CAMK2B affects the proliferation, invasion and migration of glioma cells via Ras/Raf/MEK/ERK signal pathway

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CAMK2B, a crucial kinase involved in regulating cell growth and synaptic plasticity, remains enigmatic in terms of its specific role in glioma. Methods This study comprehensively analyzed the correlation between the expression level of CAMK2B in gliomas and patient prognosis using immunohistochemistry, qRT-PCR, and Western Blot techniques. Furthermore, the study determined the role of CAMK2B in glioma cell proliferation, invasion, and migration through CCK8, EdU, wound healing, Transwell, and in vivo tumor xenograft assays. Result We observed that patients exhibiting high levels of CAMK2B exhibited superior prognostic outcomes compared to those with low levels. Furthermore, CAMK2B expression was notably lower in glioma tissues and cells compared to both normal brain tissue and human astrocyte cell lines. Notably, overexpression of CAMK2B in glioma cells suppressed their proliferative capacity by approximately 40% and attenuated their invasive and migratory abilities by 60–70%, relative to normal glioma cell lines. Conversely, knockdown of CAMK2B using siRNA-CAMK2B significantly enhanced the proliferative, invasive, and migratory capabilities of glioma cells in both in vitro and in vivo settings, increasing these abilities by 1.5 to 3 times. Remarkably, these effects could be reversed through the application of the Ras pathway inhibitor Salirasib. Western blot analysis revealed that knockdown of CAMK2B activated the Ras/Raf/MEK/ERK signaling pathway in glioma cell lines, whereas overexpression of CAMK2B exerted inhibitory effects on this pathway. Conclusion CAMK2B inhibits glioma proliferation, invasion, and migration through Ras/Raf/MEK/ERK signal pathway. CAMK2B glioma proliferation invasion migration Ras/Raf/MEK/ERK pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Glioma occurs in various brain regions and account for about 81% of brain tumors. Among glioma, 45% cases are glioblastomas, presenting as extremely malignant( 1 ). It has a higher incidence rate in males than females, and a higher prevalence in the elderly (over 65 years old). For instance, glioblastoma is about 5 times higher in the elderly than in other age patients( 2 ). Glioma is mainly treated by surgery, but due to the difficulty of surgical resection with poor prognosis. Postoperative chemotherapy and adjuvant radiotherapy can improve the prognosis of patients and slightly prolong the survival period of patients( 3 ). Temozolomide is currently a chemotherapeutic drug for glioma, but it is easy to develop tolerance( 4 ). The five-year survival period of patients with glioblastoma is 0.05–4.7%( 1 , 4 , 5 ). Thus, it is critical to find a novel mechanism underlying the glioma proliferation, invasion, and migration, and develop new therapeutic treatment for glioma. Calcium / calmodulin dependent protein kinase II (CaMKII) is a serine / threonine protein kinase and plays a central role in regulating intracellular Ca 2+ signaling pathway( 6 ). Previous studies have shown that CAMK2B is involved in the growth and development of neurons and synaptic plasticity and involved in the pathological process of many neurological disorders such as sleep disorder, memory disorder, severe intellectual disability, mental retardation, schizophrenia, neurodevelopmental disorder, Alzheimer's disease, etc.( 7 – 12 ). Through analyzing the TCGA,CCGA, Rambrandt and GEPIA database that the expression of CAMK2B in low-grade gliomas and glioblastomas is very low, make sure the relationship between the CaMK2B level and low-grade glioma, and the prognosis of glioma compared with normal brain tissues. Moreover, it has been pointed out that CAMK2A, which belongs to the homologous family with CAMK2B,its overexpression significantly inhibited the proliferation and metastasis of glioma cells induced by miR-3200-3p. The interaction between CAMK2A and miR-3200-3p regulates the progression of glioma through the Ras/Raf/MEK/ERK signaling pathway.( 13 ). CAMK2D, also belonging to the CAMK2 family, can form complexes with RNF8-MAD2. They play a certain role in the mitotic checkpoint of gliomas and can serve as mitotic checkpoint signals in gliomas, becoming a therapeutic target for gliomas .( 14 ) However, the mechanism of action and clinical significance of CAMK2B in glioma still unknown. Materials and methods Bioinformatics analysis The GEPIA database (http://gepia.cancer-pku.cn/) is an online platform for gene expression profiling interactive analysis, used to analyze the expression of CAMK2B in different types of tumor tissues and normal tissues. The expression level of CAMK2B mRNA in gliomas and its relationship with patient prognosis were analyzed using data from the Cancer Genome Atlas (TCGA) (https://tcga-data.nci.nih.gov/tcga/), Chinese Glioma Genome Atlas (CGGA) (http://www.cgga.org.cn) and Rembrandt database (http://caintegrator.nci.nih.gov/rembrandt/). All databases were accessed in August 2022. The median was employed to delineate high and low expression groups, and Kaplan-Meier analysis was conducted using the “survminer” and “survival” R packages to investigate the association between CAMK2B and the prognosis of glioma patients. Cell culture Three human glioma cells (U251,U87, and A172,) and Human Astrocytes cell (HA) were acquired from Procell Life Science & Technology Co., Ltd. The U251 cells were cultured in RPMI-1640 medium (Gibco™,11875093). The U87 cells were cultured in Minimum Essential Medium (MEM, Gibco™, 11090081). The A172 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco™, 10564011). The HA cells were cultured in Astrocyte medium (AM, Sciencell™, #1801). All mediums were contained with 10% fetal bovine serum (FBS, Gibco™, 10099141) and 1% penicillin and streptomycin (Pen-Strep Solution, BI, 2114091). All cells were cultured in a cell incubator at 37 ℃ and 5% carbon dioxide. Clinical samples Seventy-nine glioma tissues and four non-tumor brain tissues (originate from brain tissue removed during brain trauma surgery) were attained from the 2nd Hospital of Hebei Medical University on December 15, 2021. The surgical time of the sample source was from September 24, 2014 to March 9, 2019, the follow-up date for the patient's survival analysis is December 20, 2021. A portion of the samples were kept in reserve with liquid nitrogen for Western blot experiments, other samples used for IHC experiments were stored in 10% paraformaldehyde. This research was approved by the ethics committee of our hospital. All patients or their guardians obtained informed and written consent in accordance with the guidelines of the ethics committee. Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) Complete RNA was abstracted from untreated cell and transfected U251 cells and U87 cells by Trizol reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. After determining the RNA concentration, cDNA was conflated using reverse transcription kits HiScript® III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China). AceQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) was used for PCR procedure. The PCR process is divided into two steps，for first step was set as 95℃，10 min of one cycle, for second step was set as 95℃ 5s，60℃ 30s and 72℃ 30s, with a total of 40 cycles. The primer sequences of CAMK2B were as listed below: F, 5′-GCAAAGAGGCGTATGGCAAG-3′; R, 5’-GACGGGAAGTCATAGGCACC-3′. The primer sequences of GAPDH were as follow: F, 5’- GGAGCGAGATCCCTCCAAAAT-3′; R, 5′-GGCTGTTGTCATACTTCTCATGG-3′. The relative gene expression was calculated by 2-ΔΔCt method. Immunohistochemistry (IHC) The tissue was stabilized with formalin and then wrapped in paraffin，and sliced. The slices were placed in an incubator at 60℃ for 30 minutes，then they were immersed in xylene and then graded concentrations of ethanol used to remove paraffin. The slices were immersed in 2% citric acid buffer at high temperature and pressure for 25 minutes and then treated with endogenous peroxidase for antigenic repair. The repaired sections were blocked with 10% goat serum. Next, the slices were hatched with primary antibodies（1：100）at 4℃ overnight. After purified three times, the slices were hatched with biotin-labeled goat antibody Rabbit IgG antibody (sp-9001, Zhongshan Golden Bridge Bio-technology, Beijing, China) for 45 min and then incubated with streptavidin‑biotin complex containing horseradish peroxidase (sp-9001, Zhongshan Golden Bridge Bio-technology, Beijing, China) for 45min. Results were examined through avidin-biotin-peroxidase complex solution and visualized with DAB). Finally, the image was captured under an optical microscope. Western Blot Complete protein was abstracted from cells using a RIPA buffer with protease and phosphatase inhibitor. Then quantifying total protein concentration by the BCA method. Equal poundage of protein was separated by 10% SDS-PAGE and then diverted into the PVDF membranes, and blocked it use 5% bovine serum albumin (BSA) place in room temperature for forty minutes, and the incubated overnight at 4℃with the primary antibodies against CAMK2B(1:1000),GAPDH(1:7500),Ras(1:1000),RAF1(1:1000) and p-R 1（1：1000）.On the next day, after washing by TBST three times, each times for 5 minutes, the incubated the membranes by goat anti-rabbit IgG H＆L preabsorbed secondary antibody（1：10000）.The membranes were detected with the Odyssey infrared scanner. The relative expression of CAMK2B was evaluated by ImageJ. EdU assay 5000 cells were inoculated in 96-well plates and cultured overnight. Using 488 Click-iT EdU Cell Proliferation Kit (ShareBio).On the next days, cells were incubated for 2h at a concentration of 10 μM EdU. Then fix, promote infiltration, stain, and seal the cells according to the manufacturer's instructions, cell nucleus re staining using Hoechst 33342. The results were observed and obtained through a fluorescence microscope, use ImageJ for image statistics and analysis. Cell Viability Assay (CCK-8 Assay) 5000 cells were inoculated in 96-well plates and cultivated for 24,48,72,96 h，10 μl CCK-8 (5 mg/ml) was added to each well, and the cells were incubated for supplemental 2 hours at 37℃，5% carbon dioxide. Then the absorbance values of 450nm were benchmarked by a microplate reader. Transwell Assay Treated cells were inoculated in the upper compartment with serum-free medium，the bottom of the upper compartment is coated with matrix glue and the lower compartment is filled with complete medium, culture at 37℃, 5% carbon dioxide for 24 hours. Then, the migrated cells were immobilized with 4% paraformaldehyde for 30min then stained with 1% crystal violet. The invasive cells were observed with optical microscope. Wound Healing Assay The treated glioma cells were inoculated into 6-well plates. After the cells grew to a dense state, the cells were scratched with a pipette culet and fostered in serum-free medium. The laceration was captured with optical microscope at 0 th hour and 24 th hour at the same region. The wound healing area was analyzed with ImageJ. Tumor xenotransplantation Assay Transfected or untransfected U251 cells were inoculated into the right armpits of BALB/c nude mice (4 weeks old, 15-20g, male), each nude mouse was inoculated with 5×10 6 viable cells. Tumor size was checked every seven days and gauged with a slide gauge，and calculated tumor volume by equation: 0.5 × length × width 2 . The nude mice were sacrificed at 28 days, and the xenotransplantation tumors were collected, weighed and photographed. This experiment has been approved by the Ethics Committee of the 2nd Hospital of Hebei Medical University and carried out according to its guidance. Statistical Analysis All experiments were rehearsed three times, measured data were expressed as mean ± standard deviation (SD). The t-test was used to parallel data among two groups. Kaplan-Meier method with log-rank test was used to plot the survival curve of glioma patients according to the expression level of CAMK2B. GraphPad Prism9.0 was used to process all the data for statistical analysis. P < 0.05 was supposed to be statistically significant. Results The patients with high CAMK2B expression have a better prognosis Based on transcriptome data from the GEPIA database, we found that the expression level of CAMK2B is lower in glioma tissue compared to normal brain tissue (Figure1,A) . Immunohistochemical analysis of 70 patients in our hospital showed that the patient prognosis with high CAMK2B was striking better than patients with low CAMK2B (Figure1, B); Analyzing the database of the TCGA, CCGA, and Rambrandt databases obtained similar findings (Figure1,C) to findings posted on GEPIA website Figure 1A). CAMK2B is low expression in glioma cell and tissue. We analyzed CAMK2B expression in normal brain tissue and grade I-IV glioma tissue with IHC and found that CAMK2B expression levels were decreased in tissues of glioma at high grade (Figure2, A). In addition, t he mRNA levels measured by qRT-PCR and protein levels measured by Western blot (Figure2, B and C) yielded the same results, indicating that CAMK2B expression was lower in three glioma cells than in astrocytes. CAMK2B activation attenuated glioma cell proliferation To examine if CAMK2B activation affects the proliferation of glioma cells, we overexpressed CAMK2B through transfection of plasmid into U251 cells and U87 cells and transfect empty plasmids as blank control. The expression levels of CAMK2B in transfected glioma cells were assessed by qRT-PCR and Western blot (Figure 3, A and B) . The expression of CAMK2B levels were significantly increased in U251 cells and U87 cells. In addition, we used CCK-8 assay and EdU assay to determine the influence of overexpression of CAMK2B on the multiplication of U251 cells and U87 cells, CCK8 assay can be used to observe cell viability to reflect cell proliferation. We respectively divided two types of glioma cells into two groups: the CAMK2B overexpression group (group OE-CAMK2B) and the blank control group transfected with empty plasmids (group OE-NC). Inoculate cells in 96 well plates, 5,000 cells per well, and transfect plasmids into them after inoculation, the 450nm OD value of each four days was detected. Overexpression of CAMK2B reduced the viability of U251 and U87 cell line and thus inhibit its proliferation ability (Figure3, C) . The EdU assay confirmed that compared to cells transfected with control vectors, the CAMK2B overexpression group had a lower proportion of proliferating cells. (Figure3, D) . Stimulation of CAMK2B prevented the invasion and migration of glioma cells We used Transwell assay and wound healing assay to explore the influence of activated CAMK2B on the invasion and migration ability of glioma cells. Similarly, the U251 cells and U87 cells transfected with the overexpression plasmid were used for the assays. The U251 cells and U87 cells transfected with CAMK2B or control plasmids were seeded into the Transwell cell at the 0 hour and fixed and stained at the 24 hours, respectively. Then, the number of cells passing through the pores in the group OE-CAMK2B was evidently less than the group OE-NC (Figure 4, A) . In wound healing assay, the healing areas of U251 cells and U87 cells with overexpressing CAMK2B were smaller than those of the cells transfected with control plasmid (Figure 4, B) . These data indicated that overexpression of CAMK2B prevents the invasion and migration of glioma cells. Knockdown of CAMK2B promoted glioma proliferation in vivo To knockdown CAMK2B, siRNA plasmid targeting CAMK2B was transfected into glioma cell lines. The expression of CAMK2B was verified by qRT-PCR and Western blot and the expression of CAMK1D and CAMK2A was verified by qRT-PCR (Figure 5, A, B, C and D) . The expression level of CAMK2B was reduced in glioma cells by 40%. The cells of the siCAMK2B group and siNC group were injected into the armpit of nude mice , the length, width and height of the tumor were measured at 7, 14, 21 and 28 days after injection to calculate the volume of the tumor. The nude mice were executed on the 28th day, and the tumor were removed and weighed. On the 28th day, the tumor volumes were significantly more in mice subjected to siCAMK2B injection (401.9 ± 66.30 mm 3 , n =5) than in mice subjected to siCAMK2B injection (203.3 ± 82.23mm 3 , n=5). Furthermore, the tumor weight of mice received siCAMK2B injection was 463.7 ± 34.64 mg (n=5), which was significantly heavier than those 178.0 ± 91.93 mg (n = 5) in mice received scCAMK2B injection (Figure 5, E and F) . These data suggest that knockdown of CAMK2B promotes glioma proliferation and growth in vivo. Regulation of CAMK2B expression levels regulates Ras/Raf/MEK/ERK signals We regulate the expression of CAMK2B in U251 cells, and then used Western Blot to determine the protein levels in Ras/Raf/MEK/ERK pathway. Knockdown of CAMK2B with siRNA, expression levels of Ras, p-Raf, p-MEK, p-ERK were increased, an effect was inhibited by the Ras inhibitor Salirasib , which has been experimental evidence that can inhibit the proliferation of glioma both in vivo and in vitro(15, 16) (Figure 6, A) . On the other hand, overexpression of CAMK2B decreased expression levels of Ras, p-Raf, p-MEK and p-ERK in U251 cells (Figure 6, B) . Silencing CAMK2B accelerated the proliferation, invasion and migration of glioma cells through Ras/Raf/MEK/ERK pathway We conducted in vitro experiments using the U87 and U251 glioma cells, we divided each of the two cell lines into three groups for experiments, scrambled control group (siNC), siCAMK2B group(siCAMK2B) and siCAMK2B+Salirasib group (siCAMK2B+ Salirasib). CCK8 assay results showed that compared with the control group, the cell viability of the siCAMK2B group was significantly increased on the third and the fourth day, and the siCAMK2B+Salirasib group could reverse this phenomenon after adding the Ras inhibitor Salirasib; Similarly, The EdU assay obtained the same results, indicating that the siCAMK2B group had increased cell proliferation ability, which could be reversed by the Ras inhibitor Salirasib. Knocking down CAMK2B resulted in an approximately 1.5-fold increase in cell proliferation ability compared to the control group. (Figure7, A,B) Transwell experiment results showed that compared to the control group, the siCAMK2B group significantly increased the number of cells penetrating the matrix glue at 24 hours, approximately 1.2 times that of the control group. The addition of the Ras inhibitor Salirasib can reverse this phenomenon. Therefore, we believe that CAMK2B can affect the invasive ability of glioma cells. (Figure7, C) The results of the wound healing experiment showed that compared to the control group, the siCAMK2B group had a higher degree of cell migration at 24 hours, and its migration ability to U251 cells was slightly higher than that to U87 cells. This result can also be reversed by the Ras inhibitor Salirasib. (Figure7, D) These data suggest that CAMK2B affects the hyperplasia, invasion and migration of glioma cells through Ras/Raf/MEK/ERK signaling pathway. Discussion In our prior research, we have conclusively demonstrated that CAMK1D exerts a significant influence invasion and the PI3K/AKT/mTOR signaling pathway( 17 ). This study investigated whether CAMK2B (belonging to the CAMK family) exerts similar impacts on glioma invasion and migration. CAMK2B is implicated in the formation of hippocampal neurons in rats( 18 ). Simultaneously, CAMK2B can traverse the H1F-1α signaling pathway, safeguarding neurons from apoptosis triggered by homocysteine( 19 ). The Ca2+-dependent activity and CAMK2 autonomous activity are vital for survival. In mice, the absence of CAMK2B is fatal( 20 ). For instance, the locomotion of mice necessitates the activation of CAMK2B, which is mediated by calcium/calcium-binding proteins, whereas the absence of CAMK2B can result in motor impairments in mice( 21 ). The aforementioned research demonstrates that CAMK2B plays a pivotal role in the development and function of the nervous system. Furthermore, CAMK2B also contributes to the growth of various tumor types, including papillary renal cell carcinoma( 22 ),human neuroblastoma( 23 ),breast cancer( 24 )etc. Furthermore, CAMK2B also appears in EMT(Epithelial mesenchymal transition) ( 25 ) . In this study, we experimentally verified that CAMK2B plays a carcinogenic role in gliomas, and this oncogenic effect is mediated through the Ras/Raf/MEK/ERK signaling pathway. Firstly, we have confirmed that the expression of CAMK2B in gliomas is significantly lower compared to that in normal brain tissues. Furthermore, our survival analysis of 70 patients revealed that individuals with gliomas exhibiting high CAMK2B expression tend to have a longer lifespan than those with low CAMK2B expression. The experimental results obtained through modulating the expression of CAMK2B in glioma cells revealed that upon activation of CAMK2B, there was a significant reduction in the proliferation, invasion, and migration of glioma cells. Conversely, downregulation of CAMK2B led to a marked enhancement in these cellular processes. Notably, this enhancement trend could be effectively suppressed by the Ras pathway inhibitor, Salirasib, which was further corroborated at the protein expression level. Collectively, these findings underscore the antitumor role of CAMK2B in glioma, mediated primarily through the Ras/Raf/MEK/ERK signaling pathway. The Ras/Raf/MEK/ERK (MAPK) signaling pathway exhibits sensitivity in numerous tumor types. Potential reasons for its activation encompass activating mutations in the KRAS, NRAS, and BRAF genes( 26 ), precisely due to the accumulation of mutations in these crucial genes, cancer arises( 27 , 28 ). These mutations have the potential to disrupt the natural cycles of cell proliferation, differentiation, and apoptosis( 29 ). MAPK signaling transduction is one of the primary pathways in cancer biology. The disrupted MAPK signaling pathway significantly contributes to pathological research on numerous human diseases, and its activation accounts for over 40% of human cancer cases( 30 , 31 ).For instance, in colon cancer( 32 ),pancreatic cancer( 33 ),lung cancer( 34 ),gastric cancer( 35 ),breast cancer( 36 )rand other diseases are involved in the emergence and evolve of diseases. The Ras/Raf/MEK/ERK signaling pathway plays a pivotal role in the apoptotic and autophagic processes associated with glioma( 37 , 38 ), and proliferation, invasion and migration of glioma( 39 – 41 ). The Ras/Raf/MEK/ERK signaling pathway plays a pivotal role in the genesis and progression of glioma. Our findings reveal that modulating the expression level of CAMK2B effectively triggers the activation of the Ras/Raf/MEK/ERK signaling pathway. Salirasib is a widely recognized inhibitor of the Ras pathway, effectively suppressing the expression of Ras( 15 , 16 ). We discovered that his Ras pathway inhibitor effectively inhibited the pathway, thereby further corroborating the interaction between CAMK2B and the Ras/Raf/MEK/ERK signaling cascade. Furthermore, based on the KEGG database, we discovered that CAMK2B can exert an influence on the growth and proliferation of glioma cells via the PI3K/AKT/mTOR signaling pathway. Current research has revealed intricate interconnections between the Ras/Raf/MEK/ERK signaling pathway and the PI3K/AKT/mTOR signaling pathway. For instance, a study revealed that agonists implicated in Ras-ERK activation exhibit partial overlap with PI3K/mTORC1 signaling( 42 ). PI3K serves as one of the primary effectors of Ras, playing a pivotal role in regulating crucial cellular processes, including cell viability, therapeutic resistance, and angiogenesis, during oncogenic Ras activation( 43 ). Both the Ras and PI3K signaling pathways play a pivotal role in regulating cellular processes such as apoptosis, growth, differentiation, metabolism, as well as the expression of crucial genes( 44 , 45 ). In our prior research, we have established that CAMK1D, which belongs to the same CAMK family as CAMK2B, can influence the malignant progression of glioma via the PI3K signaling pathway( 17 ). Hence, we postulate that CAMK2B can potentially influence the progression of glioma via the PI3K/AKT/mTOR signaling pathway, which will be the focal point of our future in-depth investigation into CAMK2B. Simultaneously, we conducted an initial investigation into the interplay among CAMK2B, CAMD, and CAMA, all belonging to the CAMK family. Our PCR experiment revealed that suppressing the expression of CAMK2B in the U251 glioma cell line did not elicit any notable alterations in the expression patterns of CAMK1D. However, the expression level of CAMK2A has slightly increased. Some studies have revealed a closer relationship between CAMK2A and CAMK2B, indicating that they can jointly influence the development of certain diseases. Following the reduced expression level of CAMK2B, a compensatory increase in CAMK2A expression can counteract the effects of the decline, exemplified by the impact of CAMK2A and CAMK2B deletion on brain neurodevelopment, ultimately leading to intellectual disability( 8 , 9 , 46 ). This viewpoint can be regarded as a promising avenue for future investigations into the role of CAMK2B in glioma. In summary, we have demonstrated that the expression level of CAMK2B in gliomas is significantly lower compared to that in normal brain tissues, and it exerts a profound influence on the proliferative, invasive, and migratory capabilities of glioma cells both in vitro and in vivo. Furthermore, we have established that these functions of CAMK2B are mediated through the Ras/Raf/MEK/ERK signaling pathway, thereby presenting novel therapeutic targets and avenues for future research in the diagnosis and treatment of glioma. Conclusion Substantially, CAMK2B exerts its effect on the biological process of glioma by activating Ras/Raf/MEK/ERK signaling pathway. CAMK2B is down regulated in glioma and can be used as a potential target to predict the prognosis of glioma patients. Declarations Data Availability Statement The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. All data datasets generated and/or analysed during the current study are available in the following repository (http://gepia.cancer-pku.cn/, https://tcga-data.nci.nih.gov/tcga/, http://www.cgga.org.cn, http://caintegrator.nci.nih.gov/rembrandt/ ). Ethics statement The Ethics Committee of the Second Hospital of Hebei Medical University authorized this research (2021-AE047) (2021-R494) and waived the written informed consent requirement. Author Contributions SZ and JL performed the conception, design, and writing.SZ and QJ completed the experimental verification and prepared figures. QJ, SZ and YS completed the data curation. YW and ZS completed the data analysis. LL performed the Funding acquisition and supervision. All authors read and approved the final manuscript. Funding: This work was supported by the Natural Science Foundation of Hebei Province (H2021206037), the Government-funded Project on Training of Outstanding Clinical Medical Personnel of Hebei Province in the year 2021(303–16-20–06), and the Medical Research Project of Hebei Provincial Health Commission (20230031) Conflicts of Interest. No relevant financial or non-financial interests to disclose. Acknowledgments We are profoundly grateful that this study was supported by the Second Hospital of Hebei Medical University. References Ostrom QT, Bauchet L, Davis FG, Deltour I, Fisher JL, Langer CE, et al. 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Molecular cancer. 2020;19(1):17. Xu Y, Sun Q, Yuan F, Dong H, Zhang H, Geng R, et al. RND2 attenuates apoptosis and autophagy in glioblastoma cells by targeting the p38 MAPK signalling pathway. Journal of experimental & clinical cancer research : CR. 2020;39(1):174. Wang J, Yao N, Hu Y, Lei M, Wang M, Yang L, et al. PHLDA1 promotes glioblastoma cell growth via sustaining the activation state of Ras. Cellular and molecular life sciences : CMLS. 2022;79(10):520. Tang F, Wang H, Chen E, Bian E, Xu Y, Ji X, et al. LncRNA-ATB promotes TGF-β-induced glioma cells invasion through NF-κB and P38/MAPK pathway. Journal of cellular physiology. 2019;234(12):23302-14. Guo G, Yao W, Zhang Q, Bo Y. Oleanolic acid suppresses migration and invasion of malignant glioma cells by inactivating MAPK/ERK signaling pathway. PloS one. 2013;8(8):e72079. Mendoza MC, Er EE, Blenis J. The Ras-ERK and PI3K-mTOR pathways: cross-talk and compensation. Trends in biochemical sciences. 2011;36(6):320-8. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5412790","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":381761681,"identity":"4e69941e-99d6-4909-9377-ca4b1df3db45","order_by":0,"name":"Shiyang Zhang","email":"","orcid":"","institution":"The Second Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shiyang","middleName":"","lastName":"Zhang","suffix":""},{"id":381761682,"identity":"5b133ecc-474d-4632-8f46-5f11d3555159","order_by":1,"name":"Jingchen Li","email":"","orcid":"","institution":"The Second Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jingchen","middleName":"","lastName":"Li","suffix":""},{"id":381761683,"identity":"35455f1f-1fab-4f49-978c-36a9c577e7b0","order_by":2,"name":"Qianxu Jin","email":"","orcid":"","institution":"The Fourth Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qianxu","middleName":"","lastName":"Jin","suffix":""},{"id":381761684,"identity":"47884075-9267-4a27-b669-a9e42e01279a","order_by":3,"name":"Siyu Zhu","email":"","orcid":"","institution":"The Second Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Siyu","middleName":"","lastName":"Zhu","suffix":""},{"id":381761685,"identity":"66642400-9248-482c-8eb7-2005bbb936cc","order_by":4,"name":"Hongshan Yan","email":"","orcid":"","institution":"The Fourth Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hongshan","middleName":"","lastName":"Yan","suffix":""},{"id":381761686,"identity":"42baddf2-fdb5-47c2-8f9f-71d9e8eb432d","order_by":5,"name":"Yizheng Wang","email":"","orcid":"","institution":"The Fourth Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yizheng","middleName":"","lastName":"Wang","suffix":""},{"id":381761687,"identity":"34ccbaaf-550c-4b31-9fe2-65b005c96d7e","order_by":6,"name":"Zihan Song","email":"","orcid":"","institution":"The Second Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zihan","middleName":"","lastName":"Song","suffix":""},{"id":381761688,"identity":"5d13ccab-9654-4912-98f9-81c87cf2f2a1","order_by":7,"name":"Liqiang Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIie3RMWsCMRTA8XcEMqXemiNwfoU7AnYR/CoJB9flSp3ETYuQzxC/hSA4RwRdxDmig1ZwLgiO4h0dW3J2c8gf3hL48eAFwOd7wsLmQRjRv5GQAv55MjUkGorj4Xtt4kg/ShIjT+lYGZ7YRwmYRc5e1F5Od6Pl11ZB3LAiuHQdIvhUOSObs5ztl2/8XQGPrEBMOwhCpCQ9JGe2aLGSyIkVGBEHwTi8MoKRnOqPa0UGtYQQyFKtFjyhBa6ISOoIpSDLI+cxtfkrLzY0Ha+PI+YiHQvVV7ZJqLPzqei1m41VNr+4yK+t5QTDfwCfz+fz/dUdAx9RveGlPPsAAAAASUVORK5CYII=","orcid":"","institution":"The Second Hospital of Hebei Medical University","correspondingAuthor":true,"prefix":"","firstName":"Liqiang","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-11-08 01:38:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5412790/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5412790/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71100641,"identity":"abc174e6-3aae-4d86-9496-62cc9f776de2","added_by":"auto","created_at":"2024-12-11 06:52:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":991318,"visible":true,"origin":"","legend":"\u003cp\u003eCAMK2B expression in pan carcinoma and survival analysis of CAMK2B in patients with glioma. (A) CAMK2B expression in pan carcinoma. (B) Survival analysis of patients from the Second Hospital of Hebei Medical University. (C) Survival analysis of patients from TCGA, CCGA and Rambrandt databases.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5412790/v1/d2b0e46475953d8bfd2f6e7c.png"},{"id":71100389,"identity":"897fc53b-1b54-444b-8460-c5890e3eb2b9","added_by":"auto","created_at":"2024-12-11 06:44:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4518499,"visible":true,"origin":"","legend":"\u003cp\u003eCAMK2B expression in tissues and cells. (A) Immunohistochemical results showed that compared with normal brain tissue, CAMK2B expression level in glioma decreased with the increase of glioma grade. (B,C) qRT-PCR and Western Blot results shows that CAMK2B is significantly low expressed in three glioma cells(U251,U87,A172) compared with human astrocytes(HA). \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01;\u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5412790/v1/1bbce1fd9785467c33dec377.png"},{"id":71100640,"identity":"8cf461c4-5d6a-43e2-a343-39417130d9ad","added_by":"auto","created_at":"2024-12-11 06:52:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1624500,"visible":true,"origin":"","legend":"\u003cp\u003eStimulation of CAMK2B inhibits proliferation of glioma cells. (A,B) Detection of CAMK2B expression after plasmid transfection by qRT-PCR and Western Blot. (C)CCK8 assay reveals the effect of CAMK2B on U251 and U87 glioma cells viability. (D) EdU assay reveals the effect of CAMK2B on U251 and U87 glioma cells proliferation. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.05;\u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01;\u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5412790/v1/747bb2edc531fe8540a9c3d2.png"},{"id":71100386,"identity":"6d9735d8-8274-4a76-925d-b14f566770a0","added_by":"auto","created_at":"2024-12-11 06:44:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3807946,"visible":true,"origin":"","legend":"\u003cp\u003eActivating CAMK2B inhibits the invasion and migration of glioma cells. (A)Transwell assay displayed that activated CAMK2B reduced the invasion cells of U251 and U87 glioma cells. (B)Wound healing assay displayed that activated CAMK2B reduced the healing area of U251 and U87 glioma cells. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.05;\u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.001;\u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.0001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5412790/v1/9e8dcccffe616aa703c0e24a.png"},{"id":71100642,"identity":"eff8f4df-f008-4da1-a0ab-dab219d910f7","added_by":"auto","created_at":"2024-12-11 06:52:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2086465,"visible":true,"origin":"","legend":"\u003cp\u003eSilencing CAMK2B can promote the proliferation of glioma cells in vivo. (A, B) Detection of CAMK2B expression after siCAMK2B transfection by qRT-PCR and Western Blot. (C, D) Detection of CAMK1D and CAMK2A expression after siCAMK2B transfection by qRT-PCR. (E, F) Tumor volume and weight \u003cem\u003ein vivo\u003c/em\u003e were significantly promoted after subcutaneous injection of U251 cells of siCAMK2B into nude mice. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01;\u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.001;\u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.0001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5412790/v1/b64031605731436b53c5678f.png"},{"id":71100382,"identity":"e9fe95ee-06fd-4113-9fd6-aa802a516819","added_by":"auto","created_at":"2024-12-11 06:44:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":471067,"visible":true,"origin":"","legend":"\u003cp\u003eCAMK2B expression affects Ras/Raf/MEK/ERK signal pathway. (A)The Ras, p-Raf, p-MEK, p-ERK were increase after knock-down CAMK2B, nevertheless the Ras inhibitor Salirasib can reverse this change. (B) The Ras, p-Raf, p-MEK, p-ERK were decrease after activating CAMK2B.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5412790/v1/fb3bf10005ee55eaa2dc0e54.png"},{"id":71100390,"identity":"cabd08c3-04d2-446f-8391-64a1f2369e3e","added_by":"auto","created_at":"2024-12-11 06:44:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4550207,"visible":true,"origin":"","legend":"\u003cp\u003eSilencing CAMK2B can promote the proliferation, invasion and migration of glioma cells, while the Ras inhibitor Salirasib can reverse this effect. (A) CCK8 assay point out that silencing CAMK2B can enhance the viability of U251 and U87 glioma cells, and the Ras inhibitor Salirasib can reverse this result. (B) EdU assay displayed that silencing CAMK2B could enhance the proliferation of U251 and U87 glioma cells, simultaneously the Ras inhibitor Salirasib could reverse this result. (C) Transwell assay shows that knockdown of CAMK2B can improve the invasive ability of U251 and U87 glioma cells, concurrently the Ras inhibitor Salirasib can reverse this trend. (D) Wound healing assay indicated that knockdown of CAMK2B can raise the migration ability of U251 and U87 glioma cells, besides the Ras inhibitor Salirasib can reverse this phenomenon. ns, no significance; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.05;\u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01;\u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.001;\u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.0001.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5412790/v1/503d119731fbe6994006f98d.png"},{"id":72915785,"identity":"041da831-d7bb-4a06-ac72-5b631c071eba","added_by":"auto","created_at":"2025-01-03 15:47:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17431401,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5412790/v1/1f0fb980-b3ee-4919-917e-f95449f43dd0.pdf"},{"id":71100385,"identity":"0bc07fbd-5a7f-4d70-9d30-dda508f97b55","added_by":"auto","created_at":"2024-12-11 06:44:37","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":947742,"visible":true,"origin":"","legend":"","description":"","filename":"originalwesternblotimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5412790/v1/840031d43dd432992efbbf4f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"CAMK2B affects the proliferation, invasion and migration of glioma cells via Ras/Raf/MEK/ERK signal pathway","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eGlioma occurs in various brain regions and account for about 81% of brain tumors. Among glioma, 45% cases are glioblastomas, presenting as extremely malignant(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). It has a higher incidence rate in males than females, and a higher prevalence in the elderly (over 65 years old). For instance, glioblastoma is about 5 times higher in the elderly than in other age patients(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Glioma is mainly treated by surgery, but due to the difficulty of surgical resection with poor prognosis. Postoperative chemotherapy and adjuvant radiotherapy can improve the prognosis of patients and slightly prolong the survival period of patients(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Temozolomide is currently a chemotherapeutic drug for glioma, but it is easy to develop tolerance(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). The five-year survival period of patients with glioblastoma is 0.05\u0026ndash;4.7%(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Thus, it is critical to find a novel mechanism underlying the glioma proliferation, invasion, and migration, and develop new therapeutic treatment for glioma.\u003c/p\u003e \u003cp\u003eCalcium / calmodulin dependent protein kinase II (CaMKII) is a serine / threonine protein kinase and plays a central role in regulating intracellular Ca\u003csup\u003e2+\u003c/sup\u003e signaling pathway(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Previous studies have shown that CAMK2B is involved in the growth and development of neurons and synaptic plasticity and involved in the pathological process of many neurological disorders such as sleep disorder, memory disorder, severe intellectual disability, mental retardation, schizophrenia, neurodevelopmental disorder, Alzheimer's disease, etc.(\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Through analyzing the TCGA,CCGA, Rambrandt and GEPIA database that the expression of CAMK2B in low-grade gliomas and glioblastomas is very low, make sure the relationship between the CaMK2B level and low-grade glioma, and the prognosis of glioma compared with normal brain tissues. Moreover, it has been pointed out that CAMK2A, which belongs to the homologous family with CAMK2B,its overexpression significantly inhibited the proliferation and metastasis of glioma cells induced by miR-3200-3p. The interaction between CAMK2A and miR-3200-3p regulates the progression of glioma through the Ras/Raf/MEK/ERK signaling pathway.(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). CAMK2D, also belonging to the CAMK2 family, can form complexes with RNF8-MAD2. They play a certain role in the mitotic checkpoint of gliomas and can serve as mitotic checkpoint signals in gliomas, becoming a therapeutic target for gliomas .(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) However, the mechanism of action and clinical significance of CAMK2B in glioma still unknown.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eBioinformatics analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe GEPIA database (http://gepia.cancer-pku.cn/) is an online platform for gene expression profiling interactive analysis, used to analyze the expression of CAMK2B in different types of tumor tissues and normal tissues. The expression level of CAMK2B mRNA in gliomas and its relationship with patient prognosis were analyzed using data from the Cancer Genome Atlas (TCGA) (https://tcga-data.nci.nih.gov/tcga/), Chinese Glioma Genome Atlas (CGGA) (http://www.cgga.org.cn) and Rembrandt database (http://caintegrator.nci.nih.gov/rembrandt/). All databases were accessed in August 2022. The median was employed to delineate high and low expression groups, and Kaplan-Meier analysis was conducted using the \u0026ldquo;survminer\u0026rdquo; and \u0026ldquo;survival\u0026rdquo; R packages to investigate the association between CAMK2B and the prognosis of glioma patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree human glioma cells (U251,U87, and A172,) and Human Astrocytes cell (HA) were acquired from Procell Life Science \u0026amp; Technology Co., Ltd. The U251 cells were cultured in RPMI-1640 medium (Gibco\u0026trade;,11875093). The U87 cells were cultured in Minimum Essential Medium (MEM, Gibco\u0026trade;, 11090081). The A172 cells were cultured in Dulbecco\u0026apos;s modified Eagle\u0026apos;s medium (DMEM, Gibco\u0026trade;, 10564011). The HA cells were cultured in Astrocyte medium (AM, Sciencell\u0026trade;, #1801). All mediums were contained with 10% fetal bovine serum (FBS, Gibco\u0026trade;, 10099141) and 1% penicillin and streptomycin (Pen-Strep Solution, BI, 2114091). All cells were cultured in a cell incubator at 37 ℃ and 5% carbon dioxide.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeventy-nine glioma tissues and four non-tumor brain tissues (originate from brain tissue removed during brain trauma surgery) were attained from the 2nd Hospital of Hebei Medical University on December 15, 2021. The surgical time of the sample source was from September 24, 2014 to March 9, 2019, the follow-up date for the patient\u0026apos;s survival analysis is December 20, 2021. A portion of the samples were kept in reserve with liquid nitrogen for Western blot experiments, other samples used for IHC experiments were stored in 10% paraformaldehyde. This research was approved by the ethics committee of our hospital. All patients or their guardians obtained informed and written consent in accordance with the guidelines of the ethics committee.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComplete RNA was abstracted from untreated cell and transfected U251 cells and U87 cells by Trizol reagent (Thermo Fisher Scientific) according to the manufacturer\u0026apos;s instructions. After determining the RNA concentration, cDNA was conflated using reverse transcription kits HiScript\u0026reg; III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China). AceQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) was used for PCR procedure. The PCR process is divided into two steps，for first step was set as 95℃，10 min of one cycle, for second step was set as 95℃ 5s，60℃ 30s and 72℃ 30s, with a total of 40 cycles. The primer sequences of CAMK2B were as listed below: F, 5\u0026prime;-GCAAAGAGGCGTATGGCAAG-3\u0026prime;; R, 5\u0026rsquo;-GACGGGAAGTCATAGGCACC-3\u0026prime;. The primer sequences of GAPDH were as follow: F, 5\u0026rsquo;- GGAGCGAGATCCCTCCAAAAT-3\u0026prime;; R, 5\u0026prime;-GGCTGTTGTCATACTTCTCATGG-3\u0026prime;. The relative gene expression was calculated by 2-\u0026Delta;\u0026Delta;Ct method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry (IHC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tissue was stabilized with formalin and then wrapped in paraffin，and sliced. The slices were placed in an incubator at 60℃ for 30 minutes，then they were immersed in xylene and then graded concentrations of ethanol used to remove paraffin. The slices were immersed in 2% citric acid buffer at high temperature and pressure for 25 minutes and then treated with endogenous peroxidase for antigenic repair. The repaired sections were blocked with 10% goat serum. Next, the slices were hatched with primary antibodies（1：100）at 4℃ overnight. After purified three times, the slices were hatched with biotin-labeled goat antibody Rabbit IgG antibody (sp-9001, Zhongshan Golden Bridge Bio-technology, Beijing, China) for 45 min and then incubated with streptavidin‑biotin complex containing horseradish peroxidase (sp-9001, Zhongshan Golden Bridge Bio-technology, Beijing, China) for 45min. Results were examined through avidin-biotin-peroxidase complex solution and visualized with DAB). Finally, the image was captured under an optical microscope. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComplete protein was abstracted from cells using a RIPA buffer with protease and phosphatase inhibitor. Then quantifying total protein concentration by the BCA method. Equal poundage of protein was separated by 10% SDS-PAGE and then diverted into the PVDF membranes, and blocked it use 5% bovine serum albumin (BSA) place in room temperature for forty minutes, and the incubated overnight at 4℃with the primary antibodies against CAMK2B(1:1000),GAPDH(1:7500),Ras(1:1000),RAF1(1:1000) and p-R\u003c/p\u003e\n\u003cp\u003e1（1：1000）.On the next day, after washing by TBST three times, each times for 5 minutes, the incubated the membranes by goat anti-rabbit IgG H＆L preabsorbed secondary antibody（1：10000）.The membranes were detected with the Odyssey infrared scanner. The relative expression of CAMK2B was evaluated by ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEdU assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e5000 cells were inoculated in 96-well plates and cultured overnight. Using 488 Click-iT EdU Cell Proliferation Kit (ShareBio).On the next days, cells were incubated for 2h at a concentration of 10 \u0026mu;M EdU. Then fix, promote infiltration, stain, and seal the cells according to the manufacturer\u0026apos;s instructions, cell nucleus re staining using Hoechst 33342. The results were observed and obtained through a fluorescence microscope, use ImageJ for image statistics and analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Viability Assay (CCK-8 Assay)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e 5000 cells were inoculated in 96-well plates and cultivated for 24,48,72,96 h，10 \u0026mu;l CCK-8 (5 mg/ml) was added to each well, and the cells were incubated for supplemental 2 hours at 37℃，5% carbon dioxide. Then the absorbance values of 450nm were benchmarked by a microplate reader. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranswell Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTreated cells were inoculated in the upper compartment with serum-free medium，the bottom of the upper compartment is coated with matrix glue and the lower compartment is filled with complete medium, culture at 37℃, 5% carbon dioxide for 24 hours. Then, the migrated cells were immobilized with 4% paraformaldehyde for 30min then stained with 1% crystal violet. The invasive cells were observed with optical microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWound Healing Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe treated glioma cells were inoculated into 6-well plates. After the cells grew to a dense state, the cells were scratched with a pipette culet and fostered in serum-free medium. The laceration was captured with optical microscope at 0\u003csup\u003eth\u003c/sup\u003e hour and 24\u003csup\u003eth\u003c/sup\u003e hour at the same region. The wound healing area was analyzed with ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTumor xenotransplantation Assay \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransfected or untransfected U251 cells were inoculated into the right armpits of BALB/c nude mice (4 weeks old, 15-20g, male), each nude mouse was inoculated with 5\u0026times;10\u003csup\u003e6 \u003c/sup\u003eviable cells. Tumor size was checked every seven days and gauged with a slide gauge，and calculated tumor volume by equation: 0.5 \u0026times; length \u0026times; width\u003csup\u003e2\u003c/sup\u003e. The nude mice were sacrificed at 28 days, and the xenotransplantation tumors were collected, weighed and photographed. This experiment has been approved by the Ethics Committee of the 2nd Hospital of Hebei Medical University and carried out according to its guidance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were rehearsed three times, measured data were expressed as mean \u0026plusmn; standard deviation (SD). The t-test was used to parallel data among two groups. Kaplan-Meier method with log-rank test was used to plot the survival curve of glioma patients according to the expression level of CAMK2B. GraphPad Prism9.0 was used to process all the data for statistical analysis. P \u0026lt; 0.05 was supposed to be statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eThe patients with high CAMK2B expression have a better prognosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on transcriptome data from the GEPIA database, we found that the expression level of CAMK2B is lower in glioma tissue compared to normal brain tissue\u003cstrong\u003e (Figure1,A)\u003c/strong\u003e. Immunohistochemical analysis of 70 patients in our hospital showed that the patient prognosis with high CAMK2B was striking better than patients with low CAMK2B (Figure1, B); Analyzing the database of the TCGA, CCGA, and Rambrandt databases obtained similar findings \u003cstrong\u003e(Figure1,C) \u003c/strong\u003eto findings posted on GEPIA website Figure 1A). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCAMK2B is low expression in glioma cell and tissue. \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe analyzed CAMK2B expression in normal brain tissue and grade I-IV glioma tissue with IHC and found that CAMK2B expression levels were decreased in tissues of glioma at high grade\u003cstrong\u003e (Figure2, A). \u003c/strong\u003eIn addition,\u003cstrong\u003e t\u003c/strong\u003ehe mRNA levels measured by qRT-PCR and protein levels measured by Western blot \u003cstrong\u003e(Figure2, B and C)\u003c/strong\u003eyielded the same results, indicating that CAMK2B expression was lower in three glioma cells than in astrocytes. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCAMK2B activation attenuated glioma cell proliferation \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo examine if CAMK2B activation affects the proliferation of glioma cells, we overexpressed CAMK2B through transfection of plasmid into U251 cells and U87 cells and transfect empty plasmids as blank control. The expression levels of CAMK2B in transfected glioma cells were assessed by qRT-PCR and Western blot\u003cstrong\u003e (Figure 3, A and B)\u003c/strong\u003e. The expression of CAMK2B levels were significantly increased in U251 cells and U87 cells. In addition, we used CCK-8 assay and EdU assay to determine the influence of overexpression of CAMK2B on the multiplication of U251 cells and U87 cells, CCK8 assay can be used to observe cell viability to reflect cell proliferation. We respectively divided two types of glioma cells into two groups: the CAMK2B overexpression group (group OE-CAMK2B) and the blank control group transfected with empty plasmids (group OE-NC). Inoculate cells in 96 well plates, 5,000 cells per well, and transfect plasmids into them after inoculation, the 450nm OD value of each four days was detected. Overexpression of CAMK2B reduced the viability of U251 and U87 cell line and thus inhibit its proliferation ability \u003cstrong\u003e(Figure3, C)\u003c/strong\u003e. The EdU assay confirmed that compared to cells transfected with control vectors, the CAMK2B overexpression group had a lower proportion of proliferating cells. \u003cstrong\u003e(Figure3, D)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStimulation of CAMK2B prevented the invasion and migration of glioma cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe used Transwell assay and wound healing assay to explore the influence of activated CAMK2B on the invasion and migration ability of glioma cells. Similarly, the U251 cells and U87 cells transfected with the overexpression plasmid were used for the assays. The U251 cells and U87 cells transfected with CAMK2B or control plasmids were seeded into the Transwell cell at the 0 hour and fixed and stained at the 24 hours, respectively. Then, the number of cells passing through the pores in the group OE-CAMK2B was evidently less than the group OE-NC \u003cstrong\u003e(Figure 4, A)\u003c/strong\u003e. In wound healing assay, the healing areas of U251 cells and U87 cells with overexpressing CAMK2B were smaller than those of the cells transfected with control plasmid \u003cstrong\u003e(Figure 4, B)\u003c/strong\u003e. These data indicated that overexpression of CAMK2B prevents the invasion and migration of glioma cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKnockdown of CAMK2B promoted glioma proliferation in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo knockdown CAMK2B, siRNA plasmid targeting CAMK2B was transfected into glioma cell lines. The expression of CAMK2B was verified by qRT-PCR and Western blot and the expression of CAMK1D and CAMK2A was verified by qRT-PCR\u003cstrong\u003e (Figure 5, A, B, C and D)\u003c/strong\u003e. The expression level of CAMK2B was reduced in glioma cells by 40%. The cells of the siCAMK2B group and siNC group were injected into the armpit of nude mice , the length, width and height of the tumor were measured at 7, 14, 21 and 28 days after injection to calculate the volume of the tumor. The nude mice were executed on the 28th day, and the tumor were removed and weighed. On the 28th day, the tumor volumes were significantly more in mice subjected to siCAMK2B injection (401.9 \u0026plusmn; 66.30 mm\u003csup\u003e3\u003c/sup\u003e, n =5) than in mice subjected to siCAMK2B injection (203.3 \u0026plusmn; 82.23mm\u003csup\u003e3\u003c/sup\u003e, n=5). Furthermore, the tumor weight of mice received siCAMK2B injection was 463.7 \u0026plusmn; 34.64 mg (n=5), which was significantly heavier than those 178.0 \u0026plusmn; 91.93 mg (n = 5) in mice received scCAMK2B injection \u003cstrong\u003e(Figure 5, E and F)\u003c/strong\u003e. These data suggest that knockdown of CAMK2B promotes glioma proliferation and growth in vivo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRegulation of CAMK2B expression levels regulates Ras/Raf/MEK/ERK signals \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe regulate the expression of CAMK2B in U251 cells, and then used Western Blot to determine the protein levels in Ras/Raf/MEK/ERK pathway. Knockdown of CAMK2B with siRNA, expression levels of Ras, p-Raf, p-MEK, p-ERK were increased, an effect was inhibited by the Ras inhibitor Salirasib , which has been experimental evidence that can inhibit the proliferation of glioma both in vivo and in vitro(15, 16) \u003cstrong\u003e(Figure 6, A)\u003c/strong\u003e. On the other hand, overexpression of CAMK2B decreased expression levels of Ras, p-Raf, p-MEK and p-ERK in U251 cells \u003cstrong\u003e(Figure 6, B)\u003c/strong\u003e. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSilencing CAMK2B accelerated the proliferation, invasion and migration of glioma cells through Ras/Raf/MEK/ERK pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe conducted in vitro experiments using the U87 and U251 glioma cells, we divided each of the two cell lines into three groups for experiments, scrambled control group (siNC), siCAMK2B group(siCAMK2B) and siCAMK2B+Salirasib group (siCAMK2B+ Salirasib). CCK8 assay results showed that compared with the control group, the cell viability of the siCAMK2B group was significantly increased on the third and the fourth day, and the siCAMK2B+Salirasib group could reverse this phenomenon after adding the Ras inhibitor Salirasib; Similarly, The EdU assay obtained the same results, indicating that the siCAMK2B group had increased cell proliferation ability, which could be reversed by the Ras inhibitor Salirasib. Knocking down CAMK2B resulted in an approximately 1.5-fold increase in cell proliferation ability compared to the control group.\u003cstrong\u003e (Figure7, A,B)\u003c/strong\u003e Transwell experiment results showed that compared to the control group, the siCAMK2B group significantly increased the number of cells penetrating the matrix glue at 24 hours, approximately 1.2 times that of the control group. The addition of the Ras inhibitor Salirasib can reverse this phenomenon. Therefore, we believe that CAMK2B can affect the invasive ability of glioma cells. \u003cstrong\u003e(Figure7, C)\u003c/strong\u003e The results of the wound healing experiment showed that compared to the control group, the siCAMK2B group had a higher degree of cell migration at 24 hours, and its migration ability to U251 cells was slightly higher than that to U87 cells. This result can also be reversed by the Ras inhibitor Salirasib.\u003cstrong\u003e (Figure7, D)\u003c/strong\u003e These data suggest that CAMK2B affects the hyperplasia, invasion and migration of glioma cells through Ras/Raf/MEK/ERK signaling pathway.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn our prior research, we have conclusively demonstrated that CAMK1D exerts a significant influence invasion and the PI3K/AKT/mTOR signaling pathway(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). This study investigated whether CAMK2B (belonging to the CAMK family) exerts similar impacts on glioma invasion and migration. CAMK2B is implicated in the formation of hippocampal neurons in rats(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Simultaneously, CAMK2B can traverse the H1F-1α signaling pathway, safeguarding neurons from apoptosis triggered by homocysteine(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). The Ca2+-dependent activity and CAMK2 autonomous activity are vital for survival. In mice, the absence of CAMK2B is fatal(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). For instance, the locomotion of mice necessitates the activation of CAMK2B, which is mediated by calcium/calcium-binding proteins, whereas the absence of CAMK2B can result in motor impairments in mice(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). The aforementioned research demonstrates that CAMK2B plays a pivotal role in the development and function of the nervous system. Furthermore, CAMK2B also contributes to the growth of various tumor types, including papillary renal cell carcinoma(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e),human neuroblastoma(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e),breast cancer(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e)etc. Furthermore, CAMK2B also appears in EMT(Epithelial mesenchymal transition) (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) .\u003c/p\u003e \u003cp\u003eIn this study, we experimentally verified that CAMK2B plays a carcinogenic role in gliomas, and this oncogenic effect is mediated through the Ras/Raf/MEK/ERK signaling pathway. Firstly, we have confirmed that the expression of CAMK2B in gliomas is significantly lower compared to that in normal brain tissues. Furthermore, our survival analysis of 70 patients revealed that individuals with gliomas exhibiting high CAMK2B expression tend to have a longer lifespan than those with low CAMK2B expression. The experimental results obtained through modulating the expression of CAMK2B in glioma cells revealed that upon activation of CAMK2B, there was a significant reduction in the proliferation, invasion, and migration of glioma cells. Conversely, downregulation of CAMK2B led to a marked enhancement in these cellular processes. Notably, this enhancement trend could be effectively suppressed by the Ras pathway inhibitor, Salirasib, which was further corroborated at the protein expression level. Collectively, these findings underscore the antitumor role of CAMK2B in glioma, mediated primarily through the Ras/Raf/MEK/ERK signaling pathway.\u003c/p\u003e \u003cp\u003eThe Ras/Raf/MEK/ERK (MAPK) signaling pathway exhibits sensitivity in numerous tumor types. Potential reasons for its activation encompass activating mutations in the KRAS, NRAS, and BRAF genes(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), precisely due to the accumulation of mutations in these crucial genes, cancer arises(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). These mutations have the potential to disrupt the natural cycles of cell proliferation, differentiation, and apoptosis(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). MAPK signaling transduction is one of the primary pathways in cancer biology. The disrupted MAPK signaling pathway significantly contributes to pathological research on numerous human diseases, and its activation accounts for over 40% of human cancer cases(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).For instance, in colon cancer(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e),pancreatic cancer(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e),lung cancer(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e),gastric cancer(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e),breast cancer(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e)rand other diseases are involved in the emergence and evolve of diseases. The Ras/Raf/MEK/ERK signaling pathway plays a pivotal role in the apoptotic and autophagic processes associated with glioma(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), and proliferation, invasion and migration of glioma(\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). The Ras/Raf/MEK/ERK signaling pathway plays a pivotal role in the genesis and progression of glioma. Our findings reveal that modulating the expression level of CAMK2B effectively triggers the activation of the Ras/Raf/MEK/ERK signaling pathway. Salirasib is a widely recognized inhibitor of the Ras pathway, effectively suppressing the expression of Ras(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). We discovered that his Ras pathway inhibitor effectively inhibited the pathway, thereby further corroborating the interaction between CAMK2B and the Ras/Raf/MEK/ERK signaling cascade. Furthermore, based on the KEGG database, we discovered that CAMK2B can exert an influence on the growth and proliferation of glioma cells via the PI3K/AKT/mTOR signaling pathway. Current research has revealed intricate interconnections between the Ras/Raf/MEK/ERK signaling pathway and the PI3K/AKT/mTOR signaling pathway. For instance, a study revealed that agonists implicated in Ras-ERK activation exhibit partial overlap with PI3K/mTORC1 signaling(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). PI3K serves as one of the primary effectors of Ras, playing a pivotal role in regulating crucial cellular processes, including cell viability, therapeutic resistance, and angiogenesis, during oncogenic Ras activation(\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Both the Ras and PI3K signaling pathways play a pivotal role in regulating cellular processes such as apoptosis, growth, differentiation, metabolism, as well as the expression of crucial genes(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). In our prior research, we have established that CAMK1D, which belongs to the same CAMK family as CAMK2B, can influence the malignant progression of glioma via the PI3K signaling pathway(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Hence, we postulate that CAMK2B can potentially influence the progression of glioma via the PI3K/AKT/mTOR signaling pathway, which will be the focal point of our future in-depth investigation into CAMK2B. Simultaneously, we conducted an initial investigation into the interplay among CAMK2B, CAMD, and CAMA, all belonging to the CAMK family. Our PCR experiment revealed that suppressing the expression of CAMK2B in the U251 glioma cell line did not elicit any notable alterations in the expression patterns of CAMK1D. However, the expression level of CAMK2A has slightly increased. Some studies have revealed a closer relationship between CAMK2A and CAMK2B, indicating that they can jointly influence the development of certain diseases. Following the reduced expression level of CAMK2B, a compensatory increase in CAMK2A expression can counteract the effects of the decline, exemplified by the impact of CAMK2A and CAMK2B deletion on brain neurodevelopment, ultimately leading to intellectual disability(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). This viewpoint can be regarded as a promising avenue for future investigations into the role of CAMK2B in glioma.\u003c/p\u003e \u003cp\u003eIn summary, we have demonstrated that the expression level of CAMK2B in gliomas is significantly lower compared to that in normal brain tissues, and it exerts a profound influence on the proliferative, invasive, and migratory capabilities of glioma cells both in vitro and in vivo. Furthermore, we have established that these functions of CAMK2B are mediated through the Ras/Raf/MEK/ERK signaling pathway, thereby presenting novel therapeutic targets and avenues for future research in the diagnosis and treatment of glioma.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eSubstantially, CAMK2B exerts its effect on the biological process of glioma by activating Ras/Raf/MEK/ERK signaling pathway. CAMK2B is down regulated in glioma and can be used as a potential target to predict the prognosis of glioma patients.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request. All data datasets generated and/or analysed during the current study are available in the following repository (http://gepia.cancer-pku.cn/, https://tcga-data.nci.nih.gov/tcga/, http://www.cgga.org.cn, http://caintegrator.nci.nih.gov/rembrandt/\u003cstrong\u003e).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Ethics Committee of the Second Hospital of Hebei Medical University authorized this research (2021-AE047) (2021-R494) and waived the written informed consent requirement.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSZ and JL performed the conception, design, and writing.SZ and QJ completed the experimental verification\u0026nbsp;and prepared figures. QJ, SZ and YS completed the data curation. YW and ZS completed the data analysis. LL performed the Funding acquisition and supervision. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of Hebei Province (H2021206037), the Government-funded Project on Training of Outstanding Clinical Medical Personnel of Hebei Province in the year 2021(303\u0026ndash;16-20\u0026ndash;06), and the Medical Research Project of Hebei Provincial Health Commission (20230031) Conflicts of Interest.\u003c/p\u003e\n\u003cp\u003eNo relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are profoundly grateful that this study was supported by the Second Hospital of Hebei Medical University.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eOstrom QT, Bauchet L, Davis FG, Deltour I, Fisher JL, Langer CE, et al. 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The Science of the total environment. 2020;705:135809.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CAMK2B, glioma, proliferation, invasion, migration, Ras/Raf/MEK/ERK pathway","lastPublishedDoi":"10.21203/rs.3.rs-5412790/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5412790/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eGlioma exhibits a high recurrence rate and unfavorable prognosis, with the mechanisms underlying the regulation of glioma cell proliferation, invasion, and migration remaining elusive. CAMK2B, a crucial kinase involved in regulating cell growth and synaptic plasticity, remains enigmatic in terms of its specific role in glioma.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis study comprehensively analyzed the correlation between the expression level of CAMK2B in gliomas and patient prognosis using immunohistochemistry, qRT-PCR, and Western Blot techniques. Furthermore, the study determined the role of CAMK2B in glioma cell proliferation, invasion, and migration through CCK8, EdU, wound healing, Transwell, and in vivo tumor xenograft assays.\u003c/p\u003e\u003ch2\u003eResult\u003c/h2\u003e \u003cp\u003eWe observed that patients exhibiting high levels of CAMK2B exhibited superior prognostic outcomes compared to those with low levels. Furthermore, CAMK2B expression was notably lower in glioma tissues and cells compared to both normal brain tissue and human astrocyte cell lines. Notably, overexpression of CAMK2B in glioma cells suppressed their proliferative capacity by approximately 40% and attenuated their invasive and migratory abilities by 60\u0026ndash;70%, relative to normal glioma cell lines. Conversely, knockdown of CAMK2B using siRNA-CAMK2B significantly enhanced the proliferative, invasive, and migratory capabilities of glioma cells in both in vitro and in vivo settings, increasing these abilities by 1.5 to 3 times. Remarkably, these effects could be reversed through the application of the Ras pathway inhibitor Salirasib. Western blot analysis revealed that knockdown of CAMK2B activated the Ras/Raf/MEK/ERK signaling pathway in glioma cell lines, whereas overexpression of CAMK2B exerted inhibitory effects on this pathway.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eCAMK2B inhibits glioma proliferation, invasion, and migration through Ras/Raf/MEK/ERK signal pathway.\u003c/p\u003e","manuscriptTitle":"CAMK2B affects the proliferation, invasion and migration of glioma cells via Ras/Raf/MEK/ERK signal pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-11 06:44:32","doi":"10.21203/rs.3.rs-5412790/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"645f010e-16b4-4d4b-ac05-d3ae4b10f948","owner":[],"postedDate":"December 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-03T15:38:47+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-11 06:44:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5412790","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5412790","identity":"rs-5412790","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}