The Role And Mechanism Of DEC1 And RAD51 In The Progression Of Glioblastoma

【Background】Gliomas are the most commonly occurring form of brain tumor.Glioblastoma multiforme(GBM),which is the most malignant form of glioma,has a very poor prognosis with a 5-year survival rate of 4–5% and a paltry median survival of 14.6 months.Treatment with a combination of chemotherapy,radiotherapy and surgery has improved the survival rate of glioblastoma patients but often fail due to the development of chemoresistance and patients’ poor respond to chemotherapy drugs.These patients will undergo local recurrence soon.Therefore,it is of great practical significance to investigate the molecular mechanisms involved in the development of GBM and explore new potential therapeutic targets for GBM.Targeting deficient mechanisms of cellular DNA repair still represents the basis for the treatment of the majority of solid tumors,and increased DNA repair capacity is a hallmark mechanism of resistance to DNA-damaging treatments such as cytotoxic drugs and radiotherapy.RAD51 is a highly conserved protein that catalyzes DNA repair and maintains gene stability via homologous recombination,a major DNA repair pathway which directly modulates cellular sensitivity to DNA-damaging treatments.Thus,targeting RAD51 is a potential strategy for the sensitization of GBM cells to chemotherapeutic drugs by inducing DNA damage.Human differentiated embryonic chondrocyte-expressed gene 1(DEC1)is a member of the basic helix-loop-helix(b HLH)transcription factor family.Multiple cellular processes,including differentiation,regulation of circadian rhythm,tumorigenesis,and response to hypoxia,have been reported to involve DEC1.It was reported that high expression of DEC1,which was significantly associated with high pathological tumor grade and poor response to TMZ chemotherapy,was demonstrated to be an unfavorable independent prognostic factor.Studies have found that DEC1 is up-regulated in a variety of tumors and is associated with the malignant progression of tumor,and may be act as an oncogene.However,the expression and clinical significance of DEC1 in GBM remains unclear,and its biological functions and related mechanisms remain to be further studied.Based on the above fact we proposed that DEC1 and RAD51 is essential to GBM proliferation and migration and play a critical role in GBM development.Therefore,this study aims to investigate the role of DEC1 and RAD51 in glioblastoma development,drug resistance and microenvironment,so as to provide a new direction and strategy for clinical treatment of glioma.【Aims】1.To study the effects of DEC1 on the malignant biological behavior of glioblastoma.2.To explore the molecular mechanism of DEC1 in regulating the microenvironment of glioblastoma.3.To study the effects of RAD51 on the malignant biological behavior and drug resistance of glioblastoma and explore the relationship between DEC1.【Methods】1.First,the DEC1 overexpression,knockdown and corresponding control cell lines were used to study the effects of DEC1 on malignant biological behaviors such as glioma cell proliferation and migration though CCK-8 assay,colony formation assay,and wound healing assay.2.Using m RNA transcriptome sequencing to gain different gene expression profiles in LN18-sh DEC1 and LN18-Scramble cells.We used the Cluster analysis,GO function analysis,and KEGG pathway analysis to select the crucial pathway molecules regulated by DEC1.Then,q PCR assays were used to further confirm.Transwell assay were used to study the chemotaxis ability of glioma cells to tumor-associated macrophages after DEC1 was up-or down-regulated.3.TCGA database was utilized to analyze the expression changes of RAD51 in gliomas;RAD51 was overexpressed or knockdown in U251 glioma cells via lentivirus infection,or inhibited its activity by using small molecule inhibitors.Cell proliferation and migration ability were examined by CCK-8 assay,colony formation assay,and scratch wound-healing assay;CCK-8 assay and flow cytometry were utilized to assess the effect of RAD51 on temozolomide sensitivity of glioma cells upon treatment with temozolomide;Western blot was used to study the molecular mechanism of RAD51 regulating glioma development or drug resistance.【Results】1.Overexpression of DEC1 could promote the proliferation and migration of GBM cells,and downregulation of DEC1 showed opposite effects.2.According to the analysis of m RNA transcriptome sequencing,it was found that the expression of DEC1 was related to immunocyte cell chemotaxis,T cell activation,tumor immunity,angiogenesis or other functions,and CCL2/CXCL8 may be a crucial pathway molecule regulated by DEC1 in GBM cell chemotaxis.Then,it was confirmed that silencing DEC1 significantly down-regulated the m RNA level of CCL2/CXCL8.Transwell assay confirmed that the chemotactic recruitment ability of glioma cells to macrophages was enhanced after DEC1 overexpression,and this ability was decreased after DEC1 was down-regulated.3.The TCGA database showed that the expression of RAD51 was significantly increased in glioma tissues;A RAD51 overexpressing or interfering lentiviral plasmid was transfected into GBM cells to stably overexpression or knockdown RAD51,respectively.Overexpression of RAD51 can enhance the proliferation and migration ability of U251 glioma cells,and inhibition of RAD51 showed opposite effect;knockdown of RAD51 can enhance the sensitivity of U251 glioma cells to temozolomide,promoted cell apoptosis induced by temozolomide.Overexpression of RAD51 increased the expression of P53,whereas knockdown of RAD51 decreased the expression of P53.【Conclusions】1.DEC1 can enhance the proliferation and migration ability of glioma cells,and down-regulation of DEC1 inhibited cell proliferation,migration of glioma cells in vitro,DEC1 functions as an oncogene in tumor cells.3.DEC1 may affect the chemotaxis of tumor-associated macrophages by regulating the expression of CCL2 and CXCL8,and then affect the establishment and development of glioma tumor microenvironment.2.RAD51 is highly expressed in GBM tissues,and plays an oncogene function in glioma cells.RAD51 overexpression enhances the proliferation and migration of glioma cells.RAD51 knockdown increases the sensitivity of glioma cells to temozolomide.