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Locus specific TET2 alterations of DNA methylation and hydroxymethylation in glioma. (A) Schematic representation of gene organization. Exons, introns, and transcription start site (TSS) locations are shown below. The 21 CpG positions along TET2 analyzed in the Illumina methylation array are indicated by black dots (intragenic CpG sites are bounded by the orange frame). Locus-specific patterns of DNA methylation (blue to yellow) and hydroxymethylation (gray to blue) in 5 non-tumorigenic human brain and 9 glioblastoma samples are shown. Location of representative CpGs selected for study (cg12306086 and cg20586654) are indicated by the small red boxes below the heatmap, in which they are marked with asterisks. (B) Box plot showing differences between the average percentage of 5 mC and 5 hmC in both normal brain and glioma samples of the two-representative intragenic CpGs studied (cg12306086 and cg20586654) (upper part). Technical validation by bisulfite pyrosequencing of 5 mC and 5 hmC changes occurring among normal samples (n = 5), primary tumors (n = 9) and glioblastoma cell lines (n = 4) are shown below. p-values were adjusted by applying the Bonferroni correction. * p-value < 0.05; ** p-value < 0.01. (C) Scatter plots showing the percentage of 5 mC (upper panel) and 5 hmC (lower panel) in normal (blue plots) and tumoral (yellow plots) samples obtained by pyrosequencing (y-axis) and arrays (x-axis). ρ: Spearman rank correlation.
Source publication
Ten-eleven translocation (TET) enzymes are frequently deregulated in cancer, but the underlying molecular mechanisms are still poorly understood. Here we report that TET2 shows frequent epigenetic alterations in human glioblastoma including DNA hypermethylation and hypo-hydroxymethylation, as well as loss of histone acetylation. Ectopic overexpress...
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Context 1
... study the possible aberrant epigenetic regulation of TET2 in brain tumors, we first used data from the 450K Infinium Illumina methylation platform. We determined the DNA methylation and hydroxymethylation status of 21 CpG positions within TET2 promoter DNA regions and the gene body in 9 samples obtained from patients with glioblastoma and 5 from non-tumorigenic brain samples. This revealed a locus-specific pattern of DNA methylation and hydroxymethylation alterations in tumoral samples ( Figure 1A). CpG sites located at the promoter CpG island presented very low levels of 5 mC and of 5 hmC in both brain and glioblastoma samples. In contrast, intragenic CpG sites presented much higher 5 hmC levels in the non- tumorigenic samples compared with those from tumors (on average, 15% and 2%, respectively), while intragenic 5 mC levels were higher (10%) in tumoral ...
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... validate the data obtained with the methylation arrays we used bisulfite pyrosequencing to analyze the DNA methylation and hydroxymethylation status of two CpG sites located at intron 2 of TET2 (cg12306086 and cg20586654) in the same samples analyzed in the methylation arrays, as well as in additional glioblastoma cell lines (see material and methods). The results confirmed the 5 mC and 5 hmC profiles obtained in the arrays (Figure 1B- 1C). Moreover, these experiments revealed a similar pattern of 5 mC and 5 hmC at two neighboring CpG sites of the cg12306086 position (Supplementary Figure 1A). The loss of 5 hmC in glioblastoma was further validated by the results of an alternative technique which is not dependent on the oxidative bisulfite conversion and based instead on DNA immunoprecipitation with an antibody against 5 hmC (Supplementary Figure ...
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... validate the data obtained with the methylation arrays we used bisulfite pyrosequencing to analyze the DNA methylation and hydroxymethylation status of two CpG sites located at intron 2 of TET2 (cg12306086 and cg20586654) in the same samples analyzed in the methylation arrays, as well as in additional glioblastoma cell lines (see material and methods). The results confirmed the 5 mC and 5 hmC profiles obtained in the arrays (Figure 1B- 1C). Moreover, these experiments revealed a similar pattern of 5 mC and 5 hmC at two neighboring CpG sites of the cg12306086 position (Supplementary Figure 1A). The loss of 5 hmC in glioblastoma was further validated by the results of an alternative technique which is not dependent on the oxidative bisulfite conversion and based instead on DNA immunoprecipitation with an antibody against 5 hmC (Supplementary Figure ...
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... we used bisulfite pyrosequencing to analyze the 5 mC and 5 hmC levels at the cg20586654 CpG position in an independent cohort of 8 glioblastomas and 7 non- tumorigenic brains. We detected TET2 hypermethylation and hypo-hydroxymethylation in all the tumoral samples analyzed (Supplementary Figure 1B), which confirmed that this is a frequent event both in vivo and in ...
Citations
... In vivo studies showed that nude mice with TET2-transfected LN229 cells presented much lower tumorigenic potential with a considerable difference in tumor weight. Moreover, altered expression of 19 neuroectodermal markers in TET2-overexpressed cells was determined, among which the most significant changes were observed for Mash1 and Cystathionine β-synthase [48,49]. TET2 loss is associated with stem cell features and correlates with poor survival of patients with GBM [50]. ...
Glioblastoma (GBM) is a primary brain tumor arising from glial cells. The tumor is highly aggressive, the reason for which it has become the deadliest brain tumor type with the poorest prognosis. Like other cancers, it compromises molecular alteration on genetic and epigenetic levels. Epigenetics refers to changes in gene expression or cellular phenotype without the occurrence of any genetic mutations or DNA sequence alterations in the driver tumor-related genes. These epigenetic changes are reversible, making them convenient targets in cancer therapy. Therefore, we aim to review critical epigenetic dysregulation processes in glioblastoma. We will highlight the significant affected tumor-related pathways and their outcomes, such as regulation of cell cycle progression, cell growth, apoptosis, angiogenesis, cell invasiveness, immune evasion, or acquirement of drug resistance. Examples of molecular changes induced by epigenetic modifications, such as DNA epigenetic alterations, histone post-translational modifications (PTMs), and non-coding RNA (ncRNA) regulation, are highlighted. As understanding the role of epigenetic regulators and underlying molecular mechanisms in the overall pro-tumorigenic landscape of glioblastoma is essential, this literature study will provide valuable insights for establishing the prognostic or diagnostic value of various non-coding transcripts, including miRNAs.
... Abnormal expression and mutations of the TET family and ALKBH1 ″erasers" are also associated with several malignancies [22][23][24][25]. In renal cell carcinoma, high expression of ALKBH1 is correlated with malignant features of the tumor [26]. ...
5-methylcytosine modifications play a significant role in carcinogenesis; however, studies exploring 5-methylcytosine-related genes in diffuse large B-cell lymphoma patients are lacking. In this study, we aimed to understand the potential role and clinical prognostic impact of 5-methylcytosine regulators in diffuse large B-cell lymphoma and identify a prognostic biomarker based on 5-methylcytosine-associated genes. Gene expression profiles and corresponding clinical information of diffuse large B-cell lymphoma patients and normal controls were obtained from The Cancer Genome Atlas, Gene Expression Omnibus, and Genotype-Tissue Expression databases. Diffuse large B-cell lymphoma was divided into three clusters according to the 5-methylcytosine regulators, and differentially expressed genes were screened among the three clusters. Univariate Cox and Lasso-Cox regression analyses were used to screen prognostic genes and construct a prognostic risk model. Kaplan-Meier curve analysis, univariate and multivariate Cox regression analyses, and time-dependent receiver operator characteristic curve analysis were used to evaluate prognostic factors. GSVA was used to enrich potential pathways associated with 5-methylcytosine modification patterns. SsGSEA and CIBERSORT were used to assess immune cell infiltration. Six 5-methylcytosine-related genes (TUBB4A, CD3E, ZNF681, HAP1, IL22RA2, and POSTN) were used to construct a prognostic risk model, which was proved to have a good predictive effect. In addition, univariate and multivariate Cox regression risk scores were independent prognostic factors for diffuse large B-cell lymphoma. Further analysis showed that the 5-methylcytosine risk score was significantly correlated with immune cell infiltration and immune checkpoint of diffuse large B-cell lymphoma. Our study reveals for the first time a potential role for 5-methylcytosine modifications in diffuse large B-cell lymphoma, provides novel insights for future studies on diffuse large B-cell lymphoma, and offers potential prognostic biomarkers and therapeutic targets for patients with diffuse large B-cell lymphoma.
... The role of several other RNA modifications has also been described in GB occurrence and progression (Fig. 1). The teneleven translocation 1 (TET1) protein which is responsible for the oxidative processing of RNA m 5 C and formation of 5-hydroxymethylcytidine (hm 5 C) [44] was found elevated in GB [82], while TET2 is decreased [83], and TET3 is epigenetically repressed in gliomas [84]. The m 7 G methyltransferase complex METTL1/WDR4 has been also implicated in glioma pathogenesis, with METTL1 expression being augmented in gliomas and inversely correlated with glioma prognosis [85]. ...
Gliomas are highly malignant tumors accounting for the majority of brain neoplasms. They are characterized by nuclear atypia, high mitotic rate and cellular polymorphism that often contributes to aggressiveness and resistance to standard therapy. They often associate with challenging treatment approaches and poor outcomes. New treatment strategies or regimens to improve the efficacy of glioma treatment require a deeper understanding of glioma occurrence and development as well as elucidation of their molecular biological characteristics. Recent studies have revealed RNA modifications as a key regulatory mechanism involved in tumorigenesis, tumor progression, immune regulation, and response to therapy. The present review discusses research advances on several RNA modifications involved in glioma progression and tumor microenvironment (TME) immunoregulation as well as in the development of adaptive drug resistance, summarizing current progress on major RNA modification targeting strategies.
... m 5 C methylated mature miRNAs are linked to poor GBM patient outcomes through a mechanism where this modification inhibits miRNA gene silencing [112]. hm 5 C methylation was found to have a significant role in GBM formation through a mechanism involving 'eraser' TET1, while TET2 and TET3 downregulation is linked to GBM tumourigenesis [113][114][115]. Transcript levels of I 'writers' ADAR, ADARB1, and ADARB2 are reduced in different grades and types of brain tumours [116]; ADAR2 was found to inhibit GBM cell growth, and an abnormally expressed splice variant supressed adenosine-to-inosine RNA editing [117,118]. ...
Simple Summary
Glioblastoma is a complex and aggressive primary brain tumour that is rapidly fatal. Timely and accurate diagnosis is therefore crucial. Here, we explore the newly emerging field of epitranscriptomics to understand the modifications that occur on RNA molecules in the healthy and diseased brain, focusing on glioblastoma. RNA modifications are modulated by various regulators and are diverse, specific, reversible, and involved in many aspects of brain tumour biology. Epitranscriptomic biomarkers may therefore be ideal candidates for clinical diagnostic workflows. This review summarises the current understanding of epitranscriptomics and its clinical relevance in brain cancer diagnostics.
Abstract
RNA modifications are diverse, dynamic, and reversible transcript alterations rapidly gaining attention due to their newly defined RNA regulatory roles in cellular pathways and pathogenic mechanisms. The exciting emerging field of ‘epitranscriptomics’ is predominantly centred on studying the most abundant mRNA modification, N6-methyladenine (m⁶A). The m⁶A mark, similar to many other RNA modifications, is strictly regulated by so-called ‘writer’, ‘reader’, and ‘eraser’ protein species. The abundance of genes coding for the expression of these regulator proteins and m⁶A levels shows great potential as diagnostic and predictive tools across several cancer fields. This review explores our current understanding of RNA modifications in glioma biology and the potential of epitranscriptomics to develop new diagnostic and predictive classification tools that can stratify these highly complex and heterogeneous brain tumours.
... For instance, ALKBH1 and TET2 mutations are associated with myeloid and lymphoblastic leukemias [220,221], while TET1 is highly expressed in glioblastoma [222]. To the contrary, TET2 is downregulated in gliomas [200], whereas the epigenetic inhibition of TET3 may alter glioblastoma tumorigenesis [202]. While the mechanistic reasons are always related to DNA hydroxymethylation or demethylation deficiency, ALKBH1 and TET have also been indicated to oxidize m 5 C in RNA, suggesting that RNA hydroxymethylation deficiency is also associated with cancer. ...
Translation is a fundamental process in all living organisms that involves the decoding of genetic information in mRNA by ribosomes and translation factors. The dysregulation of mRNA translation is a common feature of tumorigenesis. Protein expression reflects the total outcome of multiple regulatory mechanisms that change the metabolism of mRNA pathways from synthesis to degradation. Accumulated evidence has clarified the role of an increasing amount of mRNA modifications at each phase of the pathway, resulting in translational output. Translation machinery is directly affected by mRNA modifications, influencing translation initiation, elongation, and termination or altering mRNA abundance and subcellular localization. In this review, we focus on the translation initiation factors associated with cancer as well as several important RNA modifications, for which we describe their association with cancer.
... In glioma, compared to normal brain tissue, TET2 shows reduced gene and protein expression 24,25 . TET2 expression also decreases with increasing brain tumour grade, and lower TET2 expression has been associated with worse overall survival 24 . ...
Gliomas are incurable brain cancers with poor prognosis, with epigenetic dysregulation being a distinctive feature. 5-hydroxymethylcytosine (5-hmC), an intermediate generated in the demethylation of 5-methylcytosine, is present at reduced levels in glioma tissue compared with normal brain, and that higher levels of 5-hmC are associated with improved patient survival. DNA demethylation is enzymatically driven by the ten–eleven translocation (TET) dioxygenases that require ascorbate as an essential cofactor. There is limited data on ascorbate in gliomas and the relationship between ascorbate and 5-hmC in gliomas has never been reported. Clinical glioma samples (11 low-grade, 26 high-grade) were analysed for ascorbate, global DNA methylation and hydroxymethylation, and methylation status of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter. Low-grade gliomas contained significantly higher levels of ascorbate than high-grade gliomas (p = 0.026). Levels of 5-hmC were significantly higher in low-grade than high-grade glioma (p = 0.0013). There was a strong association between higher ascorbate and higher 5-hmC (p = 0.004). Gliomas with unmethylated and methylated MGMT promoters had similar ascorbate levels (p = 0.96). One mechanism by which epigenetic modifications could occur is through ascorbate-mediated optimisation of TET activity in gliomas. These findings open the door to clinical intervention trials in patients with glioma to provide both mechanistic information and potential avenues for adjuvant ascorbate therapy.
... The "EdgeR" program package in RStudio software used applied, and "FDR <0. 1, | log2FC |> 2" was the standard initially to screen the differentially expressed m5C related lncRNA. The "DEseq2" program package was used to identify differentially expressed m5C-related lncRNA according to "Padj < 0.05 and |log2FC| > 2." Negative Matrix Factorization (NMF) Clustering of m5C Related lncRNAs Gene Sets Thirteen m5C-related genes were collected from literature mining (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Based on Pearson coefficient >0.4 and cox coefficient P<0.001. ...
Purpose
Studies reported that 5-methylcytosine (m5C) RNA transferase alters tumor progression; however, studies of m5C-related lncRNA remain lacking. This article intends to study the lncRNA modified by m5C RNA transferase in hepatocellular carcinoma using a combination of computational biology and basic experiments.
Method
We identified 13 m5C RNA transferase-related genes and selected long non-coding RNAs with a Pearson correlation coefficient greater than 0.4. Univariate Cox regression analysis was used to screen m5C RNA transferase lncRNA related to survival phenotype. We divided TCGA-LIHC into two types of m5C RNA using non-negative matrix decomposition. According to WGCNA, the co-expression models of two lncRNA regulation modes were constructed to analyze the characteristic biological processes of the two m5C RNA transferase-related lncRNA gene models. Then, a predictive model of m5C RNA transferase lncRNA was using LASSO regression. Finally, we used cell experiments, transwell experiments, and clone formation experiments to test the relationship between SNHG4 and tumor cell proliferation in Hep-G2 and Hep-3b cells line.
Results
We identified 436 m5C RNA transferase-related lncRNAs. Using univariate Cox regression analysis, 43 prognostic-related lncRNAs were determined according to P < 0.001. We divided TCGA-LIHC into two regulation modes of m5C RNA transferase using non-negative matrix factorization. The two regulation modes showed significant differences in overall and disease-free survival. We used LASSO to construct m5c-related lncRNA prognostic signature. Thus, a predictive m5C-lncRNA model was established using four lncRNAs: AC026412.3, AC010969.2, SNHG4, and AP003392.5. The score calculated by the m5C-lncRNA model significantly correlated with the overall survival of hepatocellular carcinoma. The receiver operating characteristic curve and decision curve analysis verified the accuracy of the predictive model. We observed a more robust immune response in the high-risk score group. The transwell experiments and clone formation experiments suggested that m5C RNA transferase-related lncRNA SNHG4 promotes the proliferation and migration of Hep-G2 and Hep-3b cells line.
Conclusion
Two lncRNA expression patterns regulated by m5C RNA transferase were identified. The difference between the two expression patterns and the survival phenotype in the biological process was pointed out. A 5-methylcytosine RNA methyltransferases-related lncRNA overall survival signature was constructed. These results provide some understanding of the influence of m5C transferase on hepatocellular carcinoma. The prediction model of m5C transferase lncRNA has potential clinical value in managing hepatocellular carcinoma.
... Thirteen m5C-related genes encoding for lncRNAs were retrieved from literature mining, including NOP2, NSUN2, NSUN3, NSUN4, NSUN5, NSUN7, TRDMT1, TET1, TET2, TET3, ALKBH1, YBX1, and ALYREF (Archer et al., 2016;Blanco et al., 2016;Müller et al., 2016;Cheng et al., 2018;García et al., 2018;Li et al., 2018;Chen et al., 2019;Gao et al., 2019;Janin et al., 2019;Carella et al., 2020;Mei et al., 2020;Sato et al., 2020). After excluding those with median absolute difference <0.5, the correlation of the remaining candidate genes with overall survival was analyzed by the Cox regression model using the "survival" package. ...
Purpose: Epigenetic RNA modification regulates gene expression post-transcriptionally. The aim of this study was to construct a prognostic risk model for lung adenocarcinoma (LUAD) using long non-coding RNAs (lncRNAs) related to m5C RNA methylation.
Method: The lncRNAs regulated by m5C methyltransferase were identified in TCGA-LUAD dataset using Pearson correlation analysis (coefficient > 0.4), and clustered using non-negative matrix decomposition. The co-expressing gene modules were identified by WGCNA and functionally annotated. The prognostically relevant lncRNAs were screened by LASSO regression and a risk model was constructed. LINC00628 was silenced in the NCI-H460 and NCI-H1299 cell lines using siRNA constructs, and migration and invasion were assessed by the Transwell and wound healing assays respectively.
Results: We identified 185 m5C methyltransferase-related lncRNAs in LUAD, of which 16 were significantly associated with overall survival. The lncRNAs were grouped into two clusters on the basis of m5C pattern, and were associated with significant differences in overall and disease-free survival. GSVA revealed a close relationship among m5C score, ribosomes, endolysosomes and lymphocyte migration. Using LASSO regression, we constructed a prognostic signature consisting of LINC00628, LINC02147, and MIR34AHG. The m5C-lncRNA signature score was closely related to overall survival, and the accuracy of the predictive model was verified by the receiver operating characteristic curve and decision curve analysis. Knocking down LINC00628 in NCI-H460 and NCI-H1299 cells significantly reduced their migration and invasion compared to that of control cells.
Conclusion: We constructed a prognostic risk model of LUAD using three lncRNAs regulated by m5C methyltransferase, which has potential clinical value.
... Genomic analysis of a small group of GBM patient samples suggests that the TET2 loci is prone to hypermethylation leading to repression, providing an alternative explanation for the robust loss of 5hmC observed in GBM. 34 We now show, for the first time, that forced expression of reprogramming transcription factor SOX2, which is highly expressed in GBM, reduces expression of TET2 and 5hmC (Fig. 1f, g), thus contributing to the hyper-methylated phenotype of GSCs (Fig. 1h). ...
... Ectopic expression of TET2 impairs tumor growth capacity of GBM cells and this phenotype is associated with activation of neural differentiation programs. 34,49 These findings suggest that TET2 and 5hmC regulate the tumor phenotype of GBM by controlling the stem cell phenotype of GBM cells. Consistent with these clinical and bioinformatics predictions, our results show that TET2 expression is reduced in primary GSC isolates compared to GPCs (Fig. 1b, c) and low TET2 expression correlates with poor patient outcomes in GBM (Fig. 1a). ...
... 55 In GBM, expression of transgenic TET2 activates neural differentiation programs and inhibits tumor growth capacity of non-stem-like GBM cells. 34 Likewise, TET3 expression has been reported to inhibit GSC self-renewal and tumorigenesis. 56 Despite these exciting results, developing therapeutic approaches based on TET re-expression may prove challenging due to the size of these enzymes. ...
DNA methylation is a reversible process catalyzed by the ten–eleven translocation (TET) family of enzymes (TET1, TET2, TET3) that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Altered patterns of 5hmC and 5mC are widely reported in human cancers and loss of 5hmC correlates with poor prognosis. Understanding the mechanisms leading to 5hmC loss and its role in oncogenesis will advance the development of epigenetic-based therapeutics. We show that TET2 loss associates with glioblastoma (GBM) stem cells and correlates with poor survival of GBM patients. We further identify a SOX2:miR-10b-5p:TET2 axis that represses TET2 expression, represses 5hmC, increases 5mC levels, and induces GBM cell stemness and tumor-propagating potential. In vivo delivery of a miR-10b-5p inhibitor that normalizes TET2 expression and 5hmC levels inhibits tumor growth and prolongs survival of animals bearing pre-established orthotopic GBM xenografts. These findings highlight the importance of TET2 and 5hmC loss in Sox2-driven oncogenesis and their potential for therapeutic targeting.
... Thirteen m5C-related genes were collected from literature mining (Takai et al., 2014;Archer et al., 2016;Blanco et al., 2016;Yamashita et al., 2017;Cheng et al., 2018;García et al., 2018;Chen et al., 2019;Gao et al., 2019;Janin et al., 2019;Carella et al., 2020;He et al., 2020;Mei et al., 2020;Sato et al., 2020). Then, expression data of the 13 m5C-related genes and all lncRNAs were retrieved from the TCGA dataset. ...
The effects of 5-methylcytosine in RNA (m5C) in various human cancers have been increasingly studied recently; however, the m5C regulator signature in prostate cancer (PCa) has not been well established yet. In this study, we identified and characterized a series of m5C-related long non-coding RNAs (lncRNAs) in PCa. Univariate Cox regression analysis and least absolute shrinkage and selector operation (LASSO) regression analysis were implemented to construct a m5C-related lncRNA prognostic signature. Consequently, a prognostic m5C-lnc model was established, including 17 lncRNAs: MAFG-AS1, AC012510.1, AC012065.3, AL117332.1, AC132192.2, AP001160.2, AC129510.1, AC084018.2, UBXN10-AS1, AC138956.2, ZNF32-AS2, AC017100.1, AC004943.2, SP2-AS1, Z93930.2, AP001486.2, and LINC01135. The high m5C-lnc score calculated by the model significantly relates to poor biochemical recurrence (BCR)-free survival (p < 0.0001). Receiver operating characteristic (ROC) curves and a decision curve analysis (DCA) further validated the accuracy of the prognostic model. Subsequently, a predictive nomogram combining the prognostic model with clinical features was created, and it exhibited promising predictive efficacy for BCR risk stratification. Next, the competing endogenous RNA (ceRNA) network and lncRNA–protein interaction network were established to explore the potential functions of these 17 lncRNAs mechanically. In addition, functional enrichment analysis revealed that these lncRNAs are involved in many cellular metabolic pathways. Lastly, M AFG-AS1 was selected for experimental validation; it was upregulated in PCa and probably promoted PCa proliferation and invasion in vitro. These results offer some insights into the m5C's effects on PCa and reveal a predictive model with the potential clinical value to improve the prognosis of patients with PCa.
