DNMT1 Regulates Angiogenesis Of Human Glioblastoma Stem Cells With MGMT Promoter Hypomethylation

Glioblastoma(GBM)is the most common primary malignant tumor in central nervous system with the worst treatment outcome.These days,various treatments have been performed to treat GBM,including using monoclonal antibody drugs targeting various factors,radiation therapy,alkylating agent chemotherapy drugs,tumor treatment field(TTField),etc.However,various treatments combining standard therapy failed to prolong the median survival of GBM patients.GBM is characterized as a tumor extremely rich in blood vessels.In vitro experiments including animal experiments have demonstrated that inhibition of GBM angiogenesis could effectively inhibit tumor progression and prolong survival.However,the clinical outcome of applying anti-angiogenic drugs for primary GBM treatment was not satisfied.Glioblastoma ste m cells(GSCs)are a subtype of cells with multipotent differentiation potential in glioma.They present high risistance to radiotherapy and chemotherapy,and have a higher potential to promote angiogenesis.GSCs play an important role in the progress of GBM and are also important for the recurrence of GBM.Therefore,further understanding of the unique pathophysiological characteristics of GSCs will provide more ideas for the treatment of GBM.YKL-40 and VEGF are two important secreted pro-angiogenic factors whose roles in the angiogenesis of GBM have been broadly studied,but their pro-angiogenic effects in GSCs are still unclear.In addition,GBM is a type of tumor with a high degree of heterogeneity: it is divided into four subtypes(classical,neuronal,preneural,and mesenchymal)depending on the differences in gene expression;it is also divided into glioma Cp G islands methylation phenotype(G-CIMP)positive and negative subgroups according to methylation patterns.Therefore,it is of great significance to engage precise treatment on GBM according to the different malignant characteristics of each GBM subtype.The promoter methylation status of the O6-methylguanine-DNA-methyltransferase(MGMT)has been considered as one of the indications for chemotherapy using alkylating agents.Hypomethylated MGMT promoter correlated with less sensitivities for alkylating agent treatments.However,the methylation status of MGMT promoter was also found to be related to the prognosis of GBM patients who were not treated with alkylating agents,suggesting that different methylation status of MGMT promoter might represent different GBM methylation subtypes.DNA methyltransferase-1(DNMT1)is one of the basic enzymes to maintain the methylation status of the genome.Changes of its expression level will directly affect the whole genome methylation pattern.Whether DNMT1 can affect the malignant characteristics of GSCs has not been studied yet.?Aims?1)Explore the changes of angiogenic ability in h GSCs by inducing stem cells differentiation with serum medium and investigate the effect of YKL-40 on VEGF expression in h GSCs.2)Discuss the effect of DNMT1 overexpression on the angiogenic ability in MGMT promoter hypomethylated h GSCs.Also,explore the effect of DNMT1 overexpression o n the relationship between YKL-40 and VEGF.?Methods?1)Stem cells were extracted from human GBM samples using standard stem cell isolation and screening methods.The “stemness” markers were detected using immunofluorescence staining,PCR,and Western blot.Serum-induced differentiation assay was performed and the expression and secretion of YKL-40 and VEGF was detected before and after differentiation.The MGMT promoter methylation level of the chosen h GSC cell lines in our study was detected.Then,by inhibiting the expression of YKL-40 with lentivirus,the expression of VEGF was explored.2)Using adenovirus upregulating the expression of DNMT1 in the MGMT promoter hypomethylated h GSCs,we detected the expression of YKL-40,VEGF and MGMT.After down-regulating the expression of YKL-40 and up-regulating the expression of DNMT1,the expression of VEGF was observed.?Results?The isolated h GSCs were enriched with "stemness" markers and had stronger angiogenic ability than the differentiated h DGCs.The expression level of YKL-40 and VEGF in h GSCs were higher than h DGC.In the MGMT promoter hypomethylated h GSCs,YKL-40 promoted VEGF secretion.When DNMT1 was overexpressed,the expression of MGMT,VEGF,and YKL-40 in h GSC was decreased.Meanwhile,inhibiting of YKL-40 expression promoted VEGF expression.?Conclusions?In this study,h GSCs had a stronger angiogenic ability than that of the normal differentiated GBM cells,and this ability was related to the tumor's specific methylation patterns.In MGMT promoter hypomethylated h GSCs,DNMT1 could reduce their pro-angiogenic ability and change some malignant features by increasing the genome wide methylation level.