MGMT and Whole-Genome DNA Methylation Impacts on Diagnosis, Prognosis and Therapy of Glioblastoma Multiforme.
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SUBMITTER: Della Monica R
PROVIDER: S-EPMC9266959 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Glioblastoma multiforme (GBM) is the most malignant glioma with a high death rate. N6-methyladenosine (m6A) RNA methylation plays an increasingly important role in tumors. The current study aimed to determine the function of the regulators of m6A RNA methylation in GBM. We evaluated the difference, interaction, and correlation of these regulators with TCGA database. HNRNPC, WTAP, YTHDF2 and, YTHDF1 were significantly upregulated in GBM. To explore the expression characteristics of regulators in GBM, we defined two subgroups through consensus cluster. HNRNPC, WTAP, and YTHDF2 were significantly upregulated in the cluster2 which had a good overall survival (OS). To investigate the prognostic value of regulators, we used lasso cox regression algorithm to screen an independent prognostic risk characteristic based on the expression of HNRNPC, ZC3H13, and YTHDF2. The prognostic feature between the low and high-risk groups was significantly different (P < 0.05), which could predict significance of prognosis (area under the curve (AUC) = 0.819). Moreover, we used western blot, RT-PCR, and immunohistochemical staining to verify the expression of HNRNPC was associated with malignancy and development of gliomas. Similarly, the high expression of HNRNPC had a good prognosis. In conclusion, HNRNPC is a vital participant in the malignant progression of GBM and might be valuable for prognosis.
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Project description:Glioblastoma multiforme (GBM) is one of the most lethal and damaging types of human cancer. The current study was conducted to identify differentially methylated genes (DMGs) between GBM and normal controls, and to improve our understanding of GBM at the epigenetic level. The DNA methylation profile of GBM was downloaded from the Gene Expression Omnibus (GEO) database using the accession number GSE50923. The MethyAnalysis package was applied to identify DMGs between GBM and controls, which were then analyzed by functional enrichment analysis. Protein-protein interaction (PPI) networks were constructed using the hypermethylated and hypomethylated genes. Finally, transcription factors (TFs) that can regulate the hypermethylated and hypomethylated genes were predicted, followed by construction of transcriptional regulatory networks. Furthermore, another relevant dataset, GSE22867, was downloaded from the GEO database for data validation. A total of 476 hypermethylated and 850 hypomethylated genes were identified, which were mainly associated with the functions of 'G-protein-coupled receptors ligand binding', 'cytokine activity', 'cytokine-cytokine receptor interaction', and 'D-glutamine and D-glutamate metabolism'. The hypermethylated gene neuropeptide Y (NPY) and the hypomethylated gene tumor necrosis factor (TNF) demonstrated high degrees in the PPI network. Forkhead box protein A1 (FOXA1), potassium voltage-gated channel subfamily C member 3 (KCNC3) and caspase-8 (CASP8) exhibited high degrees in the transcriptional regulatory networks. In addition, the methylation profiles of NPY, TNF, FOXA1, KCNC3 and CASP8 were confirmed by another dataset. In summary, the present study systematically analyzed the DNA methylation profile of GBM using bioinformatics approaches and identified several abnormally methylated genes, providing insight into the molecular mechanism underlying GBM.
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Project description:Glioblastoma multiforme (GBM) is the most common high-grade intracranial tumor in adults. It is characterized by uncontrolled proliferation, diffuse infiltration due to high invasive and migratory capacities, as well as intense resistance to chemo- and radiotherapy. With a five-year survival of less than 3% and an average survival rate of 12 months after diagnosis, GBM has become a focus of current research to urgently develop new therapeutic approaches in order to prolong survival of GBM patients. The methylation status of the promoter region of the O6-methylguanine-DNA methyltransferase (MGMT) is nowadays routinely analyzed since a methylated promoter region is beneficial for an effective response to temozolomide-based chemotherapy. Furthermore, several miRNAs were identified regulating MGMT expression, apart from promoter methylation, by degrading MGMT mRNA before protein translation. These miRNAs could be a promising innovative treatment approach to enhance Temozolomide (TMZ) sensitivity in MGMT unmethylated patients and to increase progression-free survival as well as long-term survival. In this review, the relevant miRNAs are systematically reviewed.
| S-EPMC7281574 | biostudies-literature
Project description:Glioblastoma (GBM) with oligodendroglioma component (GBMO) is a newly described GBM subtype in the 2007 World Health Organization classification. However, its biological and genetic characteristics are largely unknown. We investigated the clinicopathological and molecular features of 34 GBMOs and compared the survival rate of these patients with those of patients with astrocytoma, oligodendroglioma, anaplastic oligoastrocytoma (AOA), and conventional GBMs in our hospital. GBMO could be divided into two groups based on the presence of an IDH1 mutation. The IDH1 mutation was more frequently found in secondary GBMO, which had lower frequencies of EGFR amplification but higher MGMT methylation than the wild type IDH1 group, and patients with mutant IDH1 GBMO were on average younger than those with wild-type IDH1. Therefore, GBMO is a clinically and molecularly heterogeneous subtype, largely belonging to a proneural and classical subtype of GBM. The survival rate of GBMO patients itself was worse than that of AOA patients but not significantly better than that of conventional GBM patients. GBMO survival was independent of the dominant histopathological subtype i.e., astrocyte-dominant or oligodendroglioma -dominant, but it was significantly associated with the IDH1 mutation and MGMT methylation status. Therefore, GBMO should be regarded as a separate entity from AOA and must be classified as a subtype of GBM. However, further study is needed to determine whether it is a pathologic variant or a pattern of GBM because GBMO has a similar prognosis to conventional GBMs.
| S-EPMC4311043 | biostudies-literature
Project description:Few studies had investigated genome-wide methylation in glioblastoma multiforme (GBM). Our goals were to study differential methylation across the genome in gene promoters using an array-based method, as well as repetitive elements using surrogate global methylation markers. The discovery sample set for this study consisted of 54 GBM from Columbia University and Case Western Reserve University, and 24 brain controls from the New York Brain Bank. We assembled a validation dataset using methylation data of 162 TCGA GBM and 140 brain controls from dbGAP. HumanMethylation27 Analysis Bead-Chips (Illumina) were used to interrogate 26,486 informative CpG sites in both the discovery and validation datasets. Global methylation levels were assessed by analysis of L1 retrotransposon (LINE1), 5 methyl-deoxycytidine (5m-dC) and 5 hydroxylmethyl-deoxycytidine (5hm-dC) in the discovery dataset. We validated a total of 1548 CpG sites (1307 genes) that were differentially methylated in GBM compared to controls. There were more than twice as many hypomethylated genes as hypermethylated ones. Both the discovery and validation datasets found 5 tumor methylation classes. Pathway analyses showed that the top ten pathways in hypomethylated genes were all related to functions of innate and acquired immunities. Among hypermethylated pathways, transcriptional regulatory network in embryonic stem cells was the most significant. In the study of global methylation markers, 5m-dC level was the best discriminant among methylation classes, whereas in survival analyses, high level of LINE1 methylation was an independent, favorable prognostic factor in the discovery dataset. Based on a pathway approach, hypermethylation in genes that control stem cell differentiation were significant, poor prognostic factors of overall survival in both the discovery and validation datasets. Approaches that targeted these methylated genes may be a future therapeutic goal.
| S-EPMC3931727 | biostudies-literature
Project description:The repair protein O6-methylguanine-DNA methyltransferase (MGMT) is regulated epigenetically, mainly by the methylation of the MGMT promoter. MGMT promoter methylation status has emerged as a prognostic and predictive biomarker for patients with newly diagnosed glioblastoma (GBM). However, a strong negative correlation between MGMT promoter methylation and MGMT protein expression cannot be applied as a rule for all GBM patients. In order to investigate if the DNA methylation status of MGMT enhancers is associated with MGMT promoter methylation, MGMT expression, and the overall survival (OS) of GBM patients, we established assays based on high-resolution melting analysis and pyrosequencing for one intragenic and three intergenic MGMT enhancers. For CpGs in an enhancer located 560 kb upstream of the MGMT promoter, we found a significant negative correlation between the methylation status and MGMT protein levels of GBM samples expressing MGMT. The methylation status of CpGs in the intragenic enhancer (hs696) was strongly negatively correlated with MGMT promoter methylation and was significantly higher in MGMT-expressing GBM samples than in MGMT-non-expressing GBM samples. Moreover, low methylation of CpGs 01-03 and CpGs 09-13 was associated with the longer OS of the GBM patients. Our findings indicate an association between MGMT enhancer methylation and MGMT promoter methylation, MGMT protein expression, and/or OS.
| S-EPMC10297309 | biostudies-literature
Project description:The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) removes temozolomide-induced alkylation, thereby preventing DNA damage and cytotoxicity. We investigated the prognostic effect of different MGMT methylation levels on overall and progression-free survival in 327 patients with primary glioblastoma undergoing standard treatment. We obtained MGMT methylation level in 4 CpG sites using pyrosequencing. The association between MGMT methylation level and survival was investigated using Cox proportional hazards model and an extension to detect time-varying effects. We found an association between MGMT methylation level and overall survival (OS) from around 9 months after the diagnosis, with no association between MGMT methylation level and OS before that. For patients surviving at least 9 months even small increases in MGMT methylation level are significantly beneficial (HR = 0.97, 95% CI [0.96, 0.98]). The predictive ability of MGMT methylation level on OS from 9 months after diagnosis has a Harrel's C of 66%. We conclude that the MGMT methylation level is strongly associated with survival only for patients surviving beyond 9 months with considerable effects for levels much lower than previously reported. Prognostic evaluation of cut-points of MGMT methylation levels and of CpG island site selection should take the time-varying effect on overall survival into account.
| S-EPMC6581556 | biostudies-literature
Project description:The identification of molecular genetic biomarkers considerably increased our current understanding of glioma genesis, prognostic evaluation, and treatment planning. In glioblastoma, the most malignant intrinsic brain tumor entity in adults, the promoter methylation status of the gene encoding for the repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) indicates increased efficacy of current standard of care, which is concomitant and adjuvant chemoradiotherapy with the alkylating agent temozolomide. In the elderly, MGMT promoter methylation status has recently been introduced to be a predictive biomarker that can be used for stratification of treatment regimes. This review gives a short summery of epidemiological, clinical, diagnostic, and treatment aspects of patients who are currently diagnosed with glioblastoma. The most important molecular genetic markers and epigenetic alterations in glioblastoma are summarized. Special focus is given to the physiological function of DNA methylation-in particular, of the MGMT gene promoter, its clinical relevance, technical aspects of status assessment, its correlation with MGMT mRNA and protein expressions, and its place within the management cascade of glioblastoma patients.
| S-EPMC3792931 | biostudies-literature
Project description:BackgroundResistance of the highly aggressive glioblastoma multiforme (GBM) to drug therapy is a major clinical problem resulting in a poor patient's prognosis. Beside promoter methylation of the O6-methylguanine-DNA-methyltransferase (MGMT) gene the efflux transporters ABCB1 and ABCG2 have been suggested as pivotal factors contributing to drug resistance, but the methylation of ABCB1 and ABCG2 has not been assessed before in GBM.MethodsTherefore, we evaluated the proportion and prognostic significance of promoter methylation of MGMT, ABCB1 and ABCG2 in 64 GBM patient samples using pyrosequencing technology. Further, the single nucleotide polymorphisms MGMT C-56 T (rs16906252), ABCB1 C3435T (rs1045642) and ABCG2 C421A (rs2231142) were determined using the restriction fragment length polymorphism method (RFLP). To study a correlation between promoter methylation and gene expression, we analyzed MGMT, ABCB1 and ABCG2 expression in 20 glioblastoma and 7 non-neoplastic brain samples.ResultsDespite a significantly increased MGMT and ABCB1 promoter methylation in GBM tissue, multivariate regression analysis revealed no significant association between overall survival of glioblastoma patients and MGMT or ABCB1 promoter methylation. However, a significant negative correlation between promoter methylation and expression could be identified for MGMT but not for ABCB1 and ABCG2. Furthermore, MGMT promoter methylation was significantly associated with the genotypes of the MGMT C-56 T polymorphism showing a higher methylation level in the T allele bearing GBM.ConclusionsIn summary, the data of this study confirm the previous published relation of MGMT promoter methylation and gene expression, but argue for no pivotal role of MGMT, ABCB1 and ABCG2 promoter methylation in GBM patients' survival.
| S-EPMC3890604 | biostudies-literature
Project description:BackgroundLiterature reports that mature microRNA (miRNA) can be methylated at adenosine, guanosine and cytosine. However, the molecular mechanisms involved in cytosine methylation of miRNAs have not yet been fully elucidated. Here we investigated the biological role and underlying mechanism of cytosine methylation in miRNAs in glioblastoma multiforme (GBM).MethodsRNA immunoprecipitation with the anti-5methylcytosine (5mC) antibody followed by Array, ELISA, dot blot, incorporation of a radio-labelled methyl group in miRNA, and miRNA bisulfite sequencing were perfomred to detect the cytosine methylation in mature miRNA. Cross-Linking immunoprecipiation qPCR, transfection with methylation/unmethylated mimic miRNA, luciferase promoter reporter plasmid, Biotin-tagged 3'UTR/mRNA or miRNA experiments and in vivo assays were used to investigate the role of methylated miRNAs. Finally, the prognostic value of methylated miRNAs was analyzed in a cohorte of GBM pateints.ResultsOur study reveals that a significant fraction of miRNAs contains 5mC. Cellular experiments show that DNMT3A/AGO4 methylated miRNAs at cytosine residues inhibit the formation of miRNA/mRNA duplex and leading to the loss of their repressive function towards gene expression. In vivo experiments show that cytosine-methylation of miRNA abolishes the tumor suppressor function of miRNA-181a-5p miRNA for example. Our study also reveals that cytosine-methylation of miRNA-181a-5p results is associated a poor prognosis in GBM patients.ConclusionTogether, our results indicate that the DNMT3A/AGO4-mediated cytosine methylation of miRNA negatively.
| S-EPMC7041276 | biostudies-literature