The GEPIA database (http://gepia.cancer-pku.cn/), an online resource for interactive gene expression profiling, was used to analyze CAMK2B expression across various tumor and normal tissue types. Additionally, RNAseq data for glioma samples were obtained from the TCGA (https://portal.gdc.cancer.gov), CGGA (http://www.cgga.org.cn), and Rembrandt (https://gliovis.bioinfo.cnio.es/) databases, and subsequently curated for downstream analysis. The mRNA transcriptome data were converted into the FPKM format for further analysis using the log₂(value + 1) method. All prognostic data for patients were obtained from the relevant websites. Additionally, all the information from the databases was accessed in August 2022. The median expression level of CAMK2B was used as the cut-off value to classify glioma patients into high and low-expression groups. The Kaplan-Meier survival analysis was conducted using the “survminer (version 3.3.1)” and “survival (version 0.4.9)” packages in R (version 4.4.1) to investigate the association between CAMK2B expression and patient prognosis.

Human astrocytes (HA) were acquired from ScienCell (Carlsbad, CA, USA). Three human glioma cell lines (U251, U87, and A172) were acquired from Procell Life Science & Technology Co., Ltd. (Wuhan, China). All cells were identified by Short Tandem Repeat (STR) and there was no risk of mycoplasma infection. The Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco™, 11875093, Thermo Fisher Scientific, Grand Island, NY, USA) was used to culture the U251 cells. The U87 cells were cultured in Minimum Essential Medium (MEM, Gibco™, 11090081, Thermo Fisher Scientific), while the A172 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco™, 10564011, Thermo Fisher Scientific). The HA cells were cultured in an Astrocyte medium (AM, Sciencell™, #1801, Carlsbad, CA, USA). All culture media were supplemented with 10% fetal bovine serum (FBS, Gibco™, 10099141, Thermo Fisher Scientific) and 1% penicillin-streptomycin. Cell culturing was conducted in a cell incubator at 37°C and 5% carbon dioxide. The Ras pathway inhibitor salirasib (SML1166, Sigma-Aldrich Corporation, St. Louis, MO, USA) was diluted to 150 μM with DMSO, co-cultured with the cells scheduled for the experiment for 48 h, and then the subsequent experiments could be conducted.

The CAMK2B−coding sequence was synthesized and cloned into the pcDNA 3.1 vector by Hanbio Biotechnology Co., Ltd. (Shanghai, China) to construct a CAMK2B overexpression plasmid (pcCAMK2B). The blank pcDNA3.1 plasmid was used as a negative control (NC) plasmid. The siRNA for silencing CAMK2B was designed and synthesized by Thermo Fisher Scientific. The targeting sequence for siCAMK2B was CAGAAGAATGGTACAAATCCAAG. Plasmids (8 μg/10⁶ cells) or small interfering RNA (siRNA) (60 pmol/10⁶ cells) were transfected in glioma cells using Lipofectamine 3000 (L3000015, Thermo Fisher Scientific), according to the product instructions. After 48–72 h, all cells were collected and used in the follow-up experiments.

A total of 79 glioma tissue samples and 4 non-tumorous brain tissue samples (excised from patients undergoing brain trauma surgery) were attained from the Second Hospital of Hebei Medical University on 15 December 2021. The surgical collection of tissue samples was conducted between 24 September 2014, and 09 March 2019. Patient follow-up for survival analysis was completed on 20 December 2021. The remaining samples were stored in 10% paraformaldehyde for immunohistochemistry (IHC) experiments. This research was approved by the ethics committee of the Second Hospital of Hebei Medical University (Approval No. 2021-R494). Informed and written consent was obtained from either the patient or their guardian following the guidelines provided by the ethics committee.

Total RNA was extracted from untreated and transfected U251 and U87 glioma cells using Trizol reagent (15596026CN, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. After RNA quantification, cDNA was synthesized using a reverse transcription kit, HiScript^® III RT SuperMix for qPCR (+gDNA wiper) (R232-01, Vazyme, Nanjing, China), according to the manufacturer’s instructions. With GAPDH as the standardized internal reference, AceQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) was used to conduct the PCR procedure. The qPCR thermal cycling conditions were as follows: an initial denaturation step at 95°C for 10 min (1 cycle), followed by 40 amplification cycles consisting of denaturation at 95°C for 5 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. The primer sequences (Species: Human) of CAMK2B were as follows: F, 5^′-GCAAAGAGGCGTATGGCAAG-3^′; R, 5^′-GACGGGAAGTCATAGGCACC-3^′. The primer sequences (Species: Human) of GAPDH were as follows: F, 5^′-GGAGCGAGATCCCTCCAAAAT-3^′; R, 5^′-GGCTGTTGTCATACTTCTCATGG-3^′. The relative gene expression was determined using the 2^−ΔΔCt method, and GAPDH was used as the reference gene for normalization.

The tissue samples were fixed in formalin, embedded in paraffin, and subsequently sectioned for histological analysis. The tissue sections were incubated at 60°C for 30 min, immersed in xylene, and washed using graded ethanol to remove paraffin. The tissue sections were subjected to antigen retrieval by immersion in 2% citric acid buffer under high temperature and pressure conditions for 25 min, followed by treatment with endogenous peroxidase to inhibit non-specific background staining. Following antigen retrieval, the tissue sections were blocked with 10% goat serum. The sections were then incubated overnight at 4°C with primary antibodies (11533-1-AP, Proteintech, Rosemont, IL, USA) diluted at a 1:100 ratio. After three washes, the sections were then incubated with biotin-labeled goat anti-Rabbit IgG secondary antibody (sp-9001, Zhongshan Golden Bridge Bio-technology, Beijing, China), followed by incubation with streptavidin-biotin complex containing horseradish peroxidase (sp-9001, Zhongshan Golden Bridge Bio-technology, Beijing, China) for 45 min. Immunoreactivity was visualized using 3,3^′-Diaminobenzidine Tetrahydrochloride at a dilution ratio of 1:150. (DAB; sp-9001, Zhongshan Golden Bridge Bio-technology, Beijing, China). Finally, the stained sections were examined and imaged using an optical microscope (DM IL LED, Leica, Wetzlar, Germany).

Total protein extraction was conducted from the cells using RIPA buffer (R0010, Solarbio, Beijing, China) supplemented with protease and phosphatase inhibitors to prevent protein degradation and dephosphorylation. Subsequently, the concentration of total protein was quantified using the bicinchoninic acid (BCA; RW0201, Hebei Ruipate Biotechnology Co., Ltd., Hebei, China) method. Equal amounts of protein were separated by 10% SDS-PAGE and transferred onto Polyvinylidene Fluoride Membrane (PVDF) membranes (IPVH00010, Immobilon, Darmstadt, Germany). The membranes were blocked with 5% bovine serum albumin (BSA) (SB-PR079, ShareBio, Shanghai, China) at room temperature for 40 min, and incubated overnight at 4°C with primary antibodies against CAMK2B (1:1000) (11533-1-AP, Proteintech, Rosemont, IL, USA), GAPDH (1:7500) (10494-1-AP, Proteintec), Ras (1:1000) (12063-1-AP, Proteintech), Raf1 (1:1000) (26863-1-AP, Proteintech). The following day, the membranes were washed three times with TBST (pH 7.4, concentration 5%) (N8770, Solarbio, Beijing, China) (each 5 min) and then incubated with a preabsorbed goat anti-rabbit IgG H&L secondary antibody at a dilution ratio of 1:2000 (A23920, Abbkine, Wuhan, China) (1:10,000). Protein bands were detected using the Odyssey infrared scanner (Odyssey DLx, LICOR, Lincoln, NE, USA), and the relative expression of CAMK2B was evaluated using ImageJ software (v1.54d, National Institutes of Health, Bethesda, MD, USA).

A total of 5000 cells were inoculated into each well of a 96-well plate and cultured overnight. Cell proliferation was assessed using the 488 Click-iT EdU Cell Proliferation Kit (C0071S, Beyotime, Shanghai, China) according to the manufacturer’s instructions. The following day, the cells were incubated with 10 μM EdU for 2 h. Cells were fixed, permeabilized, stained, and mounted according to the manufacturer’s instructions. Nuclear counterstaining was performed using Hoechst 33342 (SB-C6043, ShareBio, Shanghai, China). The results were observed and images were captured using a fluorescence microscope (DM IL LED, Leica, Wetzlar, Germany), with ImageJ software used to conduct quantitative analysis.

A total of 5000 cells were inoculated into a 96-well plate and cultured for 24, 48, 72, and 96 h. Subsequently, 10 μL CCK-8 (SB-CCK8, ShareBio, Shanghai, China) (5 mg/mL) was added to each well and the cells were incubated for another 2 h under conditions of 37°C and 5% carbon dioxide. The OD value was assessed using a microplate reader (Multiskan FC, Thermo Fisher Scientific, Waltham, MA, USA), with the absorbance determined at 450 nm.

Treated cells (20,000 cells per well) were inoculated into the upper chamber of a Transwell insert containing serum-free medium. The bottom of the upper compartment was pre-coated with Matrigel, while the lower compartment was filled with a complete medium. Cells were cultured at 37°C, with 5% carbon dioxide for 24 h. Subsequently, the migrated cells were fixed with 4% paraformaldehyde for 30 min and then stained with 1% crystal violet. The invasive cells were then visualized with an optical microscope (DMIL LED, Leica).

The treated glioma cells were inoculated into 6-well plates. Once the cells reached the desired confluence and formed a dense monolayer, a scratch was introduced using a pipette tip. The cells were then cultured in a serum-free medium to assess migration. Images of the scratch area were captured with an optical microscope (DMIL LED, Leica) at 0 and 24 h in the same region. The wound healing area was analyzed with ImageJ software.

Four-week-old male BALB/c-nu nude mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing), with an initial body weight of 15–20 g per mouse. The mice were randomly divided into 2 groups, with 6 mice in each group, and housed in an environment maintained at 20°C–22°C, 65%–79% humidity, and a 12-h light-dark cycle, with free access to food and water. After 1 week of acclimatization to the controlled environment and regular feeding, U251 cells transfected with siCAMK2B and siNC were cultured to 90% confluence, prepared into a cell suspension at a concentration of 5 × 10⁶ cells/mL, and subcutaneously injected into the right flanks of the mice. Visible tumors typically formed subcutaneously within 3 days post-injection, and then mouse weights and tumor dimensions were measured every 7 days for 4 weeks. Tumor volume was calculated using the equation of V = ½ × length × width². At the end of the observation period, the mice were anesthetized and euthanized, and the tumors were excised, weighed, and photographed for further analysis. This experimental procedure was approved by the Ethics Committee of the Second Hospital of Hebei Medical University and performed following the established protocols (Approval No. 2021-AE047).

All experiments were conducted in triplicate. Quantitative data were expressed as mean ± standard deviation (SD), with the Student’s t-test used to compare data between two groups. The Kaplan-Meier method with a log-rank test was used to plot the survival curve for glioma patients based on the expression level of CAMK2B. The GraphPad Prism 9.0 software (v121, GraphPad Software, San Diego, CA, USA) was used to process all the data for statistical analysis. A p-value <0.05 was considered to be statistically significant.

Based on transcriptome data obtained from the GEPIA database, we found that the expression level of CAMK2B was lower in glioma tissues compared to normal brain tissues (Fig. 1A). Immunohistochemical analysis of 70 patients in our hospital showed that the patients with high levels of CAMK2B expression exhibited better prognostic outcomes compared to those with low levels of CAMK2B (Fig. 1B). Analysis results from the TCGA, CCGA, and Rambrandt databases exhibited consistent findings (Fig. 1C) with those obtained using the GEPIA database (Fig. 1A).

We analyzed CAMK2B expression in normal brain tissue and grade I-IV glioma tissue using the IHC technique. The results revealed that CAMK2B expression levels were decreased in tissues of glioma at high grades (Fig. 2A). Additionally, the mRNA levels quantified using the qRT-PCR and protein levels quantified using the Western blot (Fig. 2B,C) yielded similar results, indicating that CAMK2B expression was lower in three glioma cells compared to the astrocytes.

To examine the effects of CAMK2B activation on the proliferation of glioma cells, we overexpressed CAMK2B through the transfection of plasmid into U251 and U87 cells, with transfection of empty plasmids serving as a blank control. The expression levels of CAMK2B in transfected glioma cells (U251) were assessed using qRT-PCR and Western blot (Fig. 3A,B). The expression levels of CAMK2B were significantly upregulated in U251 cells. Additionally, we used CCK-8 and EdU assays to assess the effect of CAMK2B overexpression on the population of U251 and U87 cells; notably, the CCK-8 assay can be used to examine cell viability, which is indicative of cell proliferation. The glioma cells were categorized into two groups: the CAMK2B overexpression group (group OE-CAMK2B) and the blank control group transfected with empty plasmids (group OE-NC). The procedure for assessing cell proliferation involved the following: A total of 5000 cells per well were inoculated into 96-well plates, followed by plasmid transfection, then the OD at 450 nm was measured every four days to assess the proliferation of cells in each group. The results revealed that overexpression of CAMK2B reduced the viability of U251 and U87 cell lines, thereby inhibiting their proliferative capacity (Fig. 3C). These findings were validated through the results from the EdU assay, which revealed that the CAMK2B overexpression group had a lower proportion of proliferating cells compared to cells transfected with the control vectors (Fig. 3D).

We used Transwell and wound healing assays to assess the impact of activated CAMK2B on the invasive and migrative activity of glioma cells. The U251 and U87 cells transfected with the overexpression plasmid were used for the assays. The U251 and U87 cells transfected with CAMK2B or control plasmids were seeded into the Transwell cell at 0 h, followed by fixation and staining at 24 h. The number of cells migrating through the pores in the OE-CAMK2B group was significantly lower compared to the OE-NC group (Fig. 4A). In the wound healing assay, the healing areas of U251 and U87 cells with overexpression of CAMK2B were smaller compared to those of the cells transfected with the control plasmid (Fig. 4B). These findings indicated that overexpression of CAMK2B prevents the invasion and migration of glioma cells.

Knockdown of CAMK2B was achieved by transfecting a siRNA plasmid targeting CAMK2B into the glioma cell lines. The expression level of CAMK2B was quantified using the qRT-PCR and Western blot techniques, while the expression of CAMK1D and CAMK2A was quantified using only the qRT-PCR (Fig. 5A–D). The results revealed that the expression level of CAMK2B was downregulated in glioma cells by about 40%. The cells obtained from the siCAMK2B and siNC groups were injected into the nude mice through the armpit. Subsequently, the length, width, and height of the tumor were measured at 7, 14, 21, and 28 days after injection to determine the volume of the tumor. The nude mice were then euthanized on day 28, and the tumor was excised and weighed. Quantitative analysis on day 28 revealed that the tumor volumes were significantly higher in mice subjected to siCAMK2B injection (401.9 ± 66.30 mm³, n = 6) compared to mice subjected to siCAMK2B injection (203.3 ± 82.23 mm³, n = 6). Furthermore, the tumor weight of mice injected with siCAMK2B was 463.7 ± 34.64 mg (n = 6), which was significantly greater compared to the 178.0 ± 91.93 mg (n = 6) in mice injected with siCAMK2B (Fig. 5E,F). These findings demonstrated that the knockdown of CAMK2B promoted glioma proliferation and growth in vivo.

We regulated the expression of CAMK2B in U251 cells, then used Western blot to determine the protein levels in the Ras/Raf/MEK/ERK signaling pathway. Knockdown of CAMK2B with siRNA resulted in an increased expression level of Ras, p-Raf, p-MEK, and p-ERK; these effects were inhibited by the Ras inhibitor Salirasib, indicating that Salirasib can inhibit the proliferation of glioma cells both in vivo and in vitro [18,19] (Fig. 6A). Conversely, overexpression of CAMK2B reduced the expression levels of Ras, p-Raf, p-MEK, and p-ERK in U251 cells (Fig. 6B).

We conducted in vitro experiments using the U87 and U251 glioma cells. Each of the cell lines was divided into three groups, comprising a scrambled control group (siNC), a siCAMK2B group (siCAMK2B), and a siCAMK2B + Salirasib group (siCAMK2B + Salirasib). The CCK8 assay results showed that compared with the control group, the cell viability of the siCAMK2B group was significantly increased on days 3 and 4. However, these effects were reversed in the siCAMK2B + Salirasib group following the addition of the Ras inhibitor Salirasib. Comparable results were observed with the EdU assay, specifically, the siCAMK2B group exhibited increased cell proliferation ability, which was reversed by the Ras inhibitor Salirasib. Knocking down CAMK2B resulted in about a 1.5-fold increase in cell proliferation ability compared to the control group. (Fig. 7A,B). Transwell experiment results showed that compared to the control group, the siCAMK2B group exhibited a significantly increased number of cells penetrating the Matrigel at 24 h, approximately 1.2-fold that of the control group. However, the addition of the Ras inhibitor Salirasib reversed this phenomenon. Consequently, we hypothesize that CAMK2B possesses the ability to regulate the invasive capacity of the glioma cells (Fig. 7C). The wound healing assays showed that compared to the control group, the siCAMK2B group exhibited greater cell migration at 24 h, with its migratory effects greater in the U251 cells compared to the U87 cells. Notably, these effects observed through the wound healing assay were reversed by the Ras inhibitor Salirasib (Fig. 7D). These findings indicate that CAMK2B plays a significant role in the hyperplasia, invasion, and migration of glioma cells through the Ras/Raf/MEK/ERK signaling pathway.