Targeting CDC25A To Induce Apoptosis In Medulloblastoma Cells And The Mechanism

Background:Medulloblastoma is the most common intracranial malignancy in children.Multimodal treatment with maximal surgical resection of the tumor coupled with craniospinal irradiation and adjuvant chemotherapy has increased the five-year survival rate for medulloblastoma patients to 90% and for high-risk patients to 70%.However more than 25% of patients develop cerebellar silence,dysarthria and neurocognitive deficits after surgical resection,and most survivors exhibit long-term neurocognitive and neuroendocrine complications,in addition to a low five-year disease-free survival rate for patients with tumor spread(36%),and 18% of patients develop secondary tumors within 30 years,and the prognosis for patients with recurrent medulloblastoma remains poor.The current first-line chemotherapy regimens for medulloblastoma are the Packer and Taylor regimens,which are still under continuous research and improvement.The specific mechanisms of action of some chemotherapeutic agents in medulloblastoma are still unclear,and there are few drugs specifically targeting medulloblastoma,which need to be urgently studied and explored.To address these questions,this study used a bioinformatics approach to screen for a gene(CDC25A)that affects the prognosis of medulloblastoma,and validated the function of the gene in vitro and in vivo using a CDC25 A inhibitor(NSC663284),and performed transcriptome sequencing on cells with knockdown CDC25 A to explore the molecular mechanisms associated with CDC25 A.Based on the literature and transcriptome sequencing results,we selected the combination of sorafenib and explored the mechanism related to the combination of NSC663284 and sorafenib through in vivo and in vitro experiments,which promoted the research progress of medulloblastoma from multiple perspectives and provided new ideas for the target drug screening of medulloblastoma.Materials and Methods:1.Analysis of medulloblastoma transcriptome data by bioinformatics methods to screen for differential genes and core genes in differential genes(CDC25A)and to screen for inhibitors targeting CDC25A(NSC663284).2.MTS assay to detect the optimal drug concentration of NSC663284 in D283 and Daoy cells.3.Cell cycle assay,plate cloning assay,scratch assay,flow cytometry to detect the effect of NSC663284 on proliferation ability,migration ability,cell cycle and apoptosis of D283 and Daoy cells.4.WB,RT-PCR to detect the effect of NSC663284 on D283 and Daoy cells,CDC25 A and CDK2 protein expression and RNA transcription,and to explore the changes of NF-κB pathway-related proteins.5.Construct null and knockdown CDC25 A medulloblastoma cell lines using a lentiviral infection method.6.In vivo experiments to investigate the effect of knockdown CDC25 A cells on tumor growth rate.7.In vivo experiments were performed to investigate the effect of NSC663284 on the growth rate of medulloblastoma and the survival of tumor-bearing mice.8.Transcriptome sequencing of null and knockdown CDC25 A cell lines was performed to analyze their differential gene regulatory network changes,screen key modules and hub genes,and explore potential mechanisms of medulloblastoma peritoneal metastasis.9.Flow cytometry to detect the effect of sorafenib combined with NSC663284 on apoptosis and iron death of D283 and Daoy cells.10.WB assay to detect the changes of sorafenib combined with NSC663284 in autophagy,NF-κB pathway,and ERK and PI3 K proteins.11.In vivo experiments were performed to investigate the effects of sorafenib in combination with NSC663284 on the growth rate of medulloblastoma and the survival of tumor-bearing mice.Result:1.analyze GEO dataset(GSE74195,GSE95684)using R language limma package to obtain differential genes in medulloblastoma compared with normal cerebellar tissue,analyze the impact of candidate genes in differential genes on patient prognosis,and screen the core gene(CDC25A).2.Cell cycle,scratch,and plate cloning assays revealed that the CDC25 A inhibitor(NSC663284)blocked the cell cycle of medulloblastoma cells and inhibited the migration and proliferation of medulloblastoma cells.3.WB experiments suggested that NSC663284 blocked cell cycle by affecting the function of CDC25 A protein to dephosphorylate CDK2,and induced apoptosis by inhibiting NF-κB pathway.4.The knockdown CDC25 A medulloblastoma cell model was constructed and tested in vivo,and it was found that the tumor proliferation in sh CDC25 A mice was significantly slower than that in sh NC mice,and NSC663284 showed a tendency to slow down the tumor proliferation and improve the survival of tumor-bearing mice after acting on the tumor model constructed from D283 cells.5.After sequencing the medulloblastoma cells with knockdown of CDC25 A,it was found that the changes of gene regulatory network compared to sh NC group mainly showed that the up-regulated network was mainly involved in biological processes such as protein modification and metabolism,and the down-regulated network was mainly involved in biological processes such as mitochondrial metabolism and cell cycle,USP47,CYB5R1,RRAGA,KAT8,SCRN3 as the up-regulated network HUB genes,and GTF3C3,SARNP,CNOT10,TMEM248,BROX as the down-regulated network HUB genes regulated gene network.6.WB assay and flow cytometry detected synergistic effect of NSC663284 and sorafenib in inducing apoptosis in medulloblastoma cells;however,both did not cause apoptosis in medulloblastoma cells through iron death or autophagic pathway.7.Flow cytometry assays showed that cells with knockdown CDC25 A were more sensitive to sorafenib.8.WB experiments suggest that co-administration of sorafenib with NSC662384 promotes apoptosis by co-inhibiting NF-κB pathway.9.In vivo experiments suggested that the combination of sorafenib and NSC662384 significantly slowed tumor proliferation and tended to prolong the survival time of tumorbearing mice.Conclusion:1.CDC25 A can be used as a therapeutic target for medulloblastoma.2.Targeting CDC25 A blocks the cell cycle through the CDC25A-CDK2 pathway and affects apoptosis by inhibiting the NF-κB pathway.3.Knockdown of CDC25 A facilitates the inhibition of cell proliferation and slows down tumor proliferation in hormonal mice,and targeting CDC25 A has a tendency to slow down tumor proliferation and improve prognosis in hormonal mice.4.Transcriptome sequencing analysis after knockdown of CDC25 A suggested inhibition of regulatory networks related to energy metabolism.5.Combination of CDC25 A inhibitor with sorafenib induced apoptosis by inhibiting NF-κB pathway rather than iron death or autophagy pathway in the presence of concomitantly reduced drug concentrations.The combination of the two drugs can slow down the proliferation of medulloblastoma and has a tendency to improve the survival of hormonal mice,which has the potential to be a combination therapy for medulloblastoma.