bims-rimeca Biomed News
on RNA methylation in cancer
Issue of 2021‒09‒05
nineteen papers selected by
Sk Ramiz Islam
Saha Institute of Nuclear Physics
  1. Expression pattern and prognostic value of N6-methyladenosine RNA methylation key regulators in hepatocellular carcinoma.
  2. Methyltransferase-like 3-induced N6-methyladenosine upregulation promotes oral squamous cell carcinoma by through p38.
  3. The m6A demethylase FTO promotes the osteogenesis of mesenchymal stem cells by downregulating PPARG.
  4. METTL3 Intensifies the Progress of Oral Squamous Cell Carcinoma via Modulating the m6A Amount of PRMT5 and PD-L1.
  5. m6A regulator-mediated methylation modification patterns and tumor microenvironment infiltration characterization in hepatocellular carcinoma.
  6. N6-Methylandenosine-Related lncRNAs Predict Prognosis and Immunotherapy Response in Bladder Cancer.
  7. M6A-GSMS: Computational identification of N6-methyladenosine sites with GBDT and stacking learning in multiple species.
  8. RNA m6A methyltransferase METTL3 promotes colorectal cancer cell proliferation and invasion by regulating Snail expression.
  9. N6-methyladenosine (m6A) modification and its clinical relevance in cognitive dysfunctions.
  10. Multiplexed profiling facilitates robust m6A quantification at site, gene and sample resolution.
  11. A quantitative model predicts how m6A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions.
  12. The m6A Methyltransferase METTL14-Mediated N6-Methyladenosine Modification of PTEN mRNA Inhibits Tumor Growth and Metastasis in Stomach Adenocarcinoma.
  13. TS-m6A-DL: Tissue-specific identification of N6-methyladenosine sites using a universal deep learning model.
  14. m6A Methyltransferase METTL14-Mediated Upregulation of Cytidine Deaminase Promoting Gemcitabine Resistance in Pancreatic Cancer.
  15. Prognostic Value of an m5C RNA Methylation Regulator-Related Signature for Clear Cell Renal Cell Carcinoma.
  16. Interaction of tau with HNRNPA2B1 and N6-methyladenosine RNA mediates the progression of tauopathy.
  17. Quantifying RNA modifications by mass spectrometry: a novel source of biomarkers in oncology.
  18. Transcriptome-wide m6A methylome during osteogenic differentiation of human adipose-derived stem cells.
  19. METTL3 promotes inflammation and cell apoptosis in a pediatric pneumonia model by regulating EZH2.
  1. Mutagenesis. 2021 Sep 01. pii: geab032. [Epub ahead of print]
    Expression pattern and prognostic value of N6-methyladenosine RNA methylation key regulators in hepatocellular carcinoma.
    Min Deng, Lin Fang, Shao-Hua Li, Rong-Ce Zhao, Jie Mei, Jing-Wen Zou, Wei Wei, Rong-Ping Guo.
      Hepatocellular carcinoma (HCC) is still one of the most common malignancies worldwide. The accuracy of biomarkers for predicting the prognosis of HCC and the therapeutic effect is not satisfactory. N6-methyladenosine (m6A) methylation regulators play a crucial role in various tumors. Our research aims further to determine the predictive value of m6A methylation regulators and establish a prognostic model for HCC. In this study, the data of HCC from The Cancer Genome Atlas (TCGA) database was obtained, and the expression level of 15 genes and survival was examined. Then we identified two clusters of HCC with different clinical factors, constructed prognostic markers, and analyzed gene set enrichment, proteins' interaction, and gene co-expression. Three subgroups by consensus clustering according to the expression of the 13 genes were identified. The risk score generated by 5 genes divided HCC patients into high-risk and low-risk groups. In addition, we developed a prognostic marker that can identify high-risk HCC. Finally, a novel prognostic nomogram was developed to accurately predict HCC patients' prognosis. The expression levels of 13 m6A RNA methylation regulators were significantly up-regulated in HCC samples. The prognosis of cluster 1 and cluster 3 was worse. Patients in the high-risk group show a poor prognosis. Moreover, the risk score was an independent prognostic factor for HCC patients. In conclusion, we reveal the critical role of m6A RNA methylation modification in HCC and develop a predictive model based on the m6A RNA methylation regulators, which can accurately predict HCC patients' prognosis and provide meaningful guidance for clinical treatment.
    Keywords:  TCGA; hepatocellular carcinoma; m6A RNA methylation; nomogram; prognosis
    DOI:  https://doi.org/10.1093/mutage/geab032
  2. Oral Dis. 2021 Sep 03.
    Methyltransferase-like 3-induced N6-methyladenosine upregulation promotes oral squamous cell carcinoma by through p38.
    Ting Xu, Wenbo Zhang, Li Chai, Chao Liu, Shizhou Zhang, Tong Xu.
      BACKGROUND: Oral squamous cell carcinoma (OSCC), a main type of squamous cell cancer, is associated with considerable morbidity and mortality. Recent reports suggested methyltransferase-like 3 (METTL3)-mediated N6-methyladenosine (m6A) modification to be an essential regulator in the fate determination of stem cells. However, the functional significance of METTL3 in OSCC remains largely unknown.METHODS: METTL3 expression was examined in OSCC patient samples, followed by correlation analysis against clinical tumor features. Functional assays, such as assessment of surface marker expression, colony forming, BrdU incorporation, tumor xenograft assay, as well as m6A dot blot, were conducted to study the impact of METTL3 knock down (KD) in OSCC cells.
    RESULTS: High METTL3 expression was positively correlated with more severe clinical features of OSCC tumors. METTL3 KD caused impairment of stem-like capacities in OSCC cells, such as tumorigenicity in vivo as well as colony forming ability in vitro. Furthermore, METTL3-KD and cycloleucine, a methylation inhibitor, decreased m6A levels and down-regulated p38 expression in OSCC cells. On the other hand, the impaired cell proliferation capacity of OSCC cells after METTL3-KD was restored by exogenous expression of p38.
    CONCLUSION: Our findings identified m6A methyltransferase METTL3 as a key element in the regulation of tumorigenesis in OSCC.
    Keywords:  Methyltransferase-like 3; N6-methyladenosine; oral squamous cell carcinoma; p38; tumorigenesis
    DOI:  https://doi.org/10.1111/odi.14016
  3. Acta Pharmacol Sin. 2021 Aug 30.
    The m6A demethylase FTO promotes the osteogenesis of mesenchymal stem cells by downregulating PPARG.
    Liu-Shan Chen, Meng Zhang, Peng Chen, Xiao-Feng Xiong, Pei-Qing Liu, Hai-Bin Wang, Jun-Jian Wang, Juan Shen.
      N6-methyladenosine (m6A) is the most abundant posttranscriptional methylation modification that occurs in mRNA and modulates the fine-tuning of various biological processes in mammalian development and human diseases. In this study we investigated the role of m6A modification in the osteogenesis of mesenchymal stem cells (MSCs), and the possible mechanisms by which m6A modification regulated the processes of osteoporosis and bone necrosis. We performed systematic analysis of the differential gene signatures in patients with osteoporosis and bone necrosis and conducted m6A-RNA immunoprecipitation (m6A-RIP) sequencing to identify the potential regulatory genes involved in osteogenesis. We showed that fat mass and obesity (FTO), a primary m6A demethylase, was significantly downregulated in patients with osteoporosis and osteonecrosis. During the differentiation of human MSCs into osteoblasts, FTO was markedly upregulated. Both depletion of FTO and application of the FTO inhibitor FB23 or FB23-2 impaired osteogenic differentiation of human MSCs. Knockout of FTO in mice resulted in decreased bone mineral density and impaired bone formation. PPARG, a biomarker for osteoporosis, was identified as a critical downstream target of FTO. We further revealed that FTO mediated m6A demethylation in the 3'UTR of PPARG mRNA, and reduced PPARG mRNA stability in an YTHDF1-dependent manner. Overexpression of PPARG alleviated FTO-mediated osteogenic differentiation of MSCs, whereas knockdown of PPARG promoted FTO-induced expression of the osteoblast biomarkers ALPL and OPN during osteogenic differentiation. Taken together, this study demonstrates the functional significance of the FTO-PPARG axis in promoting the osteogenesis of human MSCs and sheds light on the role of m6A modification in mediating osteoporosis and osteonecrosis.
    Keywords:  FB23; FTO; N6-methyladenosine; PPARG; mesenchymal stem cells; osteogenesis
    DOI:  https://doi.org/10.1038/s41401-021-00756-8
  4. J Immunol Res. 2021 ;2021 6149558
    METTL3 Intensifies the Progress of Oral Squamous Cell Carcinoma via Modulating the m6A Amount of PRMT5 and PD-L1.
    Yilong Ai, Shiwei Liu, Hailing Luo, Siyuan Wu, Haigang Wei, Zhe Tang, Xia Li, Xiaozhi Lv, Chen Zou.
      N6-Methyladenosine (m6A) modification is one of the commonest chemical modifications in eukaryotic mRNAs, which has essential effects on mRNA translation, splicing, and stability. Currently, there is a rising concern on the regulatory role of m6A in tumorigenesis. As a known component in the m6A methyltransferase complex, METTL3 (methyltransferase-like 3) plays an essential role in m6A methylation. Till now, the functions of METTL3 in oral squamous cell carcinoma (OSCC) and its relative mechanism remain to be explored. In this research, through the GEPIA database, we found that high METTL3 expression has a correlation with poor prognosis of squamous cell carcinoma of head and neck. qRT-PCR displayed that METTL3 was highly expressed in OSCC cells. Functionally, METTL3 knockdown reduced the invasion, migration, and proliferation competence of OSCC cells and attenuated the activation of CD8+ T cells. In contrast, METTL3 overexpression resulted in opposite results. GEPIA, UALCAN, and SRAMP databases, PCR, western blot, and m6A RNA methylation assay confirmed the m6A modification of PRMT5 and PD-L1 mediated by METTL3. In conclusion, our results displayed that METTL3 intensified the metastasis and proliferation of OSCC by modulating the m6A amounts of PRMT5 and PD-L1, suggesting that METTL3 may be a therapeutic target for OSCC patients.
    DOI:  https://doi.org/10.1155/2021/6149558
  5. Aging (Albany NY). 2021 Aug 30. 13(undefined):
    m6A regulator-mediated methylation modification patterns and tumor microenvironment infiltration characterization in hepatocellular carcinoma.
    Xiongpei Huang, Zecheng Qiu, Liusheng Li, Bin Chen, Peiyuan Huang.
      BACKGROUND: There is increasing evidence of the epigenetic regulation of the immune response in cancer. However, the specific functions and mechanisms of RNA N6-methyladenosine (m6A) modification in the cell infiltration in the hepatocellular carcinoma (HCC) tumor microenvironment (TME) is unknown.METHODS: We systematically analyzed the m6A-modification patterns of 371 HCC samples based on 23 m6A regulators, and determined their correlation with TME cell-infiltrating characteristics. Principal-component analysis algorithms was used to calculate the m6Ascore and clarify the m6A-modification patterns of individual tumors.
    RESULTS: Three different m6A-modification patterns were identified in HCC, wherein the m6Acluster B and m6Acluster A had the best and worst prognosis, respectively. These three patterns had different TME cell infiltration characteristics and biological behavior. An m6A-scoring signature was constructed to evaluate the m6A-modification patterns within individual tumors. A low m6Ascore was associated with a low overall survival and high clinical stage. Moreover, the m6A-scoring signature was characterized by distinct immunotherapeutic landscapes; a high m6A score indicated a higher immune checkpoint inhibitor score in the anti-PD-1 treatment alone, anti-CTLA-4 treatment alone, or combined anti-CTLA-4/PD-1 treatment cohorts, which reflected significant treatment and clinical benefits.
    CONCLUSIONS: Our study highlights the significant role of the m6A modification in the HCC TME. A scoring signature to clarify the individual m6A-modification pattern would help us understand the HCC TME infiltration characterization and, thus, would guide the selection of more effective immunotherapeutic strategies.
    Keywords:  hepatocellular carcinoma; immunotherapy; m6A; methylation; tumor microenvironment
    DOI:  https://doi.org/10.18632/aging.203456
  6. Front Oncol. 2021 ;11 710767
    N6-Methylandenosine-Related lncRNAs Predict Prognosis and Immunotherapy Response in Bladder Cancer.
    Yuying Zhang, Baoyi Zhu, Minghui He, Yi Cai, Xiaoling Ying, Chonghe Jiang, Weidong Ji, Jianwen Zeng.
      Both lncRNAs and the N6-methyladenosine (m6A) modification are key regulators of tumorigenesis and innate immunity. However, little is known about the m6A modification of lncRNAs and their clinical and immune relevance in bladder cancer. In this study, we identified m6A-related lncRNAs using Pearson correlation analysis in The Cancer Genome Atlas (TCGA) and the IMvigor210 datasets. Next, univariate Cox regression was performed using the TCGA dataset to filter prognostic m6A-related lncRNAs, which were further subjected to the least absolute shrinkage and selection operator (LASSO) Cox regression to establish a 12 m6A-related lncRNA prognostic score (m6A-LRS). The m6A-LRS was validated in the IMvigor210 dataset. In addition, high m6A-LRS tumors, characterized by decreased tumor mutation load and neoantigen load, showed poorer response to immunotherapy than those with low m6A-LRS in the IMvigor210 dataset. Further, we constructed an m6A-LRS-based nomogram that demonstrated a strong ability to predict overall survival in patients with bladder cancer. Moreover, enrichment analysis revealed that tumor-associated biological processes, oncogenic signaling, and tumor hallmarks were commonly associated with a high m6A-LRS. Gene set variation analysis also indicated that high m6A-LRS was associated with activation of canonical oncogenic signatures, such as the epithelial-to-mesenchymal transition, cell cycle regulators, and DNA replication, as well as activation of immunosuppressive signatures, such as the T-cell exhaustion and pan-fibroblast-TGF-β response signatures. Furthermore, we observed distinct tumor microenvironment cell infiltration characteristics between high- and low-risk tumors. High m6A-LRS tumors showed reduced infiltration of CD8+ T-cells and enhanced infiltration of macrophages and fibroblasts. Additionally, we established a competing endogenous RNA network based on the12 m6A-related lncRNAs. Finally, three lncRNAs (SNHG16, SBF2-AS1, and BDNF-AS) were selected for further validation. The qualitative PCR assay on 10 pairs of bladder cancer and adjacent normal control samples validated the differential expression, and methylated RNA immunoprecipitation (MeRIP) analysis demonstrated a robust m6A enrichment in T24 bladder cancer cells compared with normal uroepithelial cells (SVHUC-1). In conclusion, this study introduced an m6A-related lncRNA signature that identified a subgroup of patients with poor prognoses and suboptimal immune responses, thus providing novel approaches for treatment response prediction and patient stratification in bladder cancer.
    Keywords:  N6-methyladenosine; bladder cancer; immune response; lncRNA; prognosis; tumor microenvironment
    DOI:  https://doi.org/10.3389/fonc.2021.710767
  7. J Biomol Struct Dyn. 2021 Aug 30. 1-12
    M6A-GSMS: Computational identification of N6-methyladenosine sites with GBDT and stacking learning in multiple species.
    Shengli Zhang, Jinyue Wang, Xinjie Li, Yunyun Liang.
      N6-methyladenosine (m6A) is one of the most abundant forms of RNA methylation modifications currently known. It involves a wide range of biological processes, including degradation, stability, alternative splicing, etc. Therefore, the development of convenient and efficient m6A prediction technologies are urgent. In this work, a novel predictor based on GBDT and stacking learning is developed to identify m6A sites, which is called M6A-GSMS. To achieve accurate prediction, we explore RNA sequence information from four aspects: correlation, structure, physicochemical properties and pseudo ribonucleic acid composition. After using the GBDT algorithm for feature selection, a stacking model is constructed by combining seven basic classifiers. Compared with other state-of-the-art methods, the results show that M6A-GSMS can obtain excellent performance for identifying the m6A sites. The prediction accuracy of A.thaliana, D.melanogaster, M.musculus, S.cerevisiae and Human reaches 88.4%, 60.8%, 80.5%, 92.4% and 61.8%, respectively. This method provides an effective prediction for the investigation of m6A sites. In addition, all the datasets and codes are currently available at https://github.com/Wang-Jinyue/M6A-GSMS.Communicated by Ramaswamy H. Sarma.
    Keywords:  GBDT; N6-methyladenosine site; RNA methylation; Stacking learning
    DOI:  https://doi.org/10.1080/07391102.2021.1970628
  8. Oncol Lett. 2021 Oct;22(4): 711
    RNA m6A methyltransferase METTL3 promotes colorectal cancer cell proliferation and invasion by regulating Snail expression.
    Jianfan Wen, Guowei Zhang, Yuwen Meng, Lei Zhang, Min Jiang, Zhitao Yu.
      Nitrogen 6-methyladenosine (m6A) is the result of methylation of nitrogen-6 on adenosine, and is the most abundant chemical modification of eukaryotic mRNA. Dysregulation of m6A methylation has been implicated in cancer development and progression through various mechanisms. This type of methylation is primarily regulated by methyltransferase-like 3 (METTL3). However, the molecular mechanisms underlying the role of METTL3 in colorectal cancer (CRC) have not been extensively elucidated. The present study explored m6A modification and the underlying mechanism of m6A, which serve regulatory roles in the development of CRC. It was found that METTL3 is upregulated in CRC cell lines and tissues, and its expression positively correlated with poor overall survival (OS). Mechanistically, the present study demonstrated that METTL3 methylates Snail mRNA, thus stabilizing it to promote CRC malignancy. The present findings indicate that m6A modification is involved in CRC tumorigenesis, and highlight its potential as a therapeutic target against CRC.
    Keywords:  Snail; colorectal cancer; methyltransferase-like 3; nitrogen 6-methyladenosine
    DOI:  https://doi.org/10.3892/ol.2021.12972
  9. Aging (Albany NY). 2021 Aug 30. 13(undefined):
    N6-methyladenosine (m6A) modification and its clinical relevance in cognitive dysfunctions.
    Bingying Du, Yanbo Zhang, Meng Liang, Zengkan Du, Haibo Li, Cunxiu Fan, Hailing Zhang, Yan Jiang, Xiaoying Bi.
      BACKGROUND: N6 adenosine methylation (m6A) is the most abundant internal RNA modification in eukaryotic cells. Dysregulation of m6A has been associated with the perturbations of cell proliferation and cell death in different diseases. However, the roles of m6A in the neurodegenerative process and cognitive dysfunction are unclear.METHODS: We systematically investigated the molecular alterations of m6A regulators and their clinical relevance with cognitive dysfunctions using published datasets of Alzheimer's Disease (AD), vascular dementia, and mild cognitive impairment (MCI).
    FINDINGS: The expressions of m6A regulators vary in different tissues and closely correlate with neurodegenerative pathways. We identified co-expressive m6A regulators SNRPG and SNRPD2 as potential biomarkers to predict transformation from MCI to AD. Moreover, we explored correlations between Apolipoprotein E4 and m6A methylations.
    INTERPRETATION: Collectively, these findings suggest that m6A methylations as potential biomarkers and therapeutic targets for cognitive dysfunction.
    FUNDING: This work was supported by the National Natural Science Foundation of China (81871040) and the Shanghai Health System Talent Training Program (2018BR29).
    Keywords:  KEGG pathways; WGCNA; apolipoprotein E; cognitive impairment; m6A regulator
    DOI:  https://doi.org/10.18632/aging.203457
  10. Nat Methods. 2021 Sep 03.
    Multiplexed profiling facilitates robust m6A quantification at site, gene and sample resolution.
    David Dierks, Miguel Angel Garcia-Campos, Anna Uzonyi, Modi Safra, Sarit Edelheit, Alice Rossi, Theodora Sideri, Radhika A Varier, Alexander Brandis, Yonatan Stelzer, Folkert van Werven, Ruth Scherz-Shouval, Schraga Schwartz.
      N6-methyladenosine (m6A) is the most prevalent modification of messenger RNA in mammals. To interrogate its functions and dynamics, there is a critical need to quantify m6A at three levels: site, gene and sample. Current approaches address these needs in a limited manner. Here we develop m6A-seq2, relying on multiplexed m6A-immunoprecipitation of barcoded and pooled samples. m6A-seq2 allows a big increase in throughput while reducing technical variability, requirements of input material and cost. m6A-seq2 is furthermore uniquely capable of providing sample-level relative quantitations of m6A, serving as an orthogonal alternative to mass spectrometry-based approaches. Finally, we develop a computational approach for gene-level quantitation of m6A. We demonstrate that using this metric, roughly 30% of the variability in RNA half life in mouse embryonic stem cells can be explained, establishing m6A as a main driver of RNA stability. m6A-seq2 thus provides an experimental and analytic framework for dissecting m6A-mediated regulation at three different levels.
    DOI:  https://doi.org/10.1038/s41592-021-01242-z
  11. Nat Commun. 2021 08 31. 12(1): 5201
    A quantitative model predicts how m6A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions.
    Bei Liu, Honglue Shi, Atul Rangadurai, Felix Nussbaumer, Chia-Chieh Chu, Kevin Andreas Erharter, David A Case, Christoph Kreutz, Hashim M Al-Hashimi.
      N6-methyladenosine (m6A) is a post-transcriptional modification that controls gene expression by recruiting proteins to RNA sites. The modification also slows biochemical processes through mechanisms that are not understood. Using temperature-dependent (20°C-65°C) NMR relaxation dispersion, we show that m6A pairs with uridine with the methylamino group in the anti conformation to form a Watson-Crick base pair that transiently exchanges on the millisecond timescale with a singly hydrogen-bonded low-populated (1%) mismatch-like conformation in which the methylamino group is syn. This ability to rapidly interchange between Watson-Crick or mismatch-like forms, combined with different syn:anti isomer preferences when paired (~1:100) versus unpaired (~10:1), explains how m6A robustly slows duplex annealing without affecting melting at elevated temperatures via two pathways in which isomerization occurs before or after duplex annealing. Our model quantitatively predicts how m6A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions, and provides an explanation for why the modification robustly slows diverse cellular processes.
    DOI:  https://doi.org/10.1038/s41467-021-25253-8
  12. Front Oncol. 2021 ;11 699749
    The m6A Methyltransferase METTL14-Mediated N6-Methyladenosine Modification of PTEN mRNA Inhibits Tumor Growth and Metastasis in Stomach Adenocarcinoma.
    Qi Yao, Lanzhen He, Xucan Gao, Na Tang, Lifen Lin, Xiaofang Yu, Dong Wang.
      Background: Stomach adenocarcinoma (STAD) is a common reason for tumor-related fatalities globally, as it results in distant metastasis. Methyltransferase-like 14 (METTL14), a notable RNA N6-adenosine methyltransferase (m6A), plays a significant role in the growth of tumor through controlling the RNA working. This study aims to highlight METTL14 in STAD's biological function and molecular mechanism.Methods: Bioinformatics and immunohistochemical (IHC) assays have been utilized for the detection of METTL14 expression in the STAD. METTL14's biological function has been shown while making use of HGC-27 and AGS cells in vitro experiments. MeRIP-qPCR and luciferase reporter assays were employed for the exploration of METTL14's mechanism modifying the target of phosphatase and tensin homologue (PTEN). Subcutaneous xeno transplantation model and STAD liver metastasis orthotopic tumor model were used to study METTL14 in STAD in vivo.
    Results: METTL14 expression was substantially downregulated in STAD reflecting contribution to major tumors, progressed TNM stage as well as poor overall survival (OS) in STAD. Moreover, METTL14's inhibition of STAD cells proliferation, migration and invasion has been verified in vitro assays. Furthermore, an identification of PTEN being METTL14-mediated m6A modification's substrate has been made. METTL14's overexpression highly enhanced PTEN mRNA m6A variation, stabilized PTEN mRNA and increased protein expression. Further, it has been found out that METTL14-mediated STAD cells inhibition of proliferation and invasion dependent on PTEN. At last, we demonstrated that METTL14 inhibit STAD growth and metastasis in vivo models.
    Conclusions: METTL14 inhibits tumor growth and metastasis of STAD via stabilization of PTEN mRNA expression. Therefore, METTL14 is a potential biomarker of prognosis and therapeutic targets for STAD.
    Keywords:  METTL14; Pten; STAD; m6A modification; metastasis; tumor growth
    DOI:  https://doi.org/10.3389/fonc.2021.699749
  13. Comput Struct Biotechnol J. 2021 ;19 4619-4625
    TS-m6A-DL: Tissue-specific identification of N6-methyladenosine sites using a universal deep learning model.
    Zeeshan Abbas, Hilal Tayara, Quan Zou, Kil To Chong.
      The most communal post-transcriptional modification, N6-methyladenosine (m6A), is associated with a number of crucial biological processes. The precise detection of m6A sites around the genome is critical for revealing its regulatory function and providing new insights into drug design. Although both experimental and computational models for detecting m6A sites have been introduced, but these conventional methods are laborious and expensive. Furthermore, only a handful of these models are capable of detecting m6A sites in various tissues. Therefore, a more generic and optimized computational method for detecting m6A sites in different tissues is required. In this paper, we proposed a universal model using a deep neural network (DNN) and named it TS-m6A-DL, which can classify m6A sites in several tissues of humans (Homo sapiens), mice (Mus musculus), and rats (Rattus norvegicus). To extract RNA sequence features and to convert the input into numerical format for the network, we utilized one-hot-encoding method. The model was tested using fivefold cross-validation and its stability was measured using independent datasets. The proposed model, TS-m6A-DL, achieved accuracies in the range of 75-85% using the fivefold cross-validation method and 72-84% on the independent datasets. Finally, to authenticate the generalization of the model, we performed cross-species testing and proved the generalization ability by achieving state-of-the-art results.
    Keywords:  Binary-encoding; Deep neural network; Motif; N6-methyladenosine (m6A); Tissue-specific
    DOI:  https://doi.org/10.1016/j.csbj.2021.08.014
  14. Front Oncol. 2021 ;11 696371
    m6A Methyltransferase METTL14-Mediated Upregulation of Cytidine Deaminase Promoting Gemcitabine Resistance in Pancreatic Cancer.
    Congjun Zhang, Shuangyan Ou, Yuan Zhou, Pei Liu, Peiying Zhang, Ziqian Li, Ruocai Xu, Yuqiang Li.
      Objective: Pancreatic cancer is one of the most lethal human malignancies. Gemcitabine is widely used to treat pancreatic cancer, and the resistance to chemotherapy is the major difficulty in treating the disease. N 6-methyladenosine (m6A) modification, which regulates RNA splicing, stability, translocation, and translation, plays critical roles in cancer physiological and pathological processes. METTL14, an m6A Lmethyltransferase, was found deregulated in multiple cancer types. However, its role in gemcitabine resistance in pancreatic cancer remains elusive.Methods: The mRNA and protein level of m6A modification associated genes were assessed by QRT-PCR and western blotting. Then, gemcitabine-resistant pancreatic cancer cells were established. The growth of pancreatic cancer cells were analyzed using CCK8 assay and colony formation assay. METTL14 was depleted by using shRNA. The binding of p65 on METTL14 promoter was assessed by chromatin immunoprecipitation (ChIP) assay. Protein level of deoxycytidine kinase (DCK) and cytidine deaminase (CDA) was evaluated by western blotting. In vivo experiments were conducted to further confirm the critical role of METTL14 in gemcitabine resistance.
    Results: We found that gemcitabine treatment significantly increased the expression of m6A methyltransferase METTL14, and METTL14 was up-regulated in gemcitabine-resistance human pancreatic cancer cells. Suppression of METTL14 obviously increased the sensitivity of gemcitabine in resistant cells. Moreover, we identified that transcriptional factor p65 targeted the promoter region of METTL14 and up-regulated its expression, which then increased the expression of cytidine deaminase (CDA), an enzyme inactivates gemcitabine. Furthermore, in vivo experiment showed that depletion of METTL14 rescue the response of resistance cell to gemcitabine in a xenograft model.
    Conclusion: Our study suggested that METTL14 is a potential target for chemotherapy resistance in pancreatic cancer.
    Keywords:  METTL14; N6-methyladenosine; chemotherapy; p65; pancreatic cancer
    DOI:  https://doi.org/10.3389/fonc.2021.696371
  15. Cancer Manag Res. 2021 ;13 6673-6687
    Prognostic Value of an m5C RNA Methylation Regulator-Related Signature for Clear Cell Renal Cell Carcinoma.
    Hanrong Li, Huiming Jiang, Zhicheng Huang, Zhilin Chen, Nanhui Chen.
      Purpose: Clear cell renal cell carcinoma (ccRCC) is highly heterogeneous and is one of the most lethal types of cancer within the urinary system. Aberrant expression of 5-methylcytosine (m5C) RNA methylation regulators has been shown to result in occurrence and progression of tumors. However, the role of these regulators in ccRCC remains unclear.Materials and Methods: We extracted RNA sequencing expression data with corresponding clinical information of patients with ccRCC from The Cancer Genome Atlas (TCGA) database. We then compared the expression profiles of m5C RNA methylation regulators between normal and ccRCC tissues, and determined different subtypes through consensus clustering analysis. In addition, we constructed a prognostic signature and evaluated it using a range of bioinformatics approaches. The expression of signature-related genes was subsequently verified in the clinical samples using qRT-PCR.
    Results: We identified 12 differentially expressed m5C RNA methylation regulators between cancer and normal control samples. Two clusters of patients with ccRCC and diverse clinicopathological characteristics and prognoses were then determined through consensus clustering analysis. Functional annotations revealed that m5C RNA regulators were significantly correlated with the ccRCC progression. Moreover, we constructed a four-gene risk score signature (comprised of NOP2, NSUN4, NSUN6, and TET2) and divided the patients with ccRCC into high- and low-risk groups based on the median risk score. The risk score was associated with clinicopathological features and was an independent prognostic indicator of ccRCC. Our stratified analysis results suggest that the signature has high prognostic value. Based on qRT-PCR results, the NOP2 and NSUN4 mRNA expressions were higher and those of NSUN6 and TET2 were lower in ccRCC tissues than in normal tissues.
    Conclusion: Our results demonstrate that m5C RNA methylation regulators may affect ccRCC progression and could be exploited for diagnostic and prognostic purposes.
    Keywords:  5-methylcytosine RNA methylation regulators; bioinformatics; ccRCC; epigenetics; prognostic signature
    DOI:  https://doi.org/10.2147/CMAR.S323072
  16. Mol Cell. 2021 Aug 20. pii: S1097-2765(21)00622-5. [Epub ahead of print]
    Interaction of tau with HNRNPA2B1 and N6-methyladenosine RNA mediates the progression of tauopathy.
    Lulu Jiang, Weiwei Lin, Cheng Zhang, Peter E A Ash, Mamta Verma, Julian Kwan, Emily van Vliet, Zhuo Yang, Anna Lourdes Cruz, Samantha Boudeau, Brandon F Maziuk, Shuwen Lei, Jaehyup Song, Victor E Alvarez, Stacy Hovde, Jose F Abisambra, Min-Hao Kuo, Nicholas Kanaan, Melissa E Murray, John F Crary, Jian Zhao, Ji-Xin Cheng, Leonard Petrucelli, Hu Li, Andrew Emili, Benjamin Wolozin.
      The microtubule-associated protein tau oligomerizes, but the actions of oligomeric tau (oTau) are unknown. We have used Cry2-based optogenetics to induce tau oligomers (oTau-c). Optical induction of oTau-c elicits tau phosphorylation, aggregation, and a translational stress response that includes stress granules and reduced protein synthesis. Proteomic analysis identifies HNRNPA2B1 as a principle target of oTau-c. The association of HNRNPA2B1 with endogenous oTau was verified in neurons, animal models, and human Alzheimer brain tissues. Mechanistic studies demonstrate that HNRNPA2B1 functions as a linker, connecting oTau with N6-methyladenosine (m6A) modified RNA transcripts. Knockdown of HNRNPA2B1 prevents oTau or oTau-c from associating with m6A or from reducing protein synthesis and reduces oTau-induced neurodegeneration. Levels of m6A and the m6A-oTau-HNRNPA2B1 complex are increased up to 5-fold in the brains of Alzheimer subjects and P301S tau mice. These results reveal a complex containing oTau, HNRNPA2B1, and m6A that contributes to the integrated stress response of oTau.
    Keywords:  Alzheimer's disease; METTL3; RNA methylation; RNA translation; fibrils; lamin; neurodegeneration; nuclear envelope; stress granules; tau oligomerization
    DOI:  https://doi.org/10.1016/j.molcel.2021.07.038
  17. Crit Rev Clin Lab Sci. 2021 Sep 02. 1-18
    Quantifying RNA modifications by mass spectrometry: a novel source of biomarkers in oncology.
    Amandine Amalric, Amandine Bastide, Aurore Attina, Armelle Choquet, Jerome Vialaret, Sylvain Lehmann, Alexandre David, Christophe Hirtz.
      Despite significant progress in targeted therapies, cancer recurrence remains a major cause of mortality worldwide. Identification of accurate biomarkers, through molecular profiling in healthy and cancer patient samples, will improve diagnosis and promote personalized medicine. While genetic and epigenetic alterations of DNA are currently exploited as cancer biomarkers, their robustness is limited by tumor heterogeneity. Recently, cancer-associated changes in RNA marks have emerged as a promising source of diagnostic and prognostic biomarkers. RNA epigenetics (also known as epitranscriptomics) is an emerging field in which at least 150 chemical modifications in all types of RNA (mRNA, tRNA, lncRNA, rRNA, and microRNA) have been detected. These modifications fine-tune gene expression in both physiological and pathological processes. A growing number of studies have established links between specific modified nucleoside levels in solid/liquid biopsies, and cancer onset and progression. In this review, we highlight the potential role of epitranscriptomic markers in refining cancer diagnosis and/or prognosis. RNA modification patterns may contain important information for establishing an initial diagnosis, monitoring disease evolution, and predicting response to treatment. Furthermore, recent developments in mass spectrometry allow reliable quantification of RNA marks in solid biopsies and biological fluids. We discuss the great potential of mass spectrometry for identifying epitranscriptomic biomarker signatures in cancer diagnosis. While there are various methods to quantify modified nucleosides, most are unable to detect and quantify more than one type of RNA modification at a time. Mass spectrometry analyses, especially GC-MS/MS and LC-MS/MS, overcome this limitation and simultaneously detect modified nucleosides by multiple reaction monitoring. Indeed, several groups are currently validating mass spectrometry methods that quantify several nucleosides at one time in liquid biopsies. The challenge now is to exploit these powerful analytical tools to establish epitranscriptomic signatures that should open new perspectives in personalized medicine. This review summarizes the growing clinical field of analysis of RNA modifications and discusses pre-analytical and analytical approaches, focusing in particular on the development of new mass spectrometry tools and their clinical applications.
    Keywords:  Epitranscriptomics; biomarker; cancer; mass spectrometry
    DOI:  https://doi.org/10.1080/10408363.2021.1958743
  18. Stem Cell Res Ther. 2021 Sep 01. 12(1): 489
    Transcriptome-wide m6A methylome during osteogenic differentiation of human adipose-derived stem cells.
    Wentian Sun, Yidan Song, Kai Xia, Liyuan Yu, Xinqi Huang, Zhihe Zhao, Jun Liu.
      OBJECTIVES: Adipose-derived stem cells are frequently used for bone regeneration both in vitro and in vivo. N6-methyladenosine (m6A) is the most abundant post-transcriptional modification on eukaryotic RNAs and plays multifaceted roles in development and diseases. However, the regulatory mechanisms of m6A in osteogenic differentiation of human adipose-derived stem cells (hASCs) remain elusive. The present study aimed to build the transcriptome-wide m6A methylome during the osteogenic differentiation of hASCs.MATERIALS AND METHODS: hASCs were harvested after being cultured in a basic or osteogenic medium for 7 days, and the osteogenic differentiation was validated by alkaline phosphatase (ALP) and Alizarin Red S staining, ALP activity assay, and qRT-PCR analysis of ALP, RUNX2, BGLAP, SPP1, SP7, and COL1A1 genes. The m6A level was colorimetrically measured, and the expression of m6A regulators was confirmed by qRT-PCR and western blot. Moreover, m6A MeRIP-seq and RNA-seq were performed to build the transcriptome and m6A methylome. Furthermore, bioinformatic analyses including volcano plots, Venn plots, clustering analysis, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, gene sets enrichment analysis, and protein-protein interaction analysis were conducted.
    RESULTS: In total, 1145 differentially methylated peaks, 2261 differentially expressed genes, and 671 differentially methylated and expressed genes (DMEGs) were identified. GO and KEGG pathway analyses conducted for these DMEGs revealed extensive and osteogenic biological functions. The "PI3K-Akt signaling pathway"; "MAPK signaling pathway"; "parathyroid hormone synthesis, secretion, and action"; and "p53 signaling pathway" were significantly enriched, and the DMEGs in these pathways were identified as m6A-specific key genes. A protein-protein interaction network based on DMEGs was built, and VEGFA, CD44, MMP2, HGF, and SPARC were speculated as the hub DMEGs.
    CONCLUSIONS: The total m6A level was reduced with osteogenic differentiation of hASCs. The transcriptome-wide m6A methylome built in the present study indicated quite a few signaling pathways, and hub genes were influenced by m6A modification. Future studies based on these epigenetic clues could promote understanding of the mechanisms of osteogenic differentiation of hASCs.
    Keywords:  Adipose-derived stem cell; MeRIP-seq; N6-methyladenosine; Osteogenic differentiation; RNA-seq; m6A
    DOI:  https://doi.org/10.1186/s13287-021-02508-1
  19. Allergol Immunopathol (Madr). 2021 ;49(5): 49-56
    METTL3 promotes inflammation and cell apoptosis in a pediatric pneumonia model by regulating EZH2.
    Ying Yang, Xiaoqing Yang, Yi Wu, Meijiao Fu.
      BACKGROUND: In recent times, the medical science has developed by leaps and bounds, however, the molecular mechanism of pediatric pneumonia is still unclear. Although prior researches have shown that methyltransferase-like 3 (METTL3) is up-regulated in a variety of inflammatory diseases, its role and mechanism has been rarely studied in pediatric pneumonia, and need to be defined elaborately.OBJECTIVE: In this study, the related molecular mechanism of METTL3 on inflammation and cell apoptosis in a pediatric pneumonia was investigated.
    MATERIALS AND METHODS: Quantitative real-time polymerase chain reaction (qPCR) and western blot assays were employed to examine the mRNA and protein expression level of METTL3 and EZH2 in peripheral blood monocytes from pediatric pneumonia patients or cell model (WI-38). Then, qPCR and ELISA assay were applied to verify the inflammatory response in LPS-treated WI-38 cell lines after knockdown of METTL3. Besides, MTT cell viability assays, flow cytometry, and western blot assays were applied to examine the cell viability and cell apoptosis of LPS-treated WI-38 cell after knockdown of METTL3. Further, the western blot assays were employed to examine the protein expression levels of p-JAK2, JAK2, p-STAT3, STAT3, and EZH2 in LPS-treated WI-38 cell after knockdown of METTL3. Finally, ELISA and western blot were applied to verify the inflammatory response and cell apoptosis of LPS-treated WI-38 cell after knockdown of METTL3 and overexpression of EZH2.
    RESULTS: In this study, the results showed that METTL3 and EZH2 were highly expressed in pediatric pneumonia patients and cell models (WI-38), respectively. Besides, downregulation of METTL3 inhibited LPS-induced inflammatory response and cell apoptosis. Then, the fact that METTL3 regulates the JAK2/STAT3 signaling pathway through EZH was proved. Furthermore, downregulation of METTL3 inhibits inflammation and apoptosis through EZH2.
    CONCLUSION: This study found that METTL3 promotes inflammation and cell apoptosis in a pediatric pneumonia model by regulating EZH2.
    Keywords:  EZH2; JAK2; METTL3; STAT; pediatric pneumonia
    DOI:  https://doi.org/10.15586/aei.v49i5.445
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