The Mechanism Of CEBPB Regulating P4HA2 And Affecting The Malignant Behavior Of Glioma Cells

Objective: Glioma is the most common primary malignant tumor of the nervous system,accounting for 80% of malignant brain tumors,with high recurrence and mortality rates,and a median survival period of only 2-5 years.The median survival period for patients with glioblastoma multiforme(GBM)is only about 15 months.Current treatments for glioma mainly rely on surgical resection and postoperative radiotherapy and chemotherapy,with temozolomide(TMZ)being the most used chemotherapeutic agent due to its ability to penetrate the blood-brain barrier.However,glioma often exhibits resistance to TMZ after long-term use,which is the main cause of glioma recurrence.Therefore,improving the sensitivity of glioma to TMZ is crucial for improving the survival rate of glioma patients.Increasing evidence suggests that the extracellular matrix(ECM)plays a crucial role in the development of tumors and their response to treatment,and collagen is the main protein component of the ECM.Research on collagen may provide new clues for the pathogenesis and treatment of solid tumors.Studies have shown that excessive synthesis of collagen can interfere with cell polarity and adhesion,ultimately promoting the proliferation and migration of cancer cells.In addition,the deposition of collagen in the ECM impedes the diffusion of macromolecules,thereby reducing the penetration and efficacy of drugs.Currently,there is little research on the effect of collagen on the development and prognosis of glioma,and research on collagen synthesis may provide insights for the treatment of glioma.Collagen biosynthesis involves multiple complex processes,among which prolyl4-hydroxylase is one of the key enzymes in collagen synthesis.The gene encoding the active site of this enzyme is the prolyl 4-hydroxylase subunit alpha 2(P4HA2),and overexpression of this gene can significantly increase the synthesis of its downstream product,collagen.P4HA2 has been found to be dysregulated in various cancers and is associated with cancer progression and poor prognosis.However,its impact on the TMZ resistance of glioma cells has not been reported.Transcription factors(TFs)are a group of regulatory proteins that can bind to specific DNA sequences and regulate gene transcription,and they play an important role in the occurrence and development of tumors.CCAAT enhancer binding protein beta(CEBPB),as a transcription factor,plays a crucial role in basic cellular processes and has been shown to be associated with malignant biological behavior and drug resistance in various tumors.However,research on the function of this gene is still limited,and no relevant studies have been found on its relationship with TMZ resistance and ECM construction.Isocitrate dehydrogenase(IDH)is an important molecular marker for the molecular classification of gliomas,and it has significant implications for individualized treatment and clinical prognosis of gliomas.A large amount of evidence indicates that(Isocitrate Dehydrogenase 1,IDH1)mutation type gliomas are more sensitive to TMZ treatment than IDH1 wild type gliomas.This study aims to determine the potential mechanisms behind this phenomenon and explore the impact of CEBPB and P4HA2 on the malignant biological behavior of glioma cells,TMZ resistance,and collagen synthesis,providing new targets for the treatment of gliomas.Method: 1.Bioinformatics analysis of the expression levels of CEBPB and P4HA2 in normal brain tissues(NBTs)and glioma tissues from the TCGA and GTEx databases;2.Immunohistochemistry to detect the expression levels of CEBPB and P4HA2 in NBTs and glioma tissues;3.q RT-PCR to measure the expression levels of CEBPB and P4HA2 in glioma cell lines;4.Western blot(WB)to detect the expression levels of CEBPB,P4HA2 and collagen in glioma tissues and cell lines;5.Stable transfection of CEBPB,P4HA2knockdown(KD)and overexpression(OE)plasmids in glioma cell lines(U87,U251);6.Plate colony and Ed U assays to detect cell proliferation;7.Transwell assay to detect cell migration and invasion;8.CCK-8 assay to detect cell sensitivity to TMZ;9.Stable transfection of IDH1-WT and IDH1-R132 H overexpression lentivirus in glioma cell lines(U87,U251);10.Co-immunoprecipitation(Co-IP)experiment and WB to investigate the effect of overexpressed IDH1-R132 H on the ubiquitination and degradation of CEBPB protein;11.Chromatin immunoprecipitation(Ch IP)experiments to confirme the binding of CEBPB to the promoter region of P4HA2;12.Co-transfection of full-length/truncated promoter regions of P4HA2 and overexpression plasmids of CEBPB in HEK239 T cells,as confirmed by dual luciferase reporter assays,validated the regulatory relationship between CEBPB and P4HA2;13.Subcutaneous tumor transplantation experiment in nude mice to detect tumor formation ability;14.Masson’s trichrome staining to detect collagen expression in vivo gliomas;15.Intracranial orthotopic transplantation model in nude mice to detect survival time.Results: 1.Compared to normal brain tissue and IDH1 mutation type gliomas,P4HA2 is highly expressed in IDH1 wild type gliomas and is associated with poor prognosis;2.Knockdown of P4HA2 significantly inhibits the proliferation,migration,invasion,and collagen synthesis of glioma cells and increases their sensitivity to TMZ compared to the scramble group.Overexpression of P4HA2 significantly increases the proliferation,migration,invasion,and collagen synthesis of glioma cells and decreases their sensitivity to TMZ compared to the empty vector group;3.P4HA2 and its interacting proteins are mainly enriched in the construction of the ECM and the composition of collagen;4.Compared to IDH1-mutant gliomas,collagen synthesis-related genes(P4HA2,P4 HB,COL1A1,COL3A1,COL4A2,COL5A1,COL5A2,COL8A2,COL12A1,and COL15A1)are highly expressed in IDH1 wild type gliomas;5.CEBPB expression is positively correlated with the expression of collagen synthesis-related genes and may be a transcription factor for this gene set;6.Compared to normal brain tissue and IDH1 mutation type gliomas,CEBPB is highly expressed in IDH1 wild type gliomas and is associated with poor prognosis;7.Knockdown of CEBPB significantly inhibits the proliferation,migration,invasion,and collagen synthesis of glioma cells and increases their sensitivity to TMZ compared to the scramble group.Overexpression of CEBPB significantly enhances the proliferation,migration,invasion,and collagen synthesis of glioma cells and decreases their sensitivity to TMZ compared to the empty vector group;8.CEBPB protein expression is lower in IDH1-R132H-overexpressing glioma cells,and low expression can be rescued by MG-132;9.Compared to IDH1 wild type overexpressing glioma cells,the ubiquitination level of CEBPB is higher in IDH1-R132 H overexpressing glioma cells;10.CEBPB can bind to the P4HA2 promoter region(TTTGGGCAAC)and promote the transcription of P4HA2;11.Compared to the control group,the CEBPB KD+ P4HA2 KD group significantly decreases the proliferation,migration,invasion,and collagen synthesis of glioma cells and increases their sensitivity to TMZ,while the CEBPB OE + P4HA2 OE group significantly increases the proliferation,migration,invasion,and collagen synthesis of glioma cells and decreases their sensitivity to TMZ.The CEBPB KD+ P4HA2 OE group or the CEBPB OE + P4HA2 KD group does not show a significant statistical difference in the proliferation,migration,invasion,and TMZ sensitivity compared to the control group;12.The results of the nude mouse subcutaneous transplantation tumor experiment showed that compared with the scramble group,the tumor growth rate of the CEBPB KD group,P4HA2 KD group,or CEBPB KD + P4HA2 KD group was slower,and the ultimately formed tumor volume was smaller,and collagen formation in the tumors was lower.The CEBPB KD + P4HA2 KD group had a slower tumor growth,smaller tumor volume rate and lower collagen formation than the single knockdown group.The results of the nude mouse intracranial transplantation tumor experiment showed that compared with the scramble group,the nude mice in the CEBPB KD group,P4HA2 KD group,or CEBPB KD + P4HA2 KD group had a longer survival time.Additionally,the CEBPB KD + P4HA2 KD group had a longer survival time than the single knockdown group.Conclusions: 1.CEBPB and P4HA2 are highly expressed in IDH1 wild type glioma tissues and cells,and their increased expression is associated with poor prognosis;2.Knockdown P4HA2 or CEBPB can inhibit the malignant biological behavior and collagen synthesis of glioma cells and increase the sensitivity to TMZ.Overexpression of P4HA2 or CEBPB can increase the malignant biological behavior and collagen synthesis of glioma cells,and reduce the sensitivity to TMZ;3.CEBPB promotes transcription of P4HA2 by binding to its promoter,thus exerting its pro-cancer and collagen synthesis effects;4.IDH1 mutation is negatively correlated with the transcriptional expression of collagen synthesisrelated genes;5.CEBPB is a key transcription factor for collagen synthesis,and is prone to ubiquitin-proteasomal degradation in IDH1 mutation-type glioma cells;6.Knockdown of CEBPB and P4HA2 alone or in combination can significantly inhibit the growth of xenograft tumors in nude mice and the collagen synthesis within the tumor,and prolong the survival of nude mice.