bims-rimeca Biomed News
on RNA methylation in cancer
Issue of 2021–09–19
twenty-one papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics
  1. Fusaric acid induces hepatic global m6A RNA methylation and differential expression of m6A regulatory genes in vivo - a pilot study.
  2. Emerging Roles of N6-Methyladenosine Demethylases and Its Interaction with Environmental Toxicants in Digestive System Cancers.
  3. METTL3-mediated m6A mRNA modification promotes esophageal cancer initiation and progression via Notch signaling pathway.
  4. N6-methyladenosine (m6A) regulatory gene divides hepatocellular carcinoma into three subtypes.
  5. Bioinformatical identification of key genes regulated by IGF2BP2-mediated RNA N6-methyladenosine and prediction of prognosis in hepatocellular carcinoma.
  6. METTL3 induces PLX4032 resistance in melanoma by promoting m6A-dependent EGFR translation.
  7. Mettl3 inhibits the apoptosis and autophagy of chondrocytes in inflammation through mediating Bcl2 stability via Ythdf1-mediated m6A modification.
  8. Gene and prognostic value of N6-methyladenosine (m6A) modification regulatory factors in lung adenocarcinoma.
  9. FTO-mediated demethylation of GADD45B promotes myogenesis through the activation of p38 MAPK pathway.
  10. The critical roles of m6A modification in metabolic abnormality and cardiovascular diseases.
  11. m6A Pathway Regulators Are Frequently Mutated in Breast Invasive Carcinoma and May Play an Important Role in Disease Pathogenesis.
  12. Expression Pattern and Clinical Value of Key m6A RNA Modification Regulators in Abdominal Aortic Aneurysm.
  13. VIRMA contributes to non-small cell lung cancer progression via N6-methyladenosine-dependent DAPK3 post-transcriptional modification.
  14. Cross-talk and clinical value of m[superscript 6]A regulatory gene in bladder cancer.
  15. DNAzyme based three-way junction assay for antibody-free detection of locus-specific N6-methyladenosine modifications.
  16. METTL3-dependent MALAT1 delocalization drives c-Myc induction in thymic epithelial tumors.
  17. Mass Spectrometry-Based Targeted Serum Monomethylated Ribonucleosides Profiling for Early Detection of Breast Cancer.
  18. METTL3-mediated m6A RNA methylation promotes the anti-tumour immunity of natural killer cells.
  19. YTHDF2 is a Potential Biomarker and Associated with Immune Infiltration in Kidney Renal Clear Cell Carcinoma.
  20. ac4C acetylation of RUNX2 catalyzed by NAT10 spurs osteogenesis of BMSCs and prevents ovariectomy-induced bone loss.
  21. Chemoenzymatic labeling of RNA to enrich, detect and identify methyltransferase-target sites.
  1. Epigenetics. 2021 Sep 13. 1-9
    Fusaric acid induces hepatic global m6A RNA methylation and differential expression of m6A regulatory genes in vivo - a pilot study.
    Terisha Ghazi, Savania Nagiah, Anil A Chuturgoon.
      N6-methyladenosine (m6A) is an abundant epitranscriptomic mark that regulates gene expression to execute cellular developmental programmes and environmental adaptation. Fusaric acid (FA) is a mycotoxin that contaminates agricultural foods and exerts toxicity in humans and animals; however, its epitranscriptomic effects are unclear. We investigated the effect of FA on global m6A RNA methylation and mRNA expression levels of key m6A regulatory genes in C57BL/6 mouse livers. C57BL/6 mice (n = 6/group) were orally administered 0.1 M phosphate-buffered saline (PBS) or 50 mg/kg FA. Mice were euthanized 24 h after oral administration, livers were harvested, and RNA was isolated. RNA samples were assayed for global m6A levels using an m6A RNA Methylation Quantification Kit. The mRNA expression of m6A regulators i.e. writers, erasers, and readers were measured by qRT-PCR. FA increased global m6A RNA methylation (p < 0.0001) in mouse livers. FA increased the expression of METTL3 (p = 0.0143) and METTL14 (p = 0.0281), and decreased the expression of FTO (p = 0.0036) and ALKBH5 (p = 0.0035). The expression of YTHDF2 (p = 0.0007), YTHDF3 (p = 0.0061), and YTHDC2 (p = 0.0258) were increased by FA in mouse livers. This study shows that the liver m6A epitranscriptome can be modified by FA exposure in an in vivo model and can be useful for identifying the molecular mechanisms whereby m6A RNA modifications influence the toxicological outcomes of FA exposure.
    Keywords:  epitranscriptomics; fusaric acid; m6A RNA methylation; m6A erasers; m6A readers; m6A writers; mycotoxin
    DOI:  https://doi.org/10.1080/15592294.2021.1975937
  2. Cancer Manag Res. 2021 ;13 7101-7114
    Emerging Roles of N6-Methyladenosine Demethylases and Its Interaction with Environmental Toxicants in Digestive System Cancers.
    Caiping Liu, Sheng Yang, Yanqiu Zhang, Chuntao Wang, Dandan Du, Xian Wang, Tong Liu, Geyu Liang.
      Digestive system cancers are common cancers with high cancer deaths worldwide. They have become a major threat to public health and economic burden. As one of the most universal RNA modifications in eukaryotes, the N6-methyladenosine (m6A) modification is involved in the occurrence, development, prognosis, and treatment response of various cancers, including digestive system cancers. M6A demethylases shape the m6A landscape dynamically, playing important roles in cancers. In addition, accumulating evidence reveal that many environmental toxicants are the established risk factors for digestive system cancers and associated with m6A modification. In this review, we summarize the multiple functions of M6A demethylases (fat mass and obesity-associated protein (FTO), AlkB homolog 5 (ALKBH5) and AlkB homolog 3 (ALKBH3)) in digestive system cancers, which are aberrantly expressed and affect cancer progression. We also discuss the potential roles of m6A demethylases in the assessment of environmental exposure, the signature for prevention and diagnosis of digestive system cancers.
    Keywords:  ALKBH5; FTO; digestive system cancers; environment toxicants; m6A modification
    DOI:  https://doi.org/10.2147/CMAR.S328188
  3. Mol Ther Nucleic Acids. 2021 Dec 03. 26 333-346
    METTL3-mediated m6A mRNA modification promotes esophageal cancer initiation and progression via Notch signaling pathway.
    Hui Han, Chunlong Yang, Shuishen Zhang, Maosheng Cheng, Siyao Guo, Yan Zhu, Jieyi Ma, Yu Liang, Lu Wang, Siyi Zheng, Zhaoyu Wang, Demeng Chen, Yi-Zhou Jiang, Shuibin Lin.
      Esophageal cancer is a lethal malignancy with a high mortality rate, while the molecular mechanisms underlying esophageal cancer pathogenesis are still poorly understood. Here, we found that the N6-methyladenosine (m6A) methyltransferase-like 3 (METTL3) is significantly upregulated in esophageal squamous cell carcinoma (ESCC) and associated with poor patient prognosis. Depletion of METTL3 results in decreased ESCC growth and progression in vitro and in vivo. We further established ESCC initiation and progression models using Mettl3 conditional knockout mouse and revealed that 3METTL3-mediated m6A modification promotes ESCC initiation and progression in vivo. Moreover, using METTL3 overexpression ESCC cell model and Mettl3 conditional knockin mouse model, we demonstrated the critical function of METTL3 in promoting ESCC tumorigenesis in vitro and in vivo. Mechanistically, METTL3-catalyzed m6A modification promotes NOTCH1 expression and the activation of the Notch signaling pathway. Forced activation of Notch signaling pathway successfully rescues the growth, migration, and invasion capacities of METTL3-depleted ESCC cells. Our data uncovered important mechanistical insights underlying ESCC tumorigenesis and provided molecular basis for the development of novel strategies for ESCC diagnosis and treatment.
    Keywords:  ESCC; METTL3; N6-methyladenosine; Notch signaling pathway; esophageal cancer; esophageal squamous cell carcinoma; m6A
    DOI:  https://doi.org/10.1016/j.omtn.2021.07.007
  4. J Gastrointest Oncol. 2021 Aug;12(4): 1860-1872
    N6-methyladenosine (m6A) regulatory gene divides hepatocellular carcinoma into three subtypes.
    Jie Wei, Da Lang Fang, Weijie Zhou, Yong Fei He.
       Background: The N6-methyladenosine (m6A) plays an important role in epigenetic modification and tumor progression, but the modulations of m6A in hepatocellular carcinoma (HCC) have not been determined while the relationship between m6A regulation and immune cell infiltration remains unclear.
    Methods: This study investigated the modification patterns of m6A by analyzing HCC samples from The Cancer Genome Atlas (TCGA) database and the Gene Expression Omnibus (GEO) dataset, and performed molecular typing based on the characteristics of immune cell infiltration. The m6Ascore was also constructed to quantify m6A modifications and predict the immunotherapy response and prognosis of HCC patients.
    Results: Of the 364 samples, 31 (8.52%) were genetically altered in the m6A regulatory gene, with the highest frequency of mutations in HNRNPC, ZC3H13, and LRPPRC. Three distinct molecular subtypes of m6A were identified in 590 HCC samples, which were associated with different immune cell infiltrates: immunodepletion type, immune activation type, and immune immunity type. According to the construction of the m6Ascore system in the m6A genotype, HCC patients could be divided into high and low groups. The m6A modified pattern, characterized by immune immunity and immune failure, showed a lower score and a better prognosis. However, the immune-activated type of m6A had a higher score and a poorer prognosis. Further analysis showed that the m6Ascore was correlated with tumor mutation burden (TMB), and the higher the TMB, the worse the prognosis. m6Ascore was also correlated with the expression of cytotoxic T-lymphocyte-associated protein 4 (CTAL-4), and the higher the score, the higher the expression of HCC in patients.
    Conclusions: HCC has a unique m6A modification pattern, and 3 different m6A subtypes help to classify HCC, provide knowledge of drug regimens for immunotherapy, and can be used to predict treatment response and prognosis.
    Keywords:  N6-methyladenosine (m6A); hepatocellular carcinoma (HCC); immune profiles; subtypes
    DOI:  https://doi.org/10.21037/jgo-21-378
  5. J Gastrointest Oncol. 2021 Aug;12(4): 1773-1785
    Bioinformatical identification of key genes regulated by IGF2BP2-mediated RNA N6-methyladenosine and prediction of prognosis in hepatocellular carcinoma.
    Qiang Wei.
       Background: The treatment of hepatocellular carcinoma (HCC), a malignant cancer with global spread, remains unsatisfactory, and novel prognostic biomarkers need to be identified. N6-methyladenosine (m6A) has been found to regulate tumor initiation and progression through different mechanisms. As a dynamic and reversible messenger RNA (mRNA) modification, m6A can be read by insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). IGF2BP2 targets thousands of mRNA transcripts, which may be involved in HCC progression.
    Methods: In this study, we integrated 4 classes of datasets including The Cancer Genome Atlas (TCGA)-LICH, m6A-sequencing data of HepG2 cells, and RNA-sequencing data of IGF2BP2-knockdown HepG2 cells to explore the key genes regulated by IGF2BP2-mediated m6A in HCC. The expression and m6A modification of candidates were validation in independent microarray expression profile of HCC tissue and annotated m6A database RMBase. The relationship of immune cell infiltration and the genes expression was estimated by CIBERSORT and TIMER.
    Results: A total of 89 candidate genes were filtered. Next, cluster analysis was performed base on functions and pathways to identify the enrichment pathways. By constructing a protein-protein interaction (PPI) network, we found 54 nodes. Ten significant genes were filtered from the PPI. These genes were validated in data of an independent microarray and an m6A database. We found that the upregulation of these 10 genes was associated with poor prognosis. In addition, we showed the expression of these 10 genes was associated with the infiltration of variety of immune cell and tumor purity.
    Conclusions: These identified genes may provide novel insights and facilitate the development of potential biomarkers for HCC diagnosis, as well as provide clues for IGF2BP2 inhibition therapy in HCC.
    Keywords:  IGF2BP2; N6-methyladenosine; hepatocellular carcinoma (HCC)
    DOI:  https://doi.org/10.21037/jgo-21-306
  6. Cancer Lett. 2021 Sep 13. pii: S0304-3835(21)00462-6. [Epub ahead of print]
    METTL3 induces PLX4032 resistance in melanoma by promoting m6A-dependent EGFR translation.
    Poshan Yugal Bhattarai, Garam Kim, Muna Poudel, Sung-Chul Lim, Hong Seok Choi.
      Acquired resistance often limits therapeutic efficacy of the BFAF (V600E) kinase inhibitor PLX4032 in patients with advanced melanoma. Epitranscriptomic modification of mRNAs by N (Vasan et al., 2019) [6]-methyladenosine (m6A) modification contributes to melanoma pathogenesis; however, its role in acquired PLX4032 resistance remains unexplored. Here, we showed that m6A methyltransferase METTL3 expression is upregulated in A375R cells, a PLX4032-resistant subline of A375 melanoma cells, compared with the parental cells. Moreover, METTL3 increased the m6A modification of epidermal growth factor receptor (EGFR) mRNA in A375R cells, which promoted its translation efficiency. In turn, increased EGFR expression facilitated rebound activation of the RAF/MEK/ERK pathway in A375R cells, inducing PLX4032 resistance. In contrast, knockout of METTL3 in A375R cells reduced EGFR expression and restored PLX4032 sensitivity. PLX4032 treatment following METTL3 knockout induced apoptosis and reduced colony formation in A375R cells and reduced A375R cell-derived tumor growth in BALB/c nude mice. These findings indicate that METTL3 promotes rebound activation of the RAF/MEK/ERK pathway through EGFR upregulation and highlight a critical role for METTL3-induced m6A modification in acquired PLX4032 resistance in melanoma, implicating METTL3 as a potential candidate for targeted chemotherapy.
    Keywords:  Acquired resistance; Epitranscriptomic modification; N(6)-methyladenosine; RAF/MEK/ERK pathway
    DOI:  https://doi.org/10.1016/j.canlet.2021.09.015
  7. Bone. 2021 Sep 13. pii: S8756-3282(21)00348-3. [Epub ahead of print] 116182
    Mettl3 inhibits the apoptosis and autophagy of chondrocytes in inflammation through mediating Bcl2 stability via Ythdf1-mediated m6A modification.
    Ying He, Wei Wang, Xiaoxiao Xu, Beining Yang, Xijie Yu, Yanru Wu, Jiawei Wang.
      N6-methyladenosine (m6A) methylation is one of the most common internal modifications in eukaryotic messenger RNA occurring on N6 nitrogen of adenosine. However, the roles of m6A in temporomandibular joint osteoarthritis (TMJ OA) are still elusive. Here, we investigate the function and mechanism of methyltransferase-like 3 (Mettl3) in chondrocytes in inflammation. We found that the expression of Mettl3 decreased both in vivo TMJ OA mice and in vitro inflammatory stimulation. Functionally, loss and gain studies illustrated that Mettl3 inhibited the apoptosis and autophagy of chondrocytes induced by TNF-α stimulation in vitro. Mettl3 inhibitor, S-adenosylhomocysteine (SAH) promoted the apoptosis and autophagy of chondrocytes with inflammation in vitro and aggravated the degeneration of chondrocytes and subchondral bone in monosodium iodoacetate (MIA) induced TMJ OA mice in vivo. Mechanistically, the bioinformatics analysis, m6A-RNA immunoprecipitation (MeRIP) and RNA immunoprecipitation (RIP) were used to identify that Bcl2 mRNA was the downstream target of Mettl3 for m6A modification. Furthermore, the results revealed that Yth m6A RNA binding protein 1 (Ythdf1) mediated the stability of Bcl2 mRNA catalyzed by Mettl3. Co-immunoprecipitation (Co-IP) showed that Bcl2 protein interacted with Beclin1 protein in chondrocytes induced by TNF-α stimulation. In conclusion, our findings identify that Mettl3 inhibits the apoptosis and autophagy of chondrocytes in inflammation through m6A/Ythdf1/Bcl2 signal axis which provides promising therapeutic strategy for TMJ OA.
    Keywords:  B-cell lymphoma 2; N(6)-methyladenosine; apoptosis; autophagy; chondrocyte; inflammation; methyltransferase-like 3
    DOI:  https://doi.org/10.1016/j.bone.2021.116182
  8. Eur J Cancer Prev. 2021 Sep 13.
    Gene and prognostic value of N6-methyladenosine (m6A) modification regulatory factors in lung adenocarcinoma.
    Di Zhang, DanDan Zhang, Chen Wang, XiaoLi Yang, RongQiang Zhang, Qiang Li, YongMin Xiong.
      Lung cancer is the leading cause of death worldwide, and its incidence continues to increase. The treatment of lung cancer is related to the subtypes and stages of cancer, but the therapeutic effect is still unsatisfactory. We found that 10 of the 13 genes were differentially expressed in lung cancer, YTHDF1, RBM15, HNRNPC, KIAA1429, METTL3 and YTHDF2 are high expression while METTL14, ZC3H13, FTO and WTAP are low expression. HNRNPC and METTL3 genes were associated with the risk and prognosis of LUAD and could regard as biomarkers for early diagnosis and treatment, which provides a theoretical basis for LUAD.
    DOI:  https://doi.org/10.1097/CEJ.0000000000000717
  9. Mol Ther Nucleic Acids. 2021 Dec 03. 26 34-48
    FTO-mediated demethylation of GADD45B promotes myogenesis through the activation of p38 MAPK pathway.
    Kaiping Deng, Yixuan Fan, Yaxu Liang, Yu Cai, Guomin Zhang, Mingtian Deng, Zhibo Wang, Jiawei Lu, Jianfei Shi, Feng Wang, Yanli Zhang.
      N6-methyladenosine (m6A) modification plays a critical role in mammalian development. However, the role of m6A in the skeletal muscle development remains largely unknown. Here, we report a global m6A modification pattern of goat skeletal muscle at two key development stages and identified that the m6A modification regulated the expression of the growth arrest and DNA damage-inducible 45B (GADD45B) gene, which is involved in myogenic differentiation. We showed that GADD45B expression increased during myoblast differentiation, whereas the downregulation of GADD45B inhibits myogenic differentiation and mitochondrial biogenesis. Moreover, the expression of GADD45B regulates the expression of myogenic regulatory factors and peroxisome proliferator-activated receptor gamma coactivator 1 alpha by activating the p38 mitogen-activated protein kinase (MAPK) pathway. Conversely, the inactivation of p38 MAPK abolished the GADD45B-mediated myogenic differentiation. Furthermore, we found that the knockdown of fat mass and obesity-associated protein (FTO) increases GADD45B m6A modification and decreases the stability of GADD45B mRNA, which impairs myogenic differentiation. Our results indicate that the FTO-mediated m6A modification in GADD45B mRNA drives skeletal muscle differentiation by activating the p38 MAPK pathway, which provides a molecular mechanism for the regulation of myogenesis via RNA methylation.
    Keywords:  FTO; GADD45B; goat; m6A modification; mitochondrial biogenesis; myogenic differentiation
    DOI:  https://doi.org/10.1016/j.omtn.2021.06.013
  10. Genes Dis. 2021 Nov;8(6): 746-758
    The critical roles of m6A modification in metabolic abnormality and cardiovascular diseases.
    Beijian Zhang, Hao Jiang, Zhen Dong, Aijun Sun, Junbo Ge.
      N6-methyladenosine (m6A) RNA methylation is an emerging area of epigenetics, which is a reversible and dynamic modification mediating by 'writers' (methylase, adding methyl groups, METTL3, METTL14, and WTAP), 'erasers' (demethylase, deleting methyl groups, FTO and ALKBH5), and 'readers' (YTHDF1-3, YTHDC1 and YTHDC2). Recent studies in human, animal models and cell levels have disclosed a critical role of m6A modification in regulating the homeostasis of metabolic processes and cardiovascular function. Evidence from these studies identify m6A as a candidate of biomarker and therapeutic target for metabolic abnormality and cardiovascular diseases (CVD). Comprehensive understanding of the complexity of m6A regulation in metabolic diseases and CVD will be helpful for us to understand the pathogenesis of CVD. In this review, we discuss the regulatory role of m6A in metabolic abnormality and CVD. We will emphasize the clinical relevance of m6A dysregulation in CVD.
    Keywords:  Cardiovascular disease; FTO; Heart failure; METTL3; Metabolic syndrome; Myocardial infarction; N6-methyladenosine; RNA epigenetics
    DOI:  https://doi.org/10.1016/j.gendis.2020.07.011
  11. OMICS. 2021 Sep 13.
    m6A Pathway Regulators Are Frequently Mutated in Breast Invasive Carcinoma and May Play an Important Role in Disease Pathogenesis.
    Turan Demircan, Mervenur Yavuz, Sıddıka Akgül.
      Breast invasive carcinoma (BIC) is one of the most commonly observed and the deadliest cancer among women. Studies examining the role of epigenetics and regulation of gene expression stand to make important strides in clinical management of BIC. In this context, messenger-RNA (mRNA) modification by regulatory proteins is noteworthy. Methylation of the adenosine base on the sixth nitrogen position is termed as N6-methyladenosine (m6A) modification, and this is the most abundant mRNA modification in mammals. Using several publicly available datasets, we report, in this study, comprehensive analyses and new findings on the impact of epitranscriptome regulatory factors and genetic alterations in m6A pathway genes on BIC. Accordingly, mutation frequency, type, and expression levels were determined. Importantly, we found that VIRMA, METTL14, RBM15B, EIF3B, YTHDF1, and YTHDF3 genes hold potential significance as prognostic biomarker candidates as evidenced in particular by the overall survival analysis. Enrichment of gene ontology (GO) terms and KEGG pathways for the tumor samples with genetic alterations in the epitranscriptome regulatory pathways were investigated. Dysregulation of regulatory factors in breast cancer was associated with cell division, and survival-related pathways such as "nuclear division," and "chromosome segregation." Hence, the gained overactivity of these pathways may account for BIC's poor prognosis. In conclusion, these data underscore that m6A pathway regulators are frequently mutated in BIC and likely play a significant role in disease pathogenesis. Epitranscriptome pathway genes warrant further research attention as regulators of cancer growth and biological targets in BIC, and with an eye to personalized medicine in clinical oncology.
    Keywords:  TCGA; breast invasive carcinoma; clinical oncology; epitranscriptome regulatory pathways; m6A RNA modification; personalized medicine
    DOI:  https://doi.org/10.1089/omi.2021.0114
  12. J Inflamm Res. 2021 ;14 4245-4258
    Expression Pattern and Clinical Value of Key m6A RNA Modification Regulators in Abdominal Aortic Aneurysm.
    Tan Li, Tianlong Wang, Jingjing Jing, Liping Sun.
       Background: Aberrant expression of N6-methyladenosine (m6A) RNA modification regulators plays a critical role in a variety of human diseases. However, their implication in abdominal aortic aneurysm (AAA) remains largely unknown. Herein, we sought to explore the general expression pattern and potential functions of m6A regulators in AAA.
    Methods: We analyzed gene expression data of m6A regulators in human AAA and normal tissues from public GEO database. The R package and other tools such as m6A2Target database, Gene ontology (GO) functional and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses, gene set variation analysis (GSVA), Search Tool for the Retrieval of Interacting Genes (STRING), starBase, miRDB and Cytoscape software were applied for bioinformatics analysis to investigate the downstream molecular mechanisms and upstream regulatory mechanisms for distinctly expressed regulators. Quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were performed to validate the expression of key m6A regulators in our collected human AAA specimens.
    Results: We found that METTL14 and HNRNPC were the downregulated m6A regulators, and RBM15B was the upregulated methylation transferase in human AAA. The modified genes were primarily enriched in RNA catabolic process, regulation of translation, focal adhesion, transcription coregulator activity, ribosome, RNA transport, cell cycle, et al. METTL14, HNRNPC and RBM15B levels were correlated with the immune infiltration degree of Tcm, macrophages, mast cells, Tgd and NK CD56bright cells. A total of 154 and 76 target genes of three regulators were separately involved in body metabolism and autophagy in AAA disease, and their interactive relationships and hub genes were identified. The lncRNA-miRNA-mRNA interaction regulatory networks were also constructed for METTL14, HNRNPC and RBM15B. Based on our clinical tissue and serum samples, METTL14 exhibited lower expression levels in AAA and its rupture type, and low METTL14 expression was associated with high levels of WBC and CRP (all P < 0.05).
    Conclusion: Our study presents an overview of the expression pattern and functional significance of m6A regulators in human AAA. Our findings will provide a valuable resource that may guide both mechanistic and therapeutic analyses about the role of key m6A regulators in AAA.
    Keywords:  abdominal aortic aneurysm; gene expression; m6A modification
    DOI:  https://doi.org/10.2147/JIR.S327152
  13. Cancer Lett. 2021 Sep 11. pii: S0304-3835(21)00422-5. [Epub ahead of print]
    VIRMA contributes to non-small cell lung cancer progression via N6-methyladenosine-dependent DAPK3 post-transcriptional modification.
    Yongfang Xu, Yunhao Chen, Yinan Yao, Haiyang Xie, Guohua Lu, Chengli Du, Jun Cheng, Jianying Zhou.
      N6-methyladenosine (m6A) has been reported to be abnormally expressed in non-small cell lung cancer (NSCLC), and plays a vital role in regulation of cell proliferation, invasion and metastasis. Vir-Like m6A methyltransferase associated (VIRMA, also called KIAA1429) has not been well studied in NSCLC. Thus, in this study, we investigated the biological impact and underlying mechanism of VIRMA in NSCLC. High expression of VIRMA was testified in patients with NSCLC and predicted worse prognosis in patients. VIRMA facilitated cell proliferation and tumor growth both in vitro and in vivo. Furthermore, VIRMA-regulated m6A modifications led to post-transcriptional suppression of death-associated protein kinase 3 (DAPK3, also called ZIP or ZIPK) through the YT521-B homology domain-containing family proteins 2/3(YTHDF2/3). Inhibition of DAPK3 rescued the tumor-suppressive phenotypes induced by VIRMA deficiency. In conclusion, VIRMA-guided m6A modifications promoted NSCLC progression via m6A-dependent degradation of DAPK3 mRNA. Therefore, VIRMA may be a novel therapeutic target in NSCLC.
    Keywords:  KIAA1429; N(6)-methyladenosine (m6A); YTHDF2; YTHDF3; ZIP (ZIPK)
    DOI:  https://doi.org/10.1016/j.canlet.2021.08.027
  14. BMC Urol. 2021 Sep 14. 21(1): 127
    Cross-talk and clinical value of m[superscript 6]A regulatory gene in bladder cancer.
    Ben-Zheng Zhou, Qin Luo, Ye Zhang.
       BACKGROUND: RNA modification is a regulation at the post-transcriptional level. RNA methylation modification accounts for more than 60% of all RNA modifications, and m[superscript 6]A(6-methyladenine) is the most common type of RNA methylation modification on mRNA of higher organisms. The modification level of transcription m[superscript 6]A is dynamically regulated by methyltransferase (reader), binding protein (writer) and demethylase (eraser). Furthermore, m[superscript 6]A methylation has been found to have an impact on tumor initiation and progression through various mechanisms.
    METHODS: 13 genes related m[superscript 6]A from all the gene expressions in The Cancer Genome Atlas (TCGA) were screened. Gene Ontology (GO) and KEGG analysis were applied to explore the functions of genes identified in study. We clustered the related regulators of m[superscript 6]A into three subgroups with "ConsensusClusterPlus". 13 genes were used for univariate Cox analysis to find genes associated with prognosis, and the risk model was constructed based on lasso regression. According to the median risk score of each patient, the patients were divided into high and low risk groups for survival analysis. The ROC curve evaluates the model. Then the risk group and clinical characteristics were analyzed.
    RESULTS: The three subgroups had different clinical characteristics. Our tumor clusters were related to grade, survival status. Moreover, we observed a significantly longer overall survival (OS) in the cluster 1 than the cluster 2 and cluster 3. Three m[superscript 6]A-related genes related to prognosis were used to construct a prognostic risk model. We found age are independent prognostic marker. What's more, risk score can also be an independent prognostic factor.
    CONCLUSION: Revealing the regulation and functional mechanism of cross-talk among m[superscript 6]A writers, erasers, and readers, and determine its role in bladder cancer may help in developing novel and efficient strategies for the diagnosis, prognosis and treatment of bladder cancer.
    Keywords:  Bladder cancer; Clinical feature; N6-methyladenosine (m[superscript 6]A); Prognostic; RNA modification
    DOI:  https://doi.org/10.1186/s12894-021-00880-x
  15. Biosens Bioelectron. 2021 Sep 11. pii: S0956-5663(21)00662-X. [Epub ahead of print]194 113625
    DNAzyme based three-way junction assay for antibody-free detection of locus-specific N6-methyladenosine modifications.
    Hongyan Yu, Qinli Pu, Zhi Weng, Xi Zhou, Junjie Li, Yujun Yang, Wang Luo, Yongcan Guo, Huajian Chen, Ding Wang, Guoming Xie.
      N6-methyladenosine (m6A) is the most abundant post-transcriptional modification in RNA and has important implications in physiological processes and tumor development. However, sensitive and specific quantification of locus-specific m6A modification levels remains a challenging task. In the present work, a novel m6A-sensitive DNAzyme was utilized to directly detect m6A by coupling with a three-way junction-mediated isothermal exponential CRISPR amplification reaction for the first time. This method was built on the fact that the binding arm of the DNAzyme bound to the specific site and its core structure catalyzed the selective cleavage of unmodified adenine instead of methylated adenines. Subsequently, the intact RNA was identified by the proximity effect of the three-way junction. Enormous amounts of single-stranded DNA products were generated through a combination of SDA and EXPAR for signal amplification. The specific real-time curve of products was recorded through detecting the fluorescence intensity triggered by CRISPR Cas12a. As a result, methylation target of abundance down to 1% was successfully identified. In addition, this strategy could be used for the analysis of cell RNA extracts. Combined with an electrochemical sensor for quantitative detection of RNA methylation, we demonstrated the generality of as-proposed strategy. We envision the present method would provide a new platform for the analysis of m6A in RNA and promote its application in clinical diseases.
    Keywords:  CRISPR-Cas12a; DNAzyme; Electrochemical sensor; Isothermal nucleic acid amplification; N(6)-methyladenosine
    DOI:  https://doi.org/10.1016/j.bios.2021.113625
  16. Clin Epigenetics. 2021 Sep 16. 13(1): 173
    METTL3-dependent MALAT1 delocalization drives c-Myc induction in thymic epithelial tumors.
    Alessia Iaiza, Claudia Tito, Zaira Ianniello, Federica Ganci, Valentina Laquintana, Enzo Gallo, Andrea Sacconi, Silvia Masciarelli, Luciana De Angelis, Sara Aversa, Daniele Diso, Marco Anile, Vincenzo Petrozza, Francesco Facciolo, Enrico Melis, Edoardo Pescarmona, Federico Venuta, Mirella Marino, Giovanni Blandino, Giulia Fontemaggi, Alessandro Fatica, Francesco Fazi.
       BACKGROUND: Thymic epithelial tumors (TETs) are rare neoplasms, originating from epithelial thymic cells. The oncogenic potential of these rare neoplasms is still largely undefined, and a deeper molecular characterization could result in a relevant advance in their management, greatly improving diagnosis, prognosis and treatment choice. Deregulation of N6-methyladenosine (m6A) RNA modification, catalyzed by the METTL3/METTL14 methyltransferase complex, is emerging as a relevant event in cell differentiation and carcinogenesis. Various studies have reported that altered expression of METTL3 is associated with an aggressive malignant phenotype and favors migration and invasiveness, but its role in Thymic Tumors remains unknown.
    RESULTS: In this study, we characterized that METTL3 contributes to Thymic Epithelial Tumor phenotype. We evidenced that METTL3 is overexpressed in tumor tissue compared to normal counterpart. Silencing of METTL3 expression in thymic carcinoma cells results in reduced cell proliferation and overall translation rate. Of note, METTL3 is responsible for the induction of c-MYC expression in TET cells. Specifically, high expression of c-MYC protein is enabled by lncRNA MALAT1, which is methylated and delocalized by METTL3. Interestingly, blocking of c-MYC by using JQ1 inhibitor cooperates with METTL3 depletion in the inhibition of proliferation and induction of cell death.
    CONCLUSION: This study highlighted METTL3 as a tumor promoter in Thymic tumors and c-MYC as a promising target to be exploited for the treatment of TET.
    Keywords:  JQ1 inhibitor; MALAT1; METTL3; S6K1; Thymic carcinoma; Thymic epithelial tumors; c-MYC; lncRNAs; m6A
    DOI:  https://doi.org/10.1186/s13148-021-01159-6
  17. Front Mol Biosci. 2021 ;8 741603
    Mass Spectrometry-Based Targeted Serum Monomethylated Ribonucleosides Profiling for Early Detection of Breast Cancer.
    Zhihao Fang, Yiqiu Hu, Jiani Chen, Kailun Xu, Kailai Wang, Shu Zheng, Cheng Guo.
      RNA methylation plays a significant regulatory role in various of physiological activities and it has gradually become a hotspot of epigenetics in the past decade. 2'-O-methyladenosine (Am), 2'-O-methylguanosine (Gm), 2'-O-methylcytidine (Cm), 2'-O-methyluridine (Um), N 6-methyladenosine (m6A), N 1-methylguanosine (m1G), 5-methylcytidine (m5C), and 5-methyluridine (m5U) are representative 2'-O-methylation and base-methylation modified epigenetic marks of RNA. Abnormal levels of these ribonucleosides were found to be related to various diseases including cancer. Serum is an important source of biofluid for the discovery of biomarkers, and novel tumor biomarkers can be explored by measuring these ribonucleoside modifications in human serum. Herein, we developed and applied a hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) method to determine the content of monomethylated ribonucleosides in human serum. The developed method enabled sensitive and accurate determination of these monomethylated ribonucleosides. By applying this robust method, we demonstrated the presence of Gm and Um in human serum for the first time, and we successfully quantified m6A, Gm, m1G, Cm, Um and m5U in serum samples collected from 61 patients with breast cancer and 69 healthy controls. We discovered that the levels of Gm, m1G, Cm, Um and m5U in serum were all significantly decreased in breast cancer patients whereas m6A was increased. We performed receiver operating characteristic (ROC) curve analysis, and obtained highest area under curve (AUC) value when combining these six monomethylated ribonucleosides together. These results suggest that m6A, Gm, m1G, Cm, Um and m5U might have great potential to be novel biomarkers for detection of breast cancer in the early stage. In addition, this study may stimulate future investigations about the regulatory roles of monomethylated ribonucleosides on the initiation and development of breast cancer.
    Keywords:  HILIC-MS/MS; RNA modification; breast cancer; monomethylated ribonucleosides; serum
    DOI:  https://doi.org/10.3389/fmolb.2021.741603
  18. Nat Commun. 2021 Sep 17. 12(1): 5522
    METTL3-mediated m6A RNA methylation promotes the anti-tumour immunity of natural killer cells.
    Hao Song, Jiaxi Song, Ming Cheng, Meijuan Zheng, Tian Wang, Sha Tian, Richard A Flavell, Shu Zhu, Hua-Bing Li, Chen Ding, Haiming Wei, Rui Sun, Hui Peng, Zhigang Tian.
      Natural killer (NK) cells exert critical roles in anti-tumor immunity but how their functions are regulated by epitranscriptional modification (e.g., N6-methyladenosine (m6A) methylation) is unclear. Here we report decreased expression of the m6A "writer" METTL3 in tumor-infiltrating NK cells, and a positive correlation between protein expression levels of METTL3 and effector molecules in NK cells. Deletion of Mettl3 in NK cells alters the homeostasis of NK cells and inhibits NK cell infiltration and function in the tumor microenvironment, leading to accelerated tumor development and shortened survival in mice. The gene encoding SHP-2 is m6A modified, and its protein expression is decreased in METTL3-deficient NK cells. Reduced SHP-2 activity renders NK cells hyporesponsive to IL-15, which is associated with suppressed activation of the AKT and MAPK signaling pathway in METTL3-deficient NK cells. These findings show that m6A methylation safeguards the homeostasis and tumor immunosurveillance function of NK cells.
    DOI:  https://doi.org/10.1038/s41467-021-25803-0
  19. Front Pharmacol. 2021 ;12 709548
    YTHDF2 is a Potential Biomarker and Associated with Immune Infiltration in Kidney Renal Clear Cell Carcinoma.
    Ganglin Su, Tianshu Liu, Xiaohong Han, Hao Sun, Wenan Che, Kun Hu, Junwen Xiao, Yanfeng Li, Yuchen Liu, Wujiao Li, Hongbing Mei.
      Clear cell renal cell carcinoma (ccRCC or KIRC) has a high mortality rate globally. It is necessary to identify biomarkers and investigate the mechanisms those biomarkers are associated with, to improve the prognosis of patients with KIRC. N6-Methyladenosine (m6A) affects the fate of modified RNA molecules and is involved in tumor progression. Different webservers were used in our research to investigate the mRNA transcription and clinical significance of YTHDF2 in KIRC. Survival analysis revealed that patients with elevated YTHDF2 transcription had a slightly longer OS and DFS than those with low YTHDF2 expression. YTHDF2 expression was shown to be significantly associated with the abundance of immune cells such as B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells. For a series of enrichment studies, we combined information on YTHDF2-binding molecules and expression-linked genes and identified the possible influence of "mRNA surveillance pathway," "RNA degradation," and "RNA transport" in the biology or pathogeny of KIRC. In addition, we identified multiple miRNA, kinase, and transcription factor targets of YTHDF2 in KIRC and constructed target networks. Overall, our findings show that YTHDF2 is a possible indicator of immune infiltration in the KIRC.
    Keywords:  KIRC; N6-methyladenosine (m6A) RNA methylation; YTHDF2; biomarker; immune infiltrates; prognosis
    DOI:  https://doi.org/10.3389/fphar.2021.709548
  20. Mol Ther Nucleic Acids. 2021 Dec 03. 26 135-147
    ac4C acetylation of RUNX2 catalyzed by NAT10 spurs osteogenesis of BMSCs and prevents ovariectomy-induced bone loss.
    W Yang, H Y Li, Y F Wu, R J Mi, W Z Liu, X Shen, Y X Lu, Y H Jiang, M J Ma, H Y Shen.
      N-acetyltransferase 10 (NAT10) is the key enzyme for N4-acetylcytidine (ac4C) modification of mRNA, which participates in various cellular processes and is related to many diseases. Here, we explore the relationships among osteoblast differentiation, NAT10, and ac4C, and we found that NAT0 expression and the ac4C level of total RNA were decreased in the bone tissues of bilateral ovariectomized (OVX) mice and osteoporosis patients. Adenoviruses overexpressing NAT10 reversed bone loss, and Remodelin, an NAT10 inhibitor, enhanced the loss of bone mass in OVX mice. Moreover, bone marrow-derived mesenchymal stem cells (BMSCs) with low-level ac4C modification formed fewer calcium nodules in vitro with NAT10 silencing, whereas BMSCs with high-level ac4C modification formed more calcium nodules with NAT10 overexpression. Moreover, we demonstrated that the ac4C level of runt-related transcription factor 2 (RUNX2) mRNA was increased after BMSCs were cultured in osteogenic medium (OM) and decreased after NAT10 silencing. The RUNX2 mRNA half-life and protein expression decreased after silencing NAT10 in BMSCs. Therefore, NAT10-based ac4C modification promotes the osteogenic differentiation of BMSCs by regulating the RUNX2 ac4C level. Because abnormal levels of NAT10 are probably one of the mechanisms responsible for osteoporosis, NAT10 is a new potential therapeutic target for this disease.
    Keywords:  NAT10; ac4C; bone marrow-derived mesenchymal stem cells; osteoblast differentiation; osteoporosis
    DOI:  https://doi.org/10.1016/j.omtn.2021.06.022
  21. Methods Enzymol. 2021 ;pii: S0076-6879(21)00265-2. [Epub ahead of print]658 161-190
    Chemoenzymatic labeling of RNA to enrich, detect and identify methyltransferase-target sites.
    Nadine A Kueck, Anna Ovcharenko, Katja Hartstock, Nicolas V Cornelissen, Andrea Rentmeister.
      The RNA methyltransferase (MTase) complex METTL3-METTL14 transfers methyl groups from S-adenosyl-l-methionine (AdoMet) to the N6-position of adenosines within its consensus sequence, the DRACH motif (D=A, G, U; R=A, G; H=A, C, U). Interestingly, this MTase complex shows remarkable promiscuity regarding the cosubstrate. This can be exploited to install nonnatural modifications, like clickable or photocaging groups. Clickable groups are widely used for subsequent functionalization and open a broad range of possibilities for downstream applications. Here, we elaborate on click chemistry for coupling of RNA to biotin to enrich MTase targets via streptavidin-coated magnetic beads. Importantly, after clicking and coupling to beads the modification becomes sterically demanding and stalls reverse transcriptases, leading to termination adjacent to the MTase target site. Using radioactively labeled primers in the reverse transcription, the modified position can be precisely identified on a sequencing gel via phosphor imaging.
    Keywords:  AdoMet-analoga; Chemoenzymatic RNA modification; CuAAC; METTL14; METTL3; Methyltransferase; RNA labeling; SeAdoYn
    DOI:  https://doi.org/10.1016/bs.mie.2021.06.006
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