2E). The KEGG pathway analysis revealed that upregulated genes were enriched, particularly in DNA replication and the cell cycle (Fig. 2F).

To further investigate the functional associations among the upregulated DEGs, the STRING online database was utilized to analyze the PPI network of these genes. After removing isolated and partially connected nodes, a grid network was constructed using Cytoscape software (Fig. 2G). The Cytoscape plugin cytoHubba was then exploited to determine the hub genes in the PPI network. Finally, the top 10 hub genes [cell division cycle 20 (CDC20), kinesin family member 2C (KIF2C), nucleolar and spindle associated protein 1 (NUSAP1), PBK, TTK protein kinase (TTK), kinesin family member 20A (KIF20A), DNA topoisomerase II alpha (TOP2A), CDK1, assembly factor for spindle microtubules (ASPM) and Aurora kinase A (AURKA)] were determined using the MCC algorithm in cytoHubba (Fig. 2G).

According to the GO and KEGG pathway analysis, the upregulated genes were predominantly enriched in the cell cycle, DNA replication and cell division. Among the top 10 hub genes, PBK encodes PDZ-binding kinase, also known as T-lymphokine-activated killer (T-LAK) cell-originated protein kinase (TOPK), which is a serine/threonine protein kinase belonging to the mitogen-activated protein kinase kinase (MAPKK) family and plays a vital role in mitotic progression (30,31). Furthermore, researchers have previously reported that PBK is highly expressed in cerebellar granule cell precursors of early postnatal mice and functions as a crucial regulator of progenitor proliferation and self-renewal (32). Thus, given that MBs are embryonal tumors originating from stem cells or progenitor cells in the cerebellum or posterior fossa (33), the present study focused on the possible role of PBK in MB. The significant upregulation of PBK was first verified in MB by interrogating and analyzing additional independent gene expression datasets, including data from patients with MB (GSE74195, GSE50161 and GSE42656) and a spontaneous mouse model of MB (GSE19360) (Fig. 3A-D).

Since MB is currently divided into four subgroups, the expression level of PBK was also examined in the different subgroups. Of note, PBK was highly expressed in all MB subgroups compared with normal brain samples (Fig. 3E-G), suggesting that PBK may be involved in the tumorigenesis of all subgroups. This is consistent with the function of PBK as a mitotic serine/threonine kinase and the rapid proliferating rate of MB cells. In addition, PBK expression varied among the MB subgroups, with group 4 MBs appearing to have the lowest expression level compared to the other subgroups (Fig. 3H-J). Subsequently, the prognostic significance of PBK in MB was assessed by analyzing the survival information of patients with MB from a large cohort. The overall survival curves revealed that a high PBK expression was significantly associated with poorer clinical outcomes in SHH, group 3 and group 4 MBs, apart from WNT MB (Fig. 4). Taken together, these results indicate that PBK is a crucial upregulated hub gene in MB and is likely to serve as a prognostic marker.

To further examine the potential of PBK as a therapeutic target for MB, two commonly used MB cell lines, Daoy and D341 belonging to the SHH and group 3 respectively, were employed to perform experiments in vitro. First, PBK expression was detected in Daoy and D341 cells using RT-qPCR and western blot analysis. The results revealed that both cell lines had a robust expression of PBK (Fig. 5A and B). While the D341 cells exhibited a higher PBK mRNA expression, the Daoy cells appeared to have a higher protein expression, suggesting that the translation efficiency of PBK may differ between the two cell lines. However, this inconsistency may also be caused by the different reference genes (housekeeping genes) used in the two assays. The MB cells were then treated with a widely-used PBK inhibitor (HI-TOPK-032) to observe its effects on cell viability and proliferation. In the CCK-8 assay, the viability of the Daoy and D341 cells was effectively inhibited by HI-TOPK-032 in a concentration dependent manner with an IC₅₀ value of 1.241 and 1.335 µM, respectively (Fig. 5C and D). Moreover, HI-TOPK-032 also markedly decreased MB cell growth in a manner dependent on the degree of PBK suppression, as shown in Fig. 5E and F. Consistently, the two MB cell lines treated with HI-TOPK-032 at a concentration of 1 or 2 µM exhibited a lower proliferation rate than the control group in the EdU assay (Fig. 5G). These data demonstrated that targeting PBK with its specific inhibitor significantly impaired the proliferation of MB cells in vitro.

The present study then examined the phosphorylation status of two critical downstream targets of PBK to elucidate the mechanisms underlying the effects of HI-TOPK-032 on MB cell proliferation. Western blot analysis revealed that treatment with the PBK inhibitor, HI-TOPK-032, resulted in a slight or moderate reduction in the total levels of downstream signaling molecules, including ERK1/2 and Akt (Fig. 5H). However, a considerable decrement in the phosphorylated form of downstream proteins was observed in the HI-TOPK-032-treated MB cells (Fig. 5H). These two signaling molecules have been reported as essential downstream effectors of PBK in regulating cell proliferation (34,35). Additionally, the level of PBK in the MB cells remained stable following HI-TOPK-032 treatment (Fig. 5H), in accordance with previous studies (36,37). Taken together, these results suggest that HI-TOPK-032 may inhibit the proliferation of MB cells by suppressing the phosphorylation of downstream target proteins of PBK by blocking its kinase activity.

To examine the effects of PBK inhibitor specifically on cell survival, cell apoptosis was measured by monitoring the externalization of phosphatidylserine (PS) in MB cells treated with HI-TOPK-032. Since HI-TOPK-032 would remain in the cell pellets following centrifugation and its color would interfere with the accuracy of flow cytometry detection, Annexin V/7-AAD-positive cells were observed and quantified under a fluorescence microscope. Annexin V single-positive cells represent early apoptotic cells, while Annexin V and 7-AAD double-positive cells indicate late-stage apoptotic cells and necrotic cells. The percentage of Annexin V single-positive cells was markedly higher in the HI-TOPK-032-treated group as compared with the control group, and the proportion of double-positive cells also exhibited similar results (Figs. 6A and B, and 7A and B). Subsequently, the activation of caspase-3 was examined using western blot analysis to further examine the apoptosis of MB cells. In line with PS externalization, the level of cleaved caspase-3 in the Daoy and D341 cells was substantially increased following treatment with HI-TOPK-032 (Figs. 6C and 7C). Thus, targeting PBK also induces the caspase-dependent apoptosis of MB cells.