bims-rimeca Biomed News
on RNA methylation in cancer
Issue of 2022–05–15
forty-six papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics
  1. The role of Insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs) as m6A readers in cancer.
  2. N6-methyladenosine methyltransferase KIAA1429 elevates colorectal cancer aerobic glycolysis via HK2-dependent manner.
  3. METTL3 Enhances Hepatocellular Carcinoma Progression by Regulating Polycomb Repressive Complex 1 (PRC1) Components BMI1 and RNF2.
  4. m6A Methylation Patterns and Tumor Microenvironment Infiltration Characterization in Clear-Cell Renal Cell Carcinoma.
  5. ELMo4m6A: a contextual language embedding-based predictor for detecting RNA N6-methyladenosine sites.
  6. Construction of a Comprehensive Diagnostic Scoring Model for Prostate Cancer Based on a Novel Six-Gene Panel.
  7. Methyltransferase-Like 3 Rescues the Amyloid-beta protein-Induced Reduction of Activity-Regulated Cytoskeleton Associated Protein Expression via YTHDF1-Dependent N6-Methyladenosine Modification.
  8. RNA Demethylase ALKBH5 Prevents Lung Cancer Progression by Regulating EMT and Stemness via Regulating p53.
  9. RNA methylation in gene expression regulation.
  10. Roles of the m6A Modification of RNA in the Glioblastoma Microenvironment as Revealed by Single-Cell Analyses.
  11. Role of RNA modifications in carcinogenesis and carcinogen damage response.
  12. Downregulated ALKBH5 contributes to myocardial ischemia/reperfusion injury by increasing m6A modification of Trio mRNA.
  13. N6-methyladenosine-modified TRAF1 promotes sunitinib resistance by regulating apoptosis and angiogenesis in a METTL14-dependent manner in renal cell carcinoma.
  14. SRSF3-mediated regulation of N6-methyladenosine modification-related lncRNA ANRIL splicing promotes resistance of pancreatic cancer to gemcitabine.
  15. Global N6-methyladenosine methylation analysis reveals the positive correlation between m6A modification and mRNA abundance during Apostichopus japonicus disease development.
  16. Methyltransferase-like 14 silencing relieves the development of atherosclerosis via m6A modification of p65 mRNA.
  17. Emerging Roles of FTO in Neuropsychiatric Disorders.
  18. 5-methylcytosine RNA methyltransferases and their potential roles in cancer.
  19. Multiple Phosphorylations of SR Protein SRSF3 and Its Binding to m6A Reader YTHDC1 in Human Cells.
  20. PA2G4 promotes the metastasis of hepatocellular carcinoma by stabilizing FYN mRNA in a YTHDF2-dependent manner.
  21. Roles of m6A modification in neurological diseases.
  22. The Role of N6-Methyladenosine in the Promotion of Hepatoblastoma: A Critical Review.
  23. Genome-Wide Identification, Classification and Expression Analysis of m6A Gene Family in Solanum lycopersicum.
  24. Comprehensive Analysis of N6-Methyladenosine Regulators in the Subcluster Classification and Drug Candidates Prediction of Severe Obstructive Sleep Apnea.
  25. Profiling of Transcriptome-Wide N6-Methyladenosine (m6A) Modifications and Identifying m6A Associated Regulation in Sperm Tail Formation in Anopheles sinensis.
  26. ALKBH5 regulates somatic cell reprogramming in a phase specific manner.
  27. Characteristics of N6-methyladenosine modification during sexual reproduction of Chlamydomonas reinhardtii.
  28. Activity-Based Profiling of RNA Modifying Enzymes.
  29. The transcriptome-wide N6-methyladenosine (m6A) map profiling reveals the regulatory role of m6A in the yak ovary.
  30. Pyroptosis-Related Patterns Predict Tumor Immune Landscape and Immunotherapy Response in Bladder Cancer.
  31. RNA methylation-mediated LINC01559 suppresses colorectal cancer progression by regulating the miR-106b-5p/PTEN axis.
  32. IGF2BP1 Promotes Proliferation of Neuroendocrine Neoplasms by Post-Transcriptional Enhancement of EZH2.
  33. IMP1/IGF2BP1 alters extracellular vesicle cargo in colorectal cancer.
  34. Elucidating the Kinetic Mechanism of Human METTL16.
  35. Knockdown of ARL5B Induces Mitochondrial-Mediated Apoptosis and Inhibits Glycolysis in Breast Cancer Cells by Activating MDA5 Signaling.
  36. N6-Methyladenosine dynamic changes and differential methylation in wheat grain development.
  37. Down-expression of MiR30a-5p promotes proliferation and migration of Non-Small Cell Lung Cancer cells by targetting MTDH.
  38. E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway.
  39. Defining the role of RNA acetylation via a sequence-specific RNA acetyltransferase.
  40. FAM64A promotes HNSCC tumorigenesis by mediating transcriptional autoregulation of FOXM1.
  41. FTO Alleviates CdCl2-Induced Apoptosis and Oxidative Stress via the AKT/Nrf2 Pathway in Bovine Granulosa Cells.
  42. LncRNA-PACERR induces pro-tumour macrophages via interacting with miR-671-3p and m6A-reader IGF2BP2 in pancreatic ductal adenocarcinoma.
  43. THUMPD3-AS1 facilitates cell growth and aggressiveness by the miR-218-5p/SKAP1 axis in colorectal cancer.
  44. Modification of lysine deacetylation regulates curcumol-induced necroptosis through autophagy in hepatic stellate cells.
  45. Survival of HT29 cancer cells is influenced by hepatocyte growth factor receptor inhibition through modulation of self-DNA-triggered TLR9-dependent autophagy response.
  46. MicroRNA-19a-3p Acts as an Oncogene in Gastric Cancer and Exerts the Effect by Targeting SMOC2.
  1. Int J Biol Sci. 2022 ;18(7): 2744-2758
    The role of Insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs) as m6A readers in cancer.
    Chao-Yue Sun, Di Cao, Bin-Bin Du, Cun-Wu Chen, Dong Liu.
      RNA can be modified by over 170 types of distinct chemical modifications, and the most abundant internal modification of mRNA in eukaryotes is N6-methyladenosine (m6A). The m6A modification accelerates mRNA process, including mRNA splicing, translation, transcript stability, export and decay. m6A RNA modification is installed by methyltransferase-like proteins (writers), and potentially removed by demethylases (erasers), and this process is recognized by m6A-binding proteins (readers). Notably, alterations of m6A-modified proteins (writers, erasers and readers) are involved in the tumorigenesis, progression and metastasis. Importantly, the fate of m6A-methylated mRNA is mediated mostly through m6A readers, and among these readers, insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs) are unique RNA-binding proteins (RBPs) that stabilize their targets mRNA via m6A modification. In this review, we update the writers, erasers and readers, and their cross-talks in m6A modification, and briefly discuss the oncogenic role of IGF2BPs in cancer. Most importantly, we mainly review the up-to-date knowledges of IGF2BPs (IGF2BP1/2/3) as m6A readers in an m6A-modified manner in cancer progression.
    Keywords:  Cancer; IGF2BPs; N6-methyladenosine (m6A); Reader; Stabilization
    DOI:  https://doi.org/10.7150/ijbs.70458
  2. Bioengineered. 2022 May;13(5): 11923-11932
    N6-methyladenosine methyltransferase KIAA1429 elevates colorectal cancer aerobic glycolysis via HK2-dependent manner.
    Ying Li, Liang He, Yingkai Wang, Yan Tan, Fan Zhang.
      Emerging evidence has emphasized the critical roles played by N6-methyladenosine RNA (m6A) modification in colorectal carcinoma (CRC) initiation and progression. However, the roles and mechanism of m6A and KIAA1429 in CRC progression require further clarification. Here, our research aimed to investigate the functions of KIAA1429 in CRC tumorigenesis. Results indicated that KIAA1429 up-regulation closely correlated to the poor prognosis of CRC patients. Bio-functional assays demonstrated that KIAA1429 promoted the aerobic glycolysis, including glucose uptake, lactate production, ATP generation and extracellular acidification rate (ECAR). Mechanistically, KIAA1429 positively up-regulated HK2 level via increasing its mRNA stability by binding the m6A site of HK2 mRNA via m6A-independent manner. Collectively, our work indicates that KIAA1429 has the potential to promote CRC carcinogenesis by targeting HK2 via m6A-independent manner, providing insight into the critical roles of m6A in CRC.
    Keywords:  HK2; KIAA1429; N6-methyladenosine; aerobic glycolysis; colorectal cancer
    DOI:  https://doi.org/10.1080/21655979.2022.2065952
  3. FASEB J. 2022 May;36 Suppl 1
    METTL3 Enhances Hepatocellular Carcinoma Progression by Regulating Polycomb Repressive Complex 1 (PRC1) Components BMI1 and RNF2.
    Weina Chen, Jinqiang Zhang, Wenbo Ma, Chang Han, Tong Wu.
      N6-methyladenosine (m6A) modification is the most abundant mRNA modification which plays important roles in the regulation of mRNA stability, splicing, translocation, and translation. Methyltransferase-like 3 (METTL3), a primary RNA methyltransferase that catalyzes RNA m6A modification, is implicated in several human cancers including hepatocellular carcinoma (HCC), although its mechanisms of actions remain to be further defined. In the current study, we aimed to further determine the effect and mechanism of METTL3-derived m6A in HCC. We have analyzed the TCGA database and observed that METTL3 expression is significantly upregulated in HCC patients which is associated with lower survival rate (P < 0.001). In a mouse model of HCC induced by hydrodynamic tail vein injection of hyperactive form of YAP and β-catenin in conjunction with the sleeping beauty (SB) transposon system, we observed that the expression of METTL3 was notably high in YAP/β-catenin-induced HCC. In cultured human HCC cell lines (Huh7 and PLC/PRF/5), we observed that stable knockdown of METTL3 by shRNA significantly decreased tumor cell proliferation, colony formation and migration, in vitro. When Huh and PLC/PRF/5 cells with shRNA knockdown of METTL3 were inoculated into the livers of SCID mice, we found that METTL3 knockdown significantly inhibited xenograft tumor growth, in vivo. We next delivered METTL3 and YAP expression plasmids to the livers of wild type mice via hydrodynamic tail vein injection and observed that co-expression of METTL3 plus YAP induced the development of HCC which involves almost the entire livers (20 weeks after tail vein injection). These findings provide important evidence for a tumor-promoting role of METTL3 in HCC development. Through N6-methyladenosine-sequencing (m6A-Seq) and RNA sequencing (RNA-Seq), we identified BMI1 and RNF2, two key components of the polycomb repressive complex 1 (PRC1), as direct downstream targets of METTL3 in HCC. Our further analyses revealed that both BMI1 and RNF2 were significantly elevated in HCC patients and were associated with lower survival rate and that the expression of BMI1 and RNF2 were positively correlated with METTL3 in human HCC tissues. Consistent with the above results, our further data showed that knockdown of METTL3 in Huh7 and PLC/PRF/5 significantly decreased the expression of BMI1 and RNF2. Accordingly, treatment of Huh7 and PLC/PRF/5 cells with the METTL3 inhibitor, STM2457, significantly reduced the expression of BMI1 and RNF2 and decreased tumor cell proliferation and colony formation. Moreover, our data showed that inhibition of the m6A reader YTHDF1 by siRNA significantly decreased the expression of BMI1 and RNF2 in human HCC cells. Collectively, our study provides important evidence that METTL3 promotes HCC development and progression through m6A modification of BMI1 and RNF2 mRNAs which involve a YTHDF1 dependent mechanism. It is conceivable that the METTL3-m6A-BMI1/RNF2 axis may represent a promising target for HCC treatment.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3452
  4. Front Genet. 2022 ;13 864549
    m6A Methylation Patterns and Tumor Microenvironment Infiltration Characterization in Clear-Cell Renal Cell Carcinoma.
    Tianming Ma, Jiawen Wang, Xiaodong Liu, Wei Zhang, Lingfeng Meng, Yaoguang Zhang.
      Increasing evidence suggests the essential regulation of RNA N6-methyladenosine (m6A) modification in carcinogenesis and immune response. Nevertheless, the potential impacts of these modifications on the tumor microenvironment (TME) immune cell infiltration characteristics in clear-cell renal cell carcinoma (ccRCC) remain unclear. Utilizing a consensus clustering algorithm, we determined three m6A modification patterns and identified three m6A-related gene clusters among 569 ccRCC samples, which were associated with different biological functions and clinical outcomes. Thereafter, the m6A score was constructed using m6A-associated signature genes to accurately exploit the m6A modification patterns within individual tumors. The m6A score was further demonstrated to be noticeably related to ccRCC prognosis. In addition, the m6A score was found to be strongly correlated with tumor mutational burden (TMB), microsatellite instability, immune infiltration, immune checkpoint expression, and immunotherapy response, which was also validated in the pan-cancer analyses. Our findings thoroughly elucidated that m6A modification contributes to tumor microenvironment immune-infiltrating characteristics and prognosis in ccRCC. Assessing the m6A modification patterns of individual patients with ccRCC will offer novel insights into TME infiltration and help develop more effective treatment strategies.
    Keywords:  N6-methyladenosine; clear-cell renal cell carcinoma; immune checkpoint inhibitors; pan-cancer; tumor microenvironment
    DOI:  https://doi.org/10.3389/fgene.2022.864549
  5. IEEE/ACM Trans Comput Biol Bioinform. 2022 May 10. PP
    ELMo4m6A: a contextual language embedding-based predictor for detecting RNA N6-methyladenosine sites.
    Yongxian Fan, Guicong Sun, Xiaoyong Pan.
      N6-methyladenosine (m6A) is a universal post-transcriptional modification of RNAs, and it is widely involved in various biological processes. Identifying m6A modification sites accurately is indispensable to further investigate m6A-mediated biological functions. How to better represent RNA sequences is crucial for building effective computational methods for detecting m6A modification sites. However, traditional encoding methods require complex biological prior knowledge and are time-consuming. Furthermore, most of the existing m6A sites prediction methods are limited to single species, and few methods are able to predict m6A sites across different species and tissues. Thus, it is necessary to design a more efficient computational method to predict m6A sites across multiple species and tissues. In this paper, we proposed ELMo4m6A, a contextual language embedding-based method for predicting m6A sites from RNA sequences without any prior knowledge. ELMo4m6A first learns embeddings of RNA sequences using a language model ELMo, then uses a hybrid convolutional neural network (CNN) and long short-term memory (LSTM) to identify m6A sites. The results of 5-fold cross-validation and independent testing demonstrate that ELMo4m6A is superior to state-of-the-art methods. Moreover, we applied integrated gradients to find potential sequence patterns contributing to m6A sites.
    DOI:  https://doi.org/10.1109/TCBB.2022.3173323
  6. Front Genet. 2022 ;13 831162
    Construction of a Comprehensive Diagnostic Scoring Model for Prostate Cancer Based on a Novel Six-Gene Panel.
    Yunfeng Liu, Simei Qiu, Dongshan Sun, Ting Xiong, Qiuling Xiang, Quhuan Li.
      Accumulating evidence indicates that the N6-methyladenosine (m6A) modification plays a critical role in human cancers. Given the current understanding of m6A modification, this process is believed to be dynamically regulated by m6A regulators. Although the discovery of m6A regulators has greatly enhanced our understanding of the mechanism underlying m6A modification in cancers, the function and role of m6A in the context of prostate cancer (PCa) remain unclear. Here, we aimed to establish a comprehensive diagnostic scoring model that can act as a complement to prostate-specific antigen (PSA) screening. To achieve this, we first drew the landscape of m6A regulators and constructed a LASSO-Cox model using three risk genes (METTL14, HNRNP2AB1, and YTHDF2). Particularly, METTL14 expression was found to be significantly related to overall survival, tumor T stage, relapse rate, and tumor microenvironment of PCa patients, showing that it has important prognostic value. Furthermore, for the sake of improving the predictive ability, we presented a comprehensive diagnostic scoring model based on a novel 6-gene panel by combining with genes found in our previous study, and its application potential was further validated by the whole TCGA and ICGC cohorts. Our study provides additional clues and insights regarding the treatment and diagnosis of PCa patients.
    Keywords:  METTL14; diagnostic scoring model; m6A modification; prostate cancer; tumor microenvironment
    DOI:  https://doi.org/10.3389/fgene.2022.831162
  7. Front Aging Neurosci. 2022 ;14 890134
    Methyltransferase-Like 3 Rescues the Amyloid-beta protein-Induced Reduction of Activity-Regulated Cytoskeleton Associated Protein Expression via YTHDF1-Dependent N6-Methyladenosine Modification.
    Chenhaoyi Xu, Huanghuang Huang, Min Zhang, Pei Zhang, Zezhi Li, Xueyuan Liu, Min Fang.
      Activity-regulated cytoskeleton-associated protein (ARC) is activated by the induction of long-term potentiation and plays an important role in the synaptic plasticity of memory consolidation. Previous studies have shown that abnormal expression of ARC in the brains of patients with Alzheimer's Disease (AD) leads to the disturbance of synaptic plasticity. ARC expression is mainly regulated by transcriptional and post-translational modification. However, it is unclear whether N6-methyladenosine (m6A) engages in the epigenetic modification of ARC. The AlzData database was used to analyze the brain of AD patients, and Aβ-induced cell models were used. We revealed that ARC expression was reduced in AD patients and Aβ-induced cell models. There were five m6A modification sites of ARC mRNA that were predicted by the SRAMP database, and ARC mRNA was confirmed as the target gene of methyltransferase-like 3 (METTL3) by MeRIP. Amyloid-beta protein (Aβ) repressed the m6A modification. Knockdown of METTL3 decreased ARC mRNA m6A modification and reduced ARC protein expression, while overexpression of METTL3 rescued ARC expression after Aβ treatment. Knockdown of YTH domain family, member 1 (YTHDF1) decreased ARC protein expression, while the overexpression of YTHDF1 could not rescue the loss of ARC protein expression after 3-deazaadenosine treatment or knockdown of METTL3. Our findings identify that METTL3 rescues the Aβ-induced reduction of ARC expression via YTHDF1-Dependent m6A modification, which suggests an important mechanism of epigenetic alteration in AD.
    Keywords:  Alzheimer’s Disease (AD); N6-methyladenosine (m6A); YTH domain family; activity-regulated cytoskeleton-associated protein (ARC); member 1 (YTHDF1); methyltransferase-like 3 (METTL3)
    DOI:  https://doi.org/10.3389/fnagi.2022.890134
  8. Front Oncol. 2022 ;12 858694
    RNA Demethylase ALKBH5 Prevents Lung Cancer Progression by Regulating EMT and Stemness via Regulating p53.
    Xiangli Liu, Ziyi Wang, Qiwei Yang, Xiaohai Hu, Qiang Fu, Xinyu Zhang, Wenya Li.
       Background: Although N6-methyladenosine (m6A) RNA methylation is the most abundant reversible methylation of mRNA, which plays a critical role in regulating cancer processing, few studies have examined the role of m6A in nonsmall-cell lung cancer-derived cancer stem-like cells (CSCs).
    Methods: CSCs were enriched by culturing NSCLC cells in a serum-free medium, and stem factors, including CD24, CD44, ALDH1, Nanog, Oct4, and Sox2 were detected by Western blot. ALKBH5 expression was measured by employing a tissue array. Global m6A methylation was measured after ALKBH5 knockdown. Malignances of CSCs were detected by performing CCK-8 assay, invasion assay, cell cycle analysis, and tumor formation in vitro and in vivo.
    Results: m6A demethylase ALKBH5 is highly expressed in CSCs derived from NSCLC. Knockdown of ALKBH5 increased global m6A level, and also increased E-cadherin, decreased stem hallmarkers, Nanog and Oct4, and inhibited stemness of CSCs. In lung carcinoma, ALKBH5 is found to be positively correlated with p53 by using Gene Expression Profiling Interactive Analysis (GEPIA) online tool. P53 transcriptionally regulates ALKBH5 and subsequently regulates the global m6A methylation level. Knockdown of p53 or inhibition of p53's transcriptional activity by addition of its specific inhibitor PFT-α decreased expression of ALKBH5 and CSCs' malignancies, including proliferation, invasion, and tumor formation ability, indicating that p53 may partially regulate CSC's malignancies via ALKBH5. Furthermore, we also found p53 transcriptionally regulates PRRX1, which is consistent with our previous report.
    Conclusion: Collectively, our findings indicate the pivotal role of ALKBH5 in CSCs derived from NSCLC and highlight the regulatory function of the p53/ALKBH5 axis in modulating CSC progression, which could be a promising therapeutic target for NSCLC.
    Keywords:  ALKBH5; N6-methyladenosine (m6A) methylation; cancer stem-like cells (CSCs); epithelial and mesenchymal transition (EMT); nonsmall-cell lung cancer (NSCLC)
    DOI:  https://doi.org/10.3389/fonc.2022.858694
  9. FASEB J. 2022 May;36 Suppl 1
    RNA methylation in gene expression regulation.
    Chuan He.
      Over 150 types of post-transcriptional RNA modifications have been identified in all kingdoms of life. We have discovered two RNA demethylases, FTO and ALKBH5, which catalyze oxidative demethylation of the most prevalent modifications of mammalian messenger RNA (mRNA) and other nuclear RNA, N6 -methyladenosine (m6 A). These findings indicate that reversible RNA modification could impact biological regulation analogous to the well-known reversible DNA and histone chemical modifications. I will present recent discoveries on m6 A-mediated regulation of both transcriptional and post-transcriptional events. We found m6 A methylation of chromatin-associated regulatory RNAs (promoter-associated RNAs, enhancer RNAs, and repeat RNAs), or carRNAs. carRNA methylation regulates local chromatin state and downstream transcription. Regulatory pathways and underlying mechanisms will be presented.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.0I165
  10. Front Immunol. 2022 ;13 798583
    Roles of the m6A Modification of RNA in the Glioblastoma Microenvironment as Revealed by Single-Cell Analyses.
    Feng Yuan, Xiangming Cai, Zixiang Cong, Yingshuai Wang, Yuanming Geng, Yiliyaer Aili, Chaonan Du, Junhao Zhu, Jin Yang, Chao Tang, Aifeng Zhang, Sheng Zhao, Chiyuan Ma.
       Purpose: Glioblastoma multiforme (GBM) is a common and aggressive form of brain tumor. The N6-methyladenosine (m6A) mRNA modification plays multiple roles in many biological processes and disease states. However, the relationship between m6A modifications and the tumor microenvironment in GBM remains unclear, especially at the single-cell level.
    Experimental Design: Single-cell and bulk RNA-sequencing data were acquired from the GEO and TCGA databases, respectively. We used bioinformatics and statistical tools to analyze associations between m6A regulators and multiple factors.
    Results: HNRNPA2B1 and HNRNPC were extensively expressed in the GBM microenvironment. m6A regulators promoted the stemness state in GBM cancer cells. Immune-related BP terms were enriched in modules of m6A-related genes. Cell communication analysis identified genes in the GALECTIN signaling network in GBM samples, and expression of these genes (LGALS9, CD44, CD45, and HAVCR2) correlated with that of m6A regulators. Validation experiments revealed that MDK in MK signaling network promoted migration and immunosuppressive polarization of macrophage. Expression of m6A regulators correlated with ICPs in GBM cancer cells, M2 macrophages and T/NK cells. Bulk RNA-seq analysis identified two expression patterns (low m6A/high ICP and high m6A/low ICP) with different predicted immune infiltration and responses to ICP inhibitors. A predictive nomogram model to distinguish these 2 clusters was constructed and validated with excellent performance.
    Conclusion: At the single-cell level, m6A modification facilitates the stemness state in GBM cancer cells and promotes an immunosuppressive microenvironment through ICPs and the GALECTIN signaling pathway network. And we also identified two m6A-ICP expression patterns. These findings could lead to novel treatment strategies for GBM patients.
    Keywords:  cell communication; glioblastoma; immune microenvironment; m6A; single-cell analysis
    DOI:  https://doi.org/10.3389/fimmu.2022.798583
  11. Mol Carcinog. 2022 May 13.
    Role of RNA modifications in carcinogenesis and carcinogen damage response.
    Michelle Verghese, Emma Wilkinson, Yu-Ying He.
      The field of epitranscriptomics encompasses the study of post-transcriptional RNA modifications and their regulatory enzymes. Among the numerous RNA modifications, N6 -methyladenosine (m6 A) has been identified as the most common internal modification of messenger RNA (mRNA). Although m6 A modifications were first discovered in the 1970s, advances in technology have revived interest in this field, driving an abundance of research into the role of RNA modifications in various biological processes, including cancer. As analogs to epigenetic modifications, RNA modifications also play an important role in carcinogenesis by regulating gene expression post-transcriptionally. A growing body of evidence suggests that carcinogens can modulate RNA modifications to alter the expression of oncogenes or tumor suppressors during cellular transformation. Additionally, the expression and activity of the enzymes that regulate RNA modifications can be dysregulated and contribute to carcinogenesis, making these enzymes promising targets of drug discovery. Here we summarize the roles of RNA modifications during carcinogenesis induced by exposure to various environmental carcinogens, with a main focus on the roles of the most widely studied m6 A mRNA methylation.
    Keywords:  DNA damage response; arsenic; carcinogenesis.; chemical carcinogens; environmental carcinogens; epitranscriptomics; m6A; metal; virus
    DOI:  https://doi.org/10.1002/mc.23418
  12. Ann Transl Med. 2022 Apr;10(7): 417
    Downregulated ALKBH5 contributes to myocardial ischemia/reperfusion injury by increasing m6A modification of Trio mRNA.
    Jiaxin Li, Jieshan Chen, Mingyi Zhao, Zhetao Li, Nanbo Liu, Heng Fang, Miaoxian Fang, Ping Zhu, Liming Lei, Chunbo Chen.
       Background: The modification of N6-methyladenosine (m6A) is a dynamic and reversible course that might play a role in cardiovascular disease. However, the mechanisms of m6A modification in myocardial ischemia/reperfusion injury (MIRI) remain unclear.
    Methods: A mouse model of MIRI and a cell model of oxygen-glucose deprivation/reperfusion (OGD/R) HL-1 cells were employed. In an in vivo study, the total RNA m6A modification levels were determined by dot blot, and the key genes related to m6A modification were screened by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. In an in vitro study, the effects of AlkB homolog 5 (ALKBH5), an RNA demethylase, on cell proliferation, cell injury, and apoptosis were detected by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, lactate dehydrogenase (LDH) and cardiac troponin-I (cTnI) levels, and flow cytometry. Besides, the m6A modification-changed and differentially expressed messenger RNA (mRNA) were determined by methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) in ALKBH5-overexpressed HL-1 cells. Finally, the mRNA levels of the promising targeted gene were examined by RT-qPCR and its m6A modification levels were examined by MeRIP-qPCR.
    Results: Our results showed that RNA m6A modification was involved in MIRI, in which ALKBH5 was downregulated. Functionally, by overexpressing or silencing ALKBH5 in experimental cells, we verified its protective properties on cell proliferation, cell injury, and apoptosis in the process of MIRI. Besides, we provided a mass of latent different mRNAs with m6A modification variation in ALKBH5-overexpressed HL-1 cells. Mechanistically, we further screened the most potential targeted mRNAs and suggested that triple functional domain (Trio) mRNA could be upregulated by ALKBH5 by reducing m6A level of Trio.
    Conclusions: This study demonstrated that the downregulated ALKBH5 might contribute to MIRI process by increasing the m6A modification of Trio mRNA and downregulating Trio.
    Keywords:  AlkB homolog 5 (ALKBH5); N6-methyladenosine modification (m6A modification); myocardial ischemia/reperfusion injury (MIRI)
    DOI:  https://doi.org/10.21037/atm-22-1289
  13. Mol Cancer. 2022 May 10. 21(1): 111
    N6-methyladenosine-modified TRAF1 promotes sunitinib resistance by regulating apoptosis and angiogenesis in a METTL14-dependent manner in renal cell carcinoma.
    Yuanlei Chen, Zeyi Lu, Chao Qi, Chenhao Yu, Yang Li, Wang Huan, Ruyue Wang, Wenqin Luo, Danyang Shen, Lifeng Ding, Liangliang Ren, Haiyun Xie, Dingwei Xue, Mingchao Wang, Kangxin Ni, Liqun Xia, Jun Qian, Gonghui Li.
       BACKGROUND: Sunitinib resistance can be classified into primary and secondary resistance. While accumulating research has indicated several underlying factors contributing to sunitinib resistance, the precise mechanisms in renal cell carcinoma are still unclear.
    METHODS: RNA sequencing and m6A sequencing were used to screen for functional genes involved in sunitinib resistance. In vitro and in vivo experiments were carried out and patient samples and clinical information were obtained for clinical analysis.
    RESULTS: We identified a tumor necrosis factor receptor-associated factor, TRAF1, that was significantly increased in sunitinib-resistant cells, resistant cell-derived xenograft (CDX-R) models and clinical patients with sunitinib resistance. Silencing TRAF1 increased sunitinib-induced apoptotic and antiangiogenic effects. Mechanistically, the upregulated level of TRAF1 in sunitinib-resistant cells was derived from increased TRAF1 RNA stability, which was caused by an increased level of N6-methyladenosine (m6A) in a METTL14-dependent manner. Moreover, in vivo adeno-associated virus 9 (AAV9) -mediated transduction of TRAF1 suppressed the sunitinib-induced apoptotic and antiangiogenic effects in the CDX models, whereas knockdown of TRAF1 effectively resensitized the sunitinib-resistant CDXs to sunitinib treatment.
    CONCLUSIONS: Overexpression of TRAF1 promotes sunitinib resistance by modulating apoptotic and angiogenic pathways in a METTL14-dependent manner. Targeting TRAF1 and its pathways may be a novel pharmaceutical intervention for sunitinib-treated patients.
    Keywords:  METTL14; N6-methyladenosine; RCC; Sunitinib-resistance; TRAF1
    DOI:  https://doi.org/10.1186/s12943-022-01549-1
  14. Cell Rep. 2022 May 10. pii: S2211-1247(22)00584-8. [Epub ahead of print]39(6): 110813
    SRSF3-mediated regulation of N6-methyladenosine modification-related lncRNA ANRIL splicing promotes resistance of pancreatic cancer to gemcitabine.
    Zu-Wei Wang, Jing-Jing Pan, Jian-Fei Hu, Jia-Qiang Zhang, Long Huang, Yi Huang, Cheng-Yu Liao, Can Yang, Zhi-Wen Chen, Yao-Dong Wang, Bai-Yong Shen, Yi-Feng Tian, Shi Chen.
      Serine/arginine-rich splicing factor 3 (SRSF3) regulates mRNA alternative splicing of more than 90% of protein-coding genes, providing an essential source for biological versatility. This study finds that SRSF3 expression is associated with drug resistance and poor prognosis in pancreatic cancer. We also find that SRSF3 regulates ANRIL splicing and m6A modification of ANRIL in pancreatic cancer cells. More importantly, we demonstrate that m6A methylation on lncRNA ANRIL is essential for the splicing. Moreover, our results show that SRSF3 promotes gemcitabine resistance by regulating ANRIL's splicing and ANRIL-208 (one of the ANRIL spliceosomes) can enhance DNA homologous recombination repair (HR) capacity by forming a complex with Ring1b and EZH2. In conclusion, this study establishes a link between SRSF3, m6A modification, lncRNA splicing, and DNA HR in pancreatic cancer and demonstrates that abnormal alternative splicing and m6A modification are closely related to chemotherapy resistance in pancreatic cancer.
    Keywords:  ANRIL; CP: Cancer; CP: Molecular biology; DNA repair; N6-methyladenosine; SRSF3; chemoresistance; pancreatic cancer
    DOI:  https://doi.org/10.1016/j.celrep.2022.110813
  15. Dev Comp Immunol. 2022 May 10. pii: S0145-305X(22)00096-9. [Epub ahead of print] 104434
    Global N6-methyladenosine methylation analysis reveals the positive correlation between m6A modification and mRNA abundance during Apostichopus japonicus disease development.
    Yina Shao, Xuemei Duan, Xuelin Zhao, Zhimeng Lv, Chenghua Li.
      N6-methyladenosine (m6A), the most abundant epitranscriptomic modification in eukaryotic messenger RNA (mRNA), plays important roles in regulation of gene expression for fundamental biological processes and diverse physiological functions, including combating with pathogen infection. Here, we were first profile transcriptome-wide m6A sequencing in four stages of skin ulceration syndrome-diseased Apostichopus japonicus following Vibrio splendidus infection, including Control (healthy), Early (small ulcer), Later (extensive ulcer), and Resistant (no ulcer) groups. Our results revealed that three experimental groups were all extensively methylated by m6A and the proportion of the m6A modified genes were also significantly increased to 28.90% (Early), 27.97% (Later), and 29.98% (Resistant) when compared with Control group (15.15%), indicating m6A modification could be induced by V. splendidus infection. Intriguingly, we discovered a positive correlation between the m6A methylation level and mRNA abundance, indicating a positive regulatory role of m6A in sea cucumber gene expression during V. splendidus infection. Moreover, genes with specific and differentially expressed m6A methylation in Later group were both enriched in cell adhesion, while Early and Resistant groups were both mainly involved in DNA conformation change and chromosome organization when compared with Control, suggesting the higher-methylated m6A might serve as "conformational marker" and associated to the initiation of related anti-disease genes transcription in order to improve disease resistance of sea cucumber. Subsequently, we selected the pivotal genes enriched in cell adhesion pathway and found that the IggFc-binding protein (FcGBP) and Fibrocystin-L both had higher levels of m6A methylation and higher level of mRNA expressions in Later group. Conversely, Fibrinogen C domain-containing protein 1 (F1BCD1) gene presented as an antibacterial role in sea cucumber and showed higher mRNA expression and higher m6A methylation in Resistant group and lower mRNA level in Later group. The levels of m6A methylation and mRNA abundance of FcGBP and F1BCD1 genes indicates disease occurrence or disease resistant were also verified by MeRIP-qPCR. Overall, our study presents the first comprehensive characterize of dynamic m6A methylation modification in the different stages of disease in sea cucumber. These data provide an invaluable resource for future studies of function and biological significance of m6A in mRNA in marine invertebrates.
    Keywords:  Apostichopus japonicus; N(6)-methyladenosine; RNA methylation; Skin ulceration syndrome
    DOI:  https://doi.org/10.1016/j.dci.2022.104434
  16. Bioengineered. 2022 May;13(5): 11832-11843
    Methyltransferase-like 14 silencing relieves the development of atherosclerosis via m6A modification of p65 mRNA.
    Yingjie Liu, Gang Luo, Quan Tang, Yang Song, Daxing Liu, Hongjuan Wang, Junliang Ma.
      To explore the METTL14-dependent m6A modification mechanism involved in the development of atherosclerosis. Oxidized low-density lipoprotein (ox-LDL) and the HUVEC cell line were used to establish an atherosclerosis cell model in vitro, and APOE-/- mice fed a high-fat diet were used as the animal model. Cell viability and apoptosis were assessed using MTT assays and flow cytometry. The status of m6A in HUVECs was examined using MeRIP-qPCR. Oil Red O staining was used to evaluate the lesions or plaques on aortas separated from the target mice. METTL14 and METTL3 were upregulated in HUVECs after ox-LDL treatment. After transfection with si-METTL14, the bcl-2 expression level and the viability of ox-LDL-incubated cells increased, whereas the apoptosis rate and the expressions of Bax and cleaved caspase-3 decreased. However, the effect of METTL14 knockdown was reversed by p65 overexpression. After METTL14 knockdown, there was a decrease in the total m6A content in HUVECs, m6A modification, and p65 expression. The plaques and lesion areas on the high-fat diet APOE-/- mouse aortas were smaller after METTL14 silencing. METTL14 reduced cell viability and promoted apoptosis of HUVECs, which were both induced by ox-LDL via m6A modification of p65. Knocking down METTL14 could inhibit the development of atherosclerosis in high-fat diet-treated APOE-/- mice.
    Keywords:  METTL14; aorta; atherosclerosis; m6A; p65
    DOI:  https://doi.org/10.1080/21655979.2022.2031409
  17. Biomed Res Int. 2022 ;2022 2677312
    Emerging Roles of FTO in Neuropsychiatric Disorders.
    Rui Chang, Zeyi Huang, Size Zhao, Ju Zou, Yukun Li, Sijie Tan.
      FTO (fat mass and obesity associated) is a recently discovered gene related to obesity and expressed in various tissues of the human body, especially with high expression in the brain. Earlier studies have found that FTO is involved in several biological processes, including brain development and function. In particular, recent studies have found that FTO is a demethylase of N6-methyladenosine (m6A) and it can affect neurological function through the m6A modification of mRNA. At present, a number of studies have shown that FTO is associated with many neuropsychiatric disorders. This paper reviews the discovery, structure, function, and tissue expression of FTO followed by discussing the relationship between FTO and neuropsychiatric diseases. In addition, the potential roles of FTO gene in drug addiction, major depression (MDD), and schizophrenia (SCZ) through regulating m6A modification of dopamine related genes were also highlighted.
    DOI:  https://doi.org/10.1155/2022/2677312
  18. J Transl Med. 2022 May 13. 20(1): 214
    5-methylcytosine RNA methyltransferases and their potential roles in cancer.
    Mingyang Li, Zijia Tao, Yiqiao Zhao, Lei Li, Jianyi Zheng, Zeyu Li, Xiaonan Chen.
      In recent years, 5-methylcytosine (m5C) RNA modification has emerged as a key player in regulating RNA metabolism and function through coding as well as non-coding RNAs. Accumulating evidence has shown that m5C modulates the stability, translation, transcription, nuclear export, and cleavage of RNAs to mediate cell proliferation, differentiation, apoptosis, stress responses, and other biological functions. In humans, m5C RNA modification is catalyzed by the NOL1/NOP2/sun (NSUN) family and DNA methyltransferase 2 (DNMT2). These RNA modifiers regulate the expression of multiple oncogenes such as fizzy-related-1, forkhead box protein C2, Grb associated-binding protein 2, and TEA domain transcription factor 1, facilitating the pathogenesis and progression of cancers. Furthermore, the aberrant expression of methyltransferases have been identified in various cancers and used to predict the prognosis of patients. In this review, we present a comprehensive overview of m5C RNA methyltransferases. We specifically highlight the potential mechanism of action of m5C in cancer. Finally, we discuss the prospect of m5C-relative studies.
    Keywords:  5-methylcytosine; Cancer; Molecular mechanisms; Prognosis; RNA methylation; RNA methyltransferases
    DOI:  https://doi.org/10.1186/s12967-022-03427-2
  19. Cells. 2022 Apr 26. pii: 1461. [Epub ahead of print]11(9):
    Multiple Phosphorylations of SR Protein SRSF3 and Its Binding to m6A Reader YTHDC1 in Human Cells.
    Takanori Tatsuno, Yasuhito Ishigaki.
      N6-methyladenosine (m6A) is a well-known RNA modification and has various functions with its binding proteins. Nuclear m6A reader protein YTHDC1 plays a significant role in RNA metabolism including some non-coding RNA such as LINE or circRNA. It is also known to regulate mRNA splicing through recruiting SRSF3 to the targeted mRNAs, which then mediates export of YTHDC1-bound RNA to the cytoplasm. Additionally, it has been indicated that SRSF3 binding to YHTDC1 may be mediated by its dephosphorylated status. However, their binding mechanism, including the positions of dephosphorylated residues of SRSF3, has not been sufficiently investigated. Thus, we explored the mechanism of interaction between SRSF3 and YTHDC1 in human cells. We used co-immunoprecipitation to examine the binding of YTHDC1/SRSF3 through their N- and C-terminal amino-acid residues. Furthermore, dephosphorylation-mimic serine to alanine mutants of SRSF3 indicated the position of phosphorylated residues. Cumulatively, our results demonstrate that YTHDC1 binding to SRSF3 is regulated by not only hypo-phosphorylated residues of arginine/serine-rich (RS) domain of SRSF3 but also other parts of SRSF3 via YTHDC1 N- or C-terminal residues. Our results contribute to the understanding of the complex mechanism of binding between SR protein SRSF3 and the m6A reader YTHDC1 to regulate the expression of mRNA and non-coding RNAs.
    Keywords:  N6-methyladenosine; SRSF3; YTHDC1; phosphorylation
    DOI:  https://doi.org/10.3390/cells11091461
  20. Cell Biosci. 2022 May 07. 12(1): 55
    PA2G4 promotes the metastasis of hepatocellular carcinoma by stabilizing FYN mRNA in a YTHDF2-dependent manner.
    Sheng Sun, Yiyang Liu, Meiling Zhou, Jinyuan Wen, Lin Xue, Shenqi Han, Junnan Liang, Yufei Wang, Yi Wei, Jinjin Yu, Xin Long, Xiaoping Chen, Huifang Liang, Zhao Huang, Bixiang Zhang.
       BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide with high mortality. Advanced stage upon diagnosis and cancer metastasis are the main reasons for the dismal prognosis of HCC in large part. The role of proliferation associated protein 2G4 (PA2G4) in tumorigenesis and cancer progression has been widely investigated in various cancers. However, whether and how PA2G4 participates in HCC metastasis is still underexplored.
    RESULTS: We found that the mRNA and protein levels of PA2G4 were higher in HCC samples than in normal liver tissues, and high expression of PA2G4 in HCC was correlated with a poor prognosis, by an integrative analysis of immunohistochemistry (IHC), western blot and bioinformatic approach. Moreover, the expression of PA2G4 was elevated in HCC patients with metastases than those metastasis-free. Cell migration, invasion, phalloidin staining and western blot analyses demonstrated that PA2G4 promoted epithelial to mesenchymal transition (EMT) of HCC cells in vitro. And a lung metastasis animal model exhibited that PA2G4 enhanced metastatic ability of HCC cells in vivo. RNA-sequencing combined with dual luciferase reporter assay and evaluation of mRNA half-time indicated that PA2G4 increased FYN expression by stabilizing its mRNA transcript. Recovering the impaired FYN level induced by PA2G4 knockdown rescued the impeded cell mobilities. Furthermore, endogenous immunoprecipitation (IP) and in-situ immunofluorescence (IF) showed that YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) was the endogenous binding patterner of PA2G4. In addition, RNA binding protein immunoprecipitation (RIP) and anti- N6-methyladenosine immunoprecipitation (MeRIP) assays demonstrated that FYN mRNA was N6-methyladenosine (m6A) modified and bound with PA2G4, as well as YTHDF2. Moreover, the m6A catalytic ability of YTHDF2 was found indispensable for the regulation of FYN by PA2G4. At last, the correlation of expression levels between PA2G4 and FYN in HCC tissues was verified by IHC and western blot analysis.
    CONCLUSIONS: These results indicate that PA2G4 plays a pro-metastatic role by increasing FYN expression through binding with YTHDF2 in HCC. PA2G4 may become a reliable prognostic marker or therapeutic target for HCC patients.
    Keywords:  FYN; HCC; PA2G4; YTHDF2; m6A
    DOI:  https://doi.org/10.1186/s13578-022-00788-5
  21. Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2022 Jan 28. pii: 1672-7347(2022)01-0109-07. [Epub ahead of print]47(1): 109-115
    Roles of m6A modification in neurological diseases.
    Yuan Zhang, Si Zhang, Mengmeng Shi, Menglin Li, Jiayu Zeng, Jie He.
      N6-methyladenosine (m6A) methylation modification is one of the most common epigenetic modifications for eukaryotic mRNA. Under the catalytic regulation of relevant enzymes, m6A participates in the body's pathophysiological processes via mediating RNA transcription, splicing, translation, and decay. In the past, we mainly focused on the regulation of m6A in tumors such as hematological tumors, cervical cancer, breast cancer. In recent years, it has been found that m6A is enriched in mRNAs of neurogenesis, cell cycle, and neuron differentiation. Its regulation in the nervous system is gradually being recognized. When the level of m6A modification and the expression levels of relevant enzyme proteins are changed, it will cause neurological dysfunction and participate in the occurrence and conversion of neurological diseases. Recent studies have found that the m6A modification and its associated enzymes were involved in major depressive disorder, Parkinson's disease, Alzheimer's disease, Fragile X syndrome, amyotrophic lateral sclerosis, and traumatic brain injury, and they also play a key role in the development of neurological diseases and many other neurological diseases. This paper mainly reviewed the recent progress of m6A modification-related enzymes, focusing on the impact of m6A modification and related enzyme-mediated regulation of gene expression on the central nervous system diseases, so as to provide potential targets for the prevention of neurological diseases.
    Keywords:  N6-methyladenosine; major depressive disorder; neurodegenerative diseases
    DOI:  https://doi.org/10.11817/j.issn.1672-7347.2022.200990
  22. Cells. 2022 Apr 30. pii: 1516. [Epub ahead of print]11(9):
    The Role of N6-Methyladenosine in the Promotion of Hepatoblastoma: A Critical Review.
    Finn Morgan Auld, Consolato M Sergi, Roger Leng, Fan Shen.
      Hepatoblastoma (HB) is a rare primary malignancy of the developing fetal liver. Its course is profoundly influenced by genetics, in the context of sporadic mutation or genetic syndromes. Conventionally, subtypes of HB are histologically determined based on the tissue type that is recapitulated by the tumor and the direction of its differentiation. This classification is being reevaluated based on advances on molecular pathology. The therapeutic approach comprises surgical intervention, chemotherapy (in a neoadjuvant or post-operative capacity), and in some cases, liver transplantation. Although diagnostic modalities and treatment options are evolving, some patients experience complications, including relapse, metastatic spread, and suboptimal response to chemotherapy. As yet, there is no consistent framework with which such outcomes can be predicted. N6-methyladenosine (m6A) is an RNA modification with rampant involvement in the normal processing of cell metabolism and neoplasia. It has been observed to impact the development of a variety of cancers via its governance of gene expression. M6A-associated genes appear prominently in HB. Literature data seem to underscore the role of m6A in promotion and clinical course of HB. Illuminating the pathogenetic mechanisms that drive HB are promising additions to the understanding of the clinically aggressive tumor behavior, given its potential to predict disease course and response to therapy. Implicated genes may also act as targets to facilitate the evolving personalized cancer therapy. Here, we explore the role of m6A and its genetic associates in the promotion of HB, and the impact this may have on the management of this neoplastic disease.
    Keywords:  N6-Methyladenosine; beta-catenin; hepatoblastoma; hepatoblastoma genetics; m6A; methyltransferase
    DOI:  https://doi.org/10.3390/cells11091516
  23. Int J Mol Sci. 2022 Apr 20. pii: 4522. [Epub ahead of print]23(9):
    Genome-Wide Identification, Classification and Expression Analysis of m6A Gene Family in Solanum lycopersicum.
    Hui Shen, Baobing Luo, Yunshu Wang, Jing Li, Zongli Hu, Qiaoli Xie, Ting Wu, Guoping Chen.
      Advanced knowledge of messenger RNA (mRNA) N6-methyladenosine (m6A) and DNA N6-methyldeoxyadenosine (6 mA) redefine our understanding of these epigenetic modifications. Both m6A and 6mA carry important information for gene regulation, and the corresponding catalytic enzymes sometimes belong to the same gene family and need to be distinguished. However, a comprehensive analysis of the m6A gene family in tomato remains obscure. Here, 24 putative m6A genes and their family genes in tomato were identified and renamed according to BLASTP and phylogenetic analysis. Chromosomal location, synteny, phylogenetic, and structural analyses were performed, unravelling distinct evolutionary relationships between the MT-A70, ALKBH, and YTH protein families, respectively. Most of the 24 genes had extensive tissue expression, and 9 genes could be clustered in a similar expression trend. Besides, SlYTH1 and SlYTH3A showed a different expression pattern in leaf and fruit development. Additionally, qPCR data revealed the expression variation under multiple abiotic stresses, and LC-MS/MS determination exhibited that the cold stress decreased the level of N6 2'-O dimethyladenosine (m6Am). Notably, the orthologs of newly identified single-strand DNA (ssDNA) 6mA writer-eraser-reader also existed in the tomato genome. Our study provides comprehensive information on m6A components and their family proteins in tomato and will facilitate further functional analysis of the tomato N6-methyladenosine modification genes.
    Keywords:  ALKBH; MT-A70; YTH; m6A; m6Am; tomato
    DOI:  https://doi.org/10.3390/ijms23094522
  24. Front Genet. 2022 ;13 862972
    Comprehensive Analysis of N6-Methyladenosine Regulators in the Subcluster Classification and Drug Candidates Prediction of Severe Obstructive Sleep Apnea.
    Niannian Li, Zhenfei Gao, Jinhong Shen, Yuenan Liu, Kejia Wu, Jundong Yang, Shengming Wang, Xiaoman Zhang, Yaxin Zhu, Jingyu Zhu, Jian Guan, Feng Liu, Shankai Yin.
      Background: Obstructive sleep apnea (OSA) is the most common type of sleep apnea that impacts the development or progression of many other disorders. Abnormal expression of N6-methyladenosine (m6A) RNA modification regulators have been found relating to a variety of human diseases. However, it is not yet known if m6A regulators are involved in the occurrence and development of OSA. Herein, we aim to explore the impact of m6A modification in severe OSA. Methods: We detected the differentially expressed m6A regulators in severe OSA microarray dataset GSE135917. The least absolute shrinkage and selection operator (LASSO) and support vector machines (SVM) were used to identify the severe OSA-related m6A regulators. Receiver operating characteristic (ROC) curves were performed to screen and verify the diagnostic markers. Consensus clustering algorithm was used to identify m6A patterns. And then, we explored the character of immune microenvironment, molecular functionals, protein-protein interaction networks and miRNA-TF coregulatory networks for each subcluster. Finally, the Connectivity Map (CMap) tools were used to tailor customized treatment strategies for different severe OSA subclusters. An independent dataset GSE38792 was used for validation. Results: We found that HNRNPA2B1, KIAA1429, ALKBH5, YTHDF2, FMR1, IGF2BP1 and IGF2BP3 were dysregulated in severe OSA patients. Among them, IGF2BP3 has a high diagnostic value in both independent datasets. Furthermore, severe OSA patients can be accurately classified into three m6A patterns (subcluster1, subcluster2, subcluster3). The immune response in subcluster3 was more active because it has high M0 Macrophages and M2 Macrophages infiltration and up-regulated human leukocyte antigens (HLAs) expression. Functional analysis showed that representative genes for each subcluster in severe OSA were assigned to histone methyltransferase, ATP synthesis coupled electron transport, virus replication, RNA catabolic, multiple neurodegeneration diseases pathway, et al. Moreover, our finding demonstrated cyclooxygenase inhibitors, several of adrenergic receptor antagonists and histamine receptor antagonists might have a therapeutic effect on severe OSA. Conclusion: Our study presents an overview of the expression pattern and crucial role of m6A regulators in severe OSA, which may provide critical insights for future research and help guide appropriate prevention and treatment options.
    Keywords:  RNA methylation; consensus clustering; immunity; obstructive sleep apnea; pharmacological intervention
    DOI:  https://doi.org/10.3389/fgene.2022.862972
  25. Int J Mol Sci. 2022 Apr 22. pii: 4630. [Epub ahead of print]23(9):
    Profiling of Transcriptome-Wide N6-Methyladenosine (m6A) Modifications and Identifying m6A Associated Regulation in Sperm Tail Formation in Anopheles sinensis.
    Congshan Liu, Jianping Cao, Haobing Zhang, Jiatong Wu, Jianhai Yin.
      Recent discoveries of reversible N6-methyladenosine (m6A) methylation on messenger RNA (mRNA) and mapping of m6A methylomes in many species have revealed potential regulatory functions of this RNA modification by m6A players-writers, readers, and erasers. Here, we first profile transcriptome-wide m6A in female and male Anopheles sinensis and reveal that m6A is also a highly conserved modification of mRNA in mosquitoes. Distinct from mammals and yeast but similar to Arabidopsis thaliana, m6A in An. sinensis is enriched not only around the stop codon and within 3'-untranslated regions but also around the start codon and 5'-UTR. Gene ontology analysis indicates the unique distribution pattern of m6A in An. sinensis is associated with mosquito sex-specific pathways such as tRNA wobble uridine modification and phospholipid-binding in females, and peptidoglycan catabolic process, exosome and signal recognition particle, endoplasmic reticulum targeting, and RNA helicase activity in males. The positive correlation between m6A deposition and mRNA abundance indicates that m6A can play a role in regulating gene expression in mosquitoes. Furthermore, many spermatogenesis-associated genes, especially those related to mature sperm flagellum formation, are positively modulated by m6A methylation. A transcriptional regulatory network of m6A in An. sinensis is first profiled in the present study, especially in spermatogenesis, which may provide a new clue for the control of this disease-transmitting vector.
    Keywords:  Anopheles sinensis; epigenetics; m6A; sex-specific; sperm tail; spermatogenesis
    DOI:  https://doi.org/10.3390/ijms23094630
  26. J Cell Sci. 2022 May 13. pii: jcs.259824. [Epub ahead of print]
    ALKBH5 regulates somatic cell reprogramming in a phase specific manner.
    Sherif Khodeer, Arne Klungland, John Arne Dahl.
      Establishment of the pluripotency regulatory network in somatic cells by introducing four transcription factors (octamer binding transcription factor 4 (OCT4), sex determining region Y (SRY)-box 2 (SOX2), Kruppel-like factor 4 (KLF4), and cellular myelocytomatosis (c-MYC)) provides a promising tool for cell-based therapies in regenerative medicine. Nevertheless, the mechanisms at play when generating induced pluripotent stem cells from somatic cells are only partly understood. Here, we show that the RNA specific N6-methyladenosine (m6A) demethylase ALKBH5 regulates somatic cell reprogramming in a stage-specific manner through its catalytic activity. Knockdown or knockout of Alkbh5 in the early reprogramming phase impairs reprogramming efficiency by reducing the proliferation rate through arresting the cells at G2/M phase and decreasing the upregulation of epithelial markers. On the other hand, ALKBH5 overexpression at the early reprogramming phase has no significant impact on reprogramming efficiency, while overexpression at the late phase enhances reprogramming by stabilizing Nanog transcripts, resulting in upregulated Nanog expression. Our study provides mechanistic insight into the crucial dynamic role of ALKBH5 through its catalytic activity in regulating somatic cell reprogramming at the posttranscriptional level.
    Keywords:  Alkbh5; Nanog; Reprogramming; induced pluripotent stem cells (iPSCs)
    DOI:  https://doi.org/10.1242/jcs.259824
  27. Genomics Proteomics Bioinformatics. 2022 May 09. pii: S1672-0229(22)00040-7. [Epub ahead of print]
    Characteristics of N6-methyladenosine modification during sexual reproduction of Chlamydomonas reinhardtii.
    Ying Lv, Fei Han, Mengxia Liu, Ting Zhang, Guanshen Cui, Jiaojiao Wang, Ying Yang, Yun-Gui Yang, Wenqiang Yang.
      The unicellular green alga Chlamydomonas reinhardtii (hereafter Chlamydomonas) possesses both plant and animal attributes, and it is an ideal model organism for studies of fundamental processes such as photosynthesis, sexual reproduction, and the life cycle. N6-methyladenosine (m6A) is the most prevalent mRNA modification, and it plays important roles during sexual reproduction in animals and plants. However, the pattern and function of m6A modification during the sexual reproduction of Chlamydomonas remain unknown. Here, we performed transcriptome and methylated RNA immunoprecipitation sequencing (MeRIP) sequencing on six samples from different stages during sexual reproduction of the Chlamydomonas life cycle. The results showed that m6A modification frequently occurs at the main motif of DRAC (D = G/A/U, R = A/G) in Chlamydomonas mRNAs. Moreover, m6A peaks in Chlamydomonas mRNAs are mainly enriched in the 3' untranslated regions (3' UTR) and negatively correlated with the abundance of transcripts at each stage. In particular, there is a significant negative correlation between expression levels and m6A levels of genes in the microtubule-associated pathway, indicating that m6A modification influences the sexual reproduction and life cycle of Chlamydomonas by regulating microtubule-based movement. In summary, our findings are the first to demonstrate the distributions and the functions of m6A modification in Chlamydomonas mRNAs and provide new evolutionary insights into m6A modification in the process of sexual reproduction in other plant organisms.
    Keywords:  Chlamydomonas reinhardtii; Microtubule-associated pathway; N(6)-methyladenosine (m(6)A); Photosynthesis; Sexual reproduction; m(6)A sequencing
    DOI:  https://doi.org/10.1016/j.gpb.2022.04.004
  28. FASEB J. 2022 May;36 Suppl 1
    Activity-Based Profiling of RNA Modifying Enzymes.
    Ralph Kleiner.
      Epitranscriptomic RNA modifications can regulate RNA activity, however there remains a major gap in our understanding of the scope of RNA chemistry present in biological systems, the enzymes responsible for regulating modification levels, and the ultimate function of these modifications in biological processes. To address this gap, we have developed RNA-mediated activity-based protein profiling (RNABPP), a chemoproteomic strategy relying upon metabolic RNA labeling with modified nucleoside probes, RNA-protein enrichment, and quantitative proteomics. We apply RNABPP with fluoropyrimidine nucleosides in human cells in order to investigate RNA pyrimidine-modifying enzymes acting upon mRNA. In addition to profiling 5-methylcytidine (m5 C) and 5-methyluridine (m5 U) methyltransferase activity on mRNA, we identify a novel mechanism-based RNA-protein crosslink between 5-fluorouridine-modified RNA and the uncharacterized human dihydrouridine synthase DUS3L. Further, we study the cellular substrates of DUS3L using quantitative nucleoside LC-MS/MS and catalytic cross-linking and immunoprecipitation-based sequencing. Taken together, our work provides a general approach for profiling RNA modifying enzyme activity in vivo, and reveals the existence of new pathways for the epitranscriptomic regulation of RNA behavior in human cells.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.0R330
  29. BMC Genomics. 2022 May 11. 23(1): 358
    The transcriptome-wide N6-methyladenosine (m6A) map profiling reveals the regulatory role of m6A in the yak ovary.
    Shaoke Guo, Xingdong Wang, Mengli Cao, Xiaoyun Wu, Lin Xiong, Pengjia Bao, Min Chu, Chunnian Liang, Ping Yan, Jie Pei, Xian Guo.
       BACKGROUND AND AIM: Yak estrus is a seasonal phenomenon, probably involving epigenetic regulation of synthesis and secretion of sex hormones as well as growth and development of follicles. N6-methyladenosine (m6A) is the most common internal modification of the eukaryotic mRNA. However, there are no detailed reports on the m6A transcriptome map of yak ovary. Therefore, this study aimed to collected the yak ovarian tissues at three different states of anestrus (YO-A), estrus (YO-F), and pregnancy (YO-P), and obtained the full transcriptome m6A map in yak by MeRIP-seq.
    RESULTS: The HE staining revealed that the number of growing follicles and mature follicles in the ovary during the estrus period was relatively higher than those in the anestrus period and the pregnancy period. The RT-qPCR showed that the expression of METTL3, METTL14, FTO, YTHDC1 were significantly different across different periods in the ovaries, which suggests that m6A may play a regulatory role in ovarian activity. Next, we identified 20,174, 19,747 and 13,523 m6A peaks in the three ovarian samples of YO-A, YO-F and YO-P using the methylated RNA immunoprecipitation sequencing (MeRIP-seq). The m6A peaks are highly enriched in the coding sequence (CDS) region and 3'untranslated region (3'UTR) as well as the conserved sequence of "RRACH." The GO, KEGG and GSEA analysis revealed the involvement of m6A in many physiological activities of the yak's ovary during reproductive cycle. The association analysis found that some genes such as BNC1, HOMER1, BMP15, BMP6, GPX3, and WNT11 were related to ovarian functions.
    CONCLUSIONS: The comparison of the distribution patterns of methylation peaks in the ovarian tissues across different periods further explored the m6A markers related to the regulation of ovarian ovulation and follicular development in the yak ovary. This comprehensive map provides a solid foundation for revealing the potential function of the mRNA m6A modification in the yak ovary.
    Keywords:  Anestrus; Estrus; MeRIP-seq; Pregnancy; m6A
    DOI:  https://doi.org/10.1186/s12864-022-08585-7
  30. Front Mol Biosci. 2022 ;9 815290
    Pyroptosis-Related Patterns Predict Tumor Immune Landscape and Immunotherapy Response in Bladder Cancer.
    Yilin Yan, Xiangqian Cao, Zeyi Wang, Zhengnan Huang, Jinming Cai, Pengfei Tang, Chenkai Yang, Fang Zhang, Shujie Xia, Bing Shen.
      Background: Bladder cancer (BC) is a leading cause of death from malignancy, with significant heterogeneity in the immunotherapeutic responsiveness of advanced status. Pyroptosis, a newly discovered inflammatory programmed cell death, is confirmed to play an indispensable role in tumorigenesis and anti-tumor activity. However, the effect of pyroptosis on the tumor-immune landscape remodeling and immunotherapy in BC remains elusive. Methods: We comprehensively evaluated the mRNA expression and genomic alterations of 33 pyroptosis-related genes (PRGs) in BC and evaluated the patterns of pyroptosis in publicly available BC datasets. An unsupervised clustering method was used to classify patients into distinct patterns. Then, we established a pyroptosis-related signature score (PS-score) model to quantify the pyroptosis-related patterns of individual BC patients using principal component analysis. Furthermore, we correlated the patterns with the immune landscape and response efficacy of immunotherapy. Results: Two pyroptosis-related patterns were identified in BC, and distinct patterns showed various immune characteristics. Patterns with a high expression level of PRGs exhibited a survival advantage and showed higher infiltration of cytotoxic lymphocytes. Tumors with a low PS-score were characterized by high tumor-infiltrating lymphocytes and considered "hot." Further analysis revealed that the PS-score was an independent prognostic factor and could predict the response to immunotherapy for patients with advanced BC. We found a significant positive association between AHNAK2, AHNAK nucleoprotein 2, expression, and PS-score. Functional assays showed that AHNAK2 knockdown was correlated with attenuated invasive ability. Conclusion: This work comprehensively demonstrated the potential function of pyroptosis-related patterns in the bladder tumor-immune landscape and identified their therapeutic liability in immunotherapy. Our study enhanced our understanding of the immune landscape and provided a new approach toward more effective immunotherapy strategies.
    Keywords:  bladder cancer; immunotherapy; microenvironment; pyroptosis; score
    DOI:  https://doi.org/10.3389/fmolb.2022.815290
  31. Int J Biol Sci. 2022 ;18(7): 3048-3065
    RNA methylation-mediated LINC01559 suppresses colorectal cancer progression by regulating the miR-106b-5p/PTEN axis.
    Ke Shi, Shuaixi Yang, Chen Chen, Bo Shao, Yaxin Guo, Xiaoke Wu, Luyang Zhao, Xiuxiu Yang, Qiuge Zhang, Weitang Yuan, Zhenqiang Sun.
      Long noncoding RNAs (lncRNAs) regulate multiple biological effects in cancers. Recently, RNA methylation has been found to modify not only coding RNAs but also some noncoding RNAs. How RNA methylation affects lncRNAs to affect colorectal cancer (CRC) progression remains elusive. The expression of LINC01559 was explored through RNA sequencing, quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH). The preliminary exploration of its function was performed using Western blotting (WB) and immunohistochemistry (IHC). Functional experiments in vitro and in vivo were conducted to explore the biological functions of LINC01559 in CRC. The LINC01559/miR-106-5p/PTEN axis was verified through fluorescence in situ hybridization (FISH), luciferase assays, and rescue experiments. RIP-sequencing, m6A RNA immunoprecipitation (MeRIP) assays and bioinformatic analysis were conducted to determine the upstream mechanism of LINC01559. The results showed that LINC01559 was downregulated in CRC compared with normal controls. Lower expression of LINC01559 in CRC patients predicted a poor prognosis. In addition, PTEN was found to be positively correlated with LINC01559, and miR-106b-5p could be the link between LINC01559 and PTEN. Then, silencing LINC01559 restored the malignant phenotype of CRC cells, while cotransfection of miR-106b-5p inhibitor neutralized this effect. Mechanistically, we found abundant m6A modification sites on LINC01559. Then, we uncovered these sites as potential targets of METTL3 through experiments in vivo. The results revealed a negative functional regulation of the LINC01559/miR-106b-5p/PTEN axis in CRC progression and explored a new mechanism of METTL3-mediated m6A modification on LINC01559. These results elucidate a novel potential therapeutic target for CRC treatment.
    Keywords:  LINC01559; METTL3; PTEN; colorectal cancer
    DOI:  https://doi.org/10.7150/ijbs.70630
  32. Cancers (Basel). 2022 Apr 24. pii: 2121. [Epub ahead of print]14(9):
    IGF2BP1 Promotes Proliferation of Neuroendocrine Neoplasms by Post-Transcriptional Enhancement of EZH2.
    Florian Sperling, Danny Misiak, Stefan Hüttelmaier, Patrick Michl, Heidi Griesmann.
      Neuroendocrine neoplasms (NENs) represent a heterogenous class of highly vascularized neoplasms that are increasing in prevalence and are predominantly diagnosed at a metastatic state. The molecular mechanisms leading to tumor initiation, metastasis, and chemoresistance are still under investigation. Hence, identification of novel therapeutic targets is of great interest. Here, we demonstrate that the RNA-binding Protein IGF2BP1 is a post-transcriptional regulator of components of the Polycomb repressive complex 2 (PRC2), an epigenic modifier affecting transcriptional regulation and proliferation: Comprehensive in silico analyses along with in vitro experiments showed that IGF2BP1 promotes neuroendocrine tumor cell proliferation by stabilizing the mRNA of Enhancer of Zeste 2 (EZH2), the catalytic subunit of PRC2, which represses gene expression by tri-methylation of histone H3 at lysine 27 (H3K27me3). The IGF2BP1-driven stabilization and protection of EZH2 mRNA is m6A-dependent and enhances EZH2 protein levels which stimulates cell cycle progression by silencing cell cycle arrest genes through enhanced H3K27 tri-methylation. Therapeutic inhibition of IGF2BP1 destabilizes EZH2 mRNA and results in a reduced cell proliferation, paralleled by an increase in G1 and sub-G1 phases. Combined targeting of IGF2BP1, EZH2, and Myc, a transcriptional activator of EZH2 and well-known target of IGF2BP1 cooperatively induces tumor cell apoptosis. Our data identify IGF2BP1 as an important driver of tumor progression in NEN, and indicate that disruption of the IGF2BP1-Myc-EZH2 axis represents a promising approach for targeted therapy of neuroendocrine neoplasms.
    Keywords:  EZH2; H3K27me3; IGF2BP1; NEN; RNA-binding protein; cell cycle
    DOI:  https://doi.org/10.3390/cancers14092121
  33. FASEB J. 2022 May;36 Suppl 1
    IMP1/IGF2BP1 alters extracellular vesicle cargo in colorectal cancer.
    Madeline Kuhn, Yang Zhang, Randall Armstrong, Sarah Andres.
       BACKGROUND: Colorectal cancer (CRC) is the third leading cause of cancer-related death worldwide. There is an urgent need for new therapeutic targets and methods of early detection to improve patient outcomes. Extracellular Vesicles (EVs) and EV cargo are promising new biomarkers and therapeutic targets. EVs are secreted nanoparticles that carry DNA, RNA, and proteins. EVs can target specific cell types and once taken up by a destination cell, EV cargo can shape cell gene expression and physiology. EVs can shape the tumor microenvironment (TME) (i.e., stromal, immune, and endothelial cells), which plays critical roles in patient survival and disease outcomes. Our current understanding of how EV cargo is determined and how it influences the CRC cell TME is limited. One promising but understudied molecular aspect of EVs is RNA binding proteins (RNAbps). RNAbps are post-transcriptional regulators of gene expression and could provide a link between host cell gene expression and EV cargo. Insulin-like growth factor 2 RNA binding protein 1 (IMP1, IGF2BP1) is an RNAbp with a critical role in CRC development and progression. IMP1 is expressed highly in CRC tumors, expression correlates with worse prognosis, and IMP1 binds/potently regulates tumor-associated transcripts. How EV cargo is influenced by IMP1 and effects on EV targeting to destination cells and on target cell gene expression and function is not known. Understanding how IMP1 shapes EV cargo and which TME cells are targeted, will identify new mechanisms of cancer development/progression through IMP1-EV-mediated alteration of the TME and novel EV cargo as candidates for early detection biomarkers.
    HYPOTHESIS: We hypothesize that IMP1 facilitates the packaging of tumor-promoting EV cargo that interact and communicate with the TME to enhance tumor progression.
    METHODS: EVs were isolated from IMP1 null and IMP1-overexpressing (IMP1OE ) CRC cell lines, HT-29, SW480, as well as wildtype Caco2 cells (express high levels of IMP1) by size-exclusion chromatography. EVs were assessed by nanoparticle tracking analysis, western blot, electron microscopy, and qPCR.
    RESULTS: We found that IMP1 enters EVs from Caco2 and SW480 IMP1OE cell lines, but not HT-29 IMP1OE or corresponding null controls. IMP1 did not alter EV secretion in CRC cell lines. We found that IMP1 target mRNAs, such as KRAS, cMYC are present within EVs, but not the IMP1 target PTGS2.
    CONCLUSIONS: Our novel findings suggest that IMP1 enters the EV to directly influence EV cargo in specific CRC cell lines. Our findings have implications for the development of novel early detection biomarkers and therapeutic approaches in CRC.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R2392
  34. FASEB J. 2022 May;36 Suppl 1
    Elucidating the Kinetic Mechanism of Human METTL16.
    Kurtis Breger, Jessica A Brown.
      Over 140 RNA modifications have been discovered, yet only recently have they been studied in depth due to recent technological advancements. N6 -methyladenosine (m6 A) is an abundant RNA modification in messenger RNA (mRNA) and long non-coding RNA that affects various cellular functions such as mRNA stability, phase-separation of RNAs, and others. Methyltransferase-like protein 16 (METTL16) is one of four catalytically active m6 A RNA methyltransferases in humans. Two well-known methylation targets of METTL16 are U6 spliceosomal RNA and hairpins in the 3' untranslated region of MAT2A mRNA. However, METTL16 binds to many other RNAs, including the 3' triple helix of MALAT1. Using in vitro assays, we have started to investigate the kinetic mechanism and other fundamental properties of METTL16. Thus far, we have determined that METTL16 is a monomer in complex with either U6 snRNA or the MALAT1 triple helix. The METTL16•RNA complex has a dissociation constant (KD ) of 18 nM with the U6 snRNA and 31 nM with the MALAT1 triple helix. The apparent dissociation constant for S-adenosylmethionine (SAM), the methyl donor, with the METTL16•U6 snRNA binary complex is 112 µM. Under in vitro conditions, the cancer-associated MALAT1 triple helix is not a substrate of METTL16 at position A8290 and other adenosine residues. Preincubation assays suggest that there is an ordered mechanism by which U6 snRNA binds to METTL16 before SAM. Steady-state assays established a kcat of 0.074 min-1 and single-turnover assays established a kchem of 0.56 min-1 . This difference in the rates suggests conformational rearrangements and/or product release may be rate limiting. Ongoing work includes the characterization of METTL16 mutants. Mutations in the METTL16 K-loop led to a 7-fold increase of SAM binding to METTL16•U6 snRNA. Future studies will focus on more METTL16 mutants, including critical residues in other structures and those identified in cancer patients, to ascertain how these residues affect the kinetic mechanism of METTL16. This study will enable future research on METTL16 as a therapeutic target.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3413
  35. Curr Cancer Drug Targets. 2022 May 11.
    Knockdown of ARL5B Induces Mitochondrial-Mediated Apoptosis and Inhibits Glycolysis in Breast Cancer Cells by Activating MDA5 Signaling.
    Lei Zhang, Xuqiao Hu, Huaiyu Wu, Hongtian Tian, Jieying Zeng, Di Song, Keen Yang, Jing Chen, Jinfeng Xu, Fajin Dong.
       AIM: Mitochondria are essential for energy metabolism in the tumor microenvironment and the survival of cancer cells.
    BACKGROUND: ADP-ribosylation factor-like GTPase 5b (ARL5B) has been found to be associated with mitochondrial dysfunction and breast cancer (BC) progression, but the underlying mechanism needs to be further understood.
    OBJECTIVE: We investigated the effects of ARL5B on the apoptosis and glycolysis of breast cancer cells and its underlying mechanisms.
    METHOD: Quantitative reverse transcription-PCR (qRT-PCR) and western blot assays were used to detect the expression of ARL5B in breast cancer tissues and cells. An ARL5B loss-of-function assay was performed to verify its role in BC development.
    RESULTS: ARL5B was upregulated in breast cancer tissues and cell lines. ARL5B knockdown induced apoptosis and activated the mitochondrial pathway in breast cancer cells. Interestingly, the inhibition of ARL5B repressed the aerobic glycolysis of breast cancer cells. The role of ARL5B in breast cancer cells was exerted by mediating the activation of viral RNA sensor MDA5-evoked signaling. Silencing ARL5B triggered MDA5 signaling by upregulating the key proteins involved in the MDA5 pathway. Importantly, MDA5 silencing reversed the effects of ARL5B knockdown on mitochondrial-mediated apoptosis and glycolysis, whereas poly(I:C), as a ligand for MDA5, further enhanced ARL5B knockdown-facilitated mitochondrial apoptosis and the inhibition of glycolysis. Conclusion The knockdown of ARL5B activated MDA5 signaling and thus led to the enhanced mitochondrial-mediated apoptosis and glycolysis inhibition in breast cancer cells. Our study suggested that ARL5B might be a potential therapy target for BC.
    Keywords:  ARL5B; apoptosis; breast cancer; energy metabolism
    DOI:  https://doi.org/10.2174/1568009622666220511112538
  36. Planta. 2022 May 14. 255(6): 125
    N6-Methyladenosine dynamic changes and differential methylation in wheat grain development.
    Wenxiang Li, Yi Yu, Xuanrong Chen, Qian Fang, Anqi Yang, Xinyu Chen, Lei Wu, Chengyu Wang, Dechuan Wu, Sihong Ye, Dexiang Wu, Genlou Sun.
       MAIN CONCLUSION: More methylation changes occur in late interval than in early interval of wheat seed development with protein and the starch synthesis-related pathway enriched in the later stages. Wheat seed development is a critical process to determining wheat yield and quality, which is controlled by genetics, epigenetics and environments. The N6-methyladenosine (m6A) modification is a reversible and dynamic process and plays regulatory role in plant development and stress responses. To better understand the role of m6A in wheat grain development, we characterized the m6A modification at 10 day post-anthesis (DPA), 20 DPA and 30 DPA in wheat grain development. m6A-seq identified 30,615, 30,326, 27,676 high confidence m6A peaks from the 10DPA, 20DPA, and 30DPA, respectively, and enriched at 3'UTR. There were 29,964, 29,542 and 26,834 unique peaks identified in AN0942_10d, AN0942_20d and AN0942_30d. One hundred and forty-two genes were methylated by m6A throughout seed development, 940 genes methylated in early grain development (AN0942_20d vs AN0942_10d), 1542 genes in late grain development (AN0942_30d vs AN0942_20d), and 1190 genes between early and late development stage (AN0942_30d vs AN0942_10d). KEGG enrichment analysis found that protein-related pathways and the starch synthesis-related pathway were significantly enriched in the later stages of seed development. Our results provide novel knowledge on m6A dynamic changes and its roles in wheat grain development.
    Keywords:  Differential methylation; Genome; Grain development; Regulation; Triticum aestivum; m6A methylation
    DOI:  https://doi.org/10.1007/s00425-022-03893-4
  37. FASEB J. 2022 May;36 Suppl 1
    Down-expression of MiR30a-5p promotes proliferation and migration of Non-Small Cell Lung Cancer cells by targetting MTDH.
    Souraka Tapara Dramani Maman.
       OBJECTIVE: MTDH has been identified to play a critical role in many cancer types, but its potential mechanism in Non-Small Cell Lung Cancer (NSCLC) still remains unclear. We investigated the role of miRNA30a-5p in the regulation of MTDH expression, which is known as an essential biomarker for NSCLC initiation and progression.
    MATERIALS AND METHODS: Quantitative real-time PCR (qRT-PCR) has been performed to evaluate the expression level of MTDH and miR30a-5p. Then, we transfected miR30a-5p mimic and inhibitor into H1299 and A549 cells to investigate their impact in cell proliferation, migration, and invasion by using CCK-8 assay, wound-healing assay, and transwell assay respectively. The evidence of miR30a-5p as a specific target of the MTDH gene was explored through luciferase report assay. The expression level of MTDH was evaluated by its mRNA and protein level using a western blot assay.
    RESULTS: Results revealed that miR30a-5p was upregulated whereas MTDH was downregulated. Down-expression of miR30a-5p increases the expression level of MTDH and promotes NSCLC cell proliferation and invasion. Luciferase report assay confirmed the binding of miR30a-5p on MTDH mRNA 3'UTR region.
    CONCLUSIONS: This funding highlights the silencing mechanism of MTDH in lung cancer and further revealed that MTDH can be used as a potential diagnostic and therapeutic biomarker.
    Keywords:  3′UTR region; Luciferase report assay; MTDH; Non-Small Cell Lung Cancer; miR30a-5p; silencing mechanism
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.00R60
  38. Cell Commun Signal. 2022 May 09. 20(1): 62
    E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway.
    Manman Liang, Lijing Wang, Zhengui Sun, Xingwu Chen, Hanli Wang, Lilong Qin, Wenying Zhao, Biao Geng.
       BACKGROUND: Recent studies have indicated that some members of the tripartite motif (TRIM) proteins function as important regulators for non-small cell lung cancer (NSCLC), However, the regulatory mechanism underpinning aberrant expression of TRIM in NSCLC remains unclear. Here we report that TRIM15 plays important roles in NSCLC progression through modulating Keap1-Nrf2 signaling pathway.
    METHODS: TRIM15 expression was evaluated by western blot analysis, tissue microarray-based immunohistochemistry analysis. The interactions between TRIM15 and Keap1 were analyzed by co-immunoprecipitation (Co-IP) and immunofluorescence co-localization assay. The correlation between TRIM15 and Keap1 was measured by Co-IP and ubiquitination analysis in vitro. Gain- and lost-of-function experiments were used to detect TRIM15 promotes proliferation and invasion of NSCLC cells both in vitro and vivo.
    RESULTS: Here, we revealed that TRIM15 was frequently upregulated in NSCLC samples and associated with poor prognosis. Functionally, TRIM15 knockdown resulted in decreased cancer cell proliferation and metastasis, whereas ectopic TRIM15 expression facilitated tumor cancer cell proliferation and metastasis in vitro and in vivo. Moreover, TRIM15 promoted cell proliferation and metastasis depends on its E3 ubiquitin ligase. Mechanistically, TRIM15 directly targeted Keap1 by ubiquitination and degradation, the principal regulator of Nrf2 degradation, leading to Nrf2 escaping from Keap1-mediated degradation, subsequently promoting antioxidant response and tumor progression.
    CONCLUSIONS: Therefore, our study characterizes the pivotal roles of TRIM15 promotes NSCLC progression via Nrf2 stability mediated by promoting Keap1 ubiquitination and degradation and could be a valuable prognostic biomarker and a potential therapeutic target in NSCLC. Video Abstract.
    Keywords:  Keap1; NSCLC; Nrf2; TRIM15; Ubiquitination and degradation
    DOI:  https://doi.org/10.1186/s12964-022-00875-7
  39. FASEB J. 2022 May;36 Suppl 1
    Defining the role of RNA acetylation via a sequence-specific RNA acetyltransferase.
    Samantha R-A Nance, Jordan L Meier, Raj Chari, Christine N Evans.
      RNA modifications play a critical role in protein abundance, biology, and disease. However, methods for testing the causal effects of individual RNA modifications remain in their infancy. Here we report the preliminary development of an epitranscriptome engineering strategy to manipulate RNA cytidine acetylation in living cells. Our approach fuses a eukaryotic cytidine acetyltransferase to a sequence-specific RNA-binding protein. First, we optimize the ectopic expression of these constructs in human embryonic kidney cells. Next, we develop and apply assays to assess guide-dependent and independent acetylation of a model substrate. Finally, we demonstrate the dependence of modification on consensus sequence, guide/target proximity, and linker protein fusion geometry. These studies provide a foundation for site-specific rescue of individual cytidine acetylation events in model organisms, and in so doing will help illuminate the function of RNA modifications in disease.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R4033
  40. Int J Oral Sci. 2022 May 10. 14(1): 25
    FAM64A promotes HNSCC tumorigenesis by mediating transcriptional autoregulation of FOXM1.
    Xinyuan Zhao, Huan Chen, Yu Qiu, Li Cui.
      Head and neck squamous cell carcinoma (HNSCC) still lacks effective targeted treatment. Therefore, exploring novel and robust molecular targets is critical for improving the clinical outcome of HNSCC. Here, we reported that the expression levels of family with sequence similarity 64, member A (FAM64A) were significantly higher in HNSCC tissues and cell lines. In addition, FAM64A overexpression was found to be strongly associated with an unfavorable prognosis of HNSCC. Both in vitro and in vivo evidence showed that FAM64A depletion suppressed the malignant activities of HNSCC cells, and vice versa. Moreover, we found that the FAM64A level was progressively increased from normal to dysplastic to cancerous tissues in a carcinogenic 4-nitroquinoline-1-oxide mouse model. Mechanistically, a physical interaction was found between FAM64A and forkhead box protein M1 (FOXM1) in HNSCC cells. FAM64A promoted HNSCC tumorigenesis not only by enhancing the transcriptional activity of FOXM1, but also, more importantly, by modulating FOXM1 expression via the autoregulation loop. Furthermore, a positive correlation between FAM64A and FOXM1 was found in multiple independent cohorts. Taken together, our findings reveal a previously unknown mechanism behind the activation of FOXM1 in HNSCC, and FAM64A might be a promising molecular therapeutic target for treating HNSCC.
    DOI:  https://doi.org/10.1038/s41368-022-00174-4
  41. Int J Mol Sci. 2022 Apr 29. pii: 4948. [Epub ahead of print]23(9):
    FTO Alleviates CdCl2-Induced Apoptosis and Oxidative Stress via the AKT/Nrf2 Pathway in Bovine Granulosa Cells.
    He Ding, Zhiqiang Li, Xin Li, Xiaorui Yang, Jing Zhao, Jing Guo, Wenfa Lu, Hongyu Liu, Jun Wang.
      Cadmium (Cd) is a common environmental heavy metal contaminant of reproduction toxicity. Cd accumulation in animals leads to the damage of granulosa cells. However, its mechanism needs to be elucidated. This research found that treating granulosa cells with Cd resulted in reduced cell viability. The flow cytometry results showed that Cd increased the degree of apoptosis and level of superoxide anion (O2-) in granulosa cells. Further analysis showed that Cd treatment resulted in reduced expression levels of nuclear factor erythroid 2-related factor-2 (Nrf2), superoxide dismutase (SOD), catalase (CAT) and NAD(P)H: quinone oxidoreductase 1 (NQO1), and an increased expression level of malondialdehyde (MDA); the expression levels of Bcl-2 associated X (Bax) and caspase-3 increased, whereas that of B-cell lymphoma 2 (Bcl-2) decreased. Changes in m6A methylation-related enzymes were noted with Cd-induced damage to granulosa cells. The results of transcriptome and MeRIP sequencing revealed that the AKT pathway participated in Cd-induced damage in granulosa cells, and the MAX network transcriptional repressor (MNT) may be a potential target gene of fat mass and obesity-associated protein (FTO). FTO and YTH domain family member 2 (YTHDF2) regulated MNT expression through m6A modification. FTO overexpression alleviated Cd-induced apoptosis and oxidative stress through the activation of the AKT/Nrf2 pathway; this process could be reversed using siMNT. Overall, these findings associated m6A with Cd-induced damage to granulosa cells and provided insights into Cd-induced granulosa cell cytotoxicity from a new perspective centered on m6A modification.
    Keywords:  CdCl2; FTO; apoptosis; granulosa cells; m6A
    DOI:  https://doi.org/10.3390/ijms23094948
  42. J Hematol Oncol. 2022 May 07. 15(1): 52
    LncRNA-PACERR induces pro-tumour macrophages via interacting with miR-671-3p and m6A-reader IGF2BP2 in pancreatic ductal adenocarcinoma.
    Yihao Liu, Minmin Shi, Xingfeng He, Yizhi Cao, Pengyi Liu, Fanlu Li, Siyi Zou, Chenlei Wen, Qian Zhan, Zhiwei Xu, Jiancheng Wang, Baofa Sun, Baiyong Shen.
       BACKGROUND: LncRNA-PACERR plays critical role in the polarization of tissue-associated macrophages (TAMs). In this study, we found the function and molecular mechanism of PACERR in TAMs to regulate pancreatic ductal adenocarcinoma (PDAC) progression.
    METHODS: We used qPCR to analyse the expression of PACERR in TAMs and M1-tissue-resident macrophages (M1-NTRMs) which were isolated from 46 PDAC tissues. The function of PACERR on macrophages polarization and PDAC proliferation, migration and invasion were confirmed through in vivo and in vitro assays. The molecular mechanism of PACERR was discussed via fluorescence in situ hybridization (FISH), RNA pull-down, ChIP-qPCR, RIP-qPCR and luciferase assays.
    RESULTS: LncRNA-PACERR was high expression in TAMs and associated with poor prognosis in PDAC patients. Our finding validated that LncRNA-PACERR increased the number of M2-polarized cells and facilized cell proliferation, invasion and migration in vitro and in vivo. Mechanistically, LncRNA-PACERR activate KLF12/p-AKT/c-myc pathway by binding to miR-671-3p. And LncRNA-PACERR which bound to IGF2BP2 acts as an m6A-dependent manner to enhance the stability of KLF12 and c-myc in cytoplasm. In addition, the promoter of LncRNA-PACERR was a target of KLF12 and LncRNA-PACERR recruited EP300 to increase the acetylation of histone by interacting with KLF12 in nucleus.
    CONCLUSIONS: This study found that LncRNA-PACERR functions as key regulator of TAMs in PDAC microenvironment and revealed the novel mechanisms in cytoplasm and in nucleus.
    Keywords:  IGF2BP2; KLF12; LncRNA-PACERR; PDAC; TAMs; m6A; miR-671-3p
    DOI:  https://doi.org/10.1186/s13045-022-01272-w
  43. Cell Biochem Biophys. 2022 May 10.
    THUMPD3-AS1 facilitates cell growth and aggressiveness by the miR-218-5p/SKAP1 axis in colorectal cancer.
    Yuwei Pu, Jinrong Wei, Yong Wu, Kui Zhao, Yongyou Wu, Shu Wu, Xiaodong Yang, Chungen Xing.
       BACKGROUND: Colorectal cancer (CRC) is a malignant cancer with a high mortality. Accumulating studies have revealed that mRNAs involved in ceRNA (competing endogenous RNA) network are implicated in the tumorigenesis and development of CRC. Here, we aimed to elucidate the ceRNA network involving Src kinase associated phosphoprotein 1 (SKAP1) in the biological characteristics of CRC.
    METHODS: Expression levels of genes in colon adenocarcinoma (COAD) samples and prognosis of COAD patients were predicted using publicly available online tool. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), clony formation and Transwell assays were conducted to test the biological functions of SKAP1 and THUMPD3 antisense RNA 1 (THUMPD3-AS1) in CRC cells. Western blot was used to measure the protein levels of SKAP1. Gene expression in CRC cells was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The interaction between miR-218-5p and THUMPD3-AS1 (or SKAP1) was verified by RNA pulldown and luciferase reporter assays.
    RESULTS: SKAP1 was upregulated in COAD tissues and CRC cells and it reflected a poor prognosis in patients with COAD. SKAP1 knockdown inhibited CRC (HT-29 and HCT-116) cell proliferation, migration and invasion. Mechanistically, THUMPD3-AS1 acted as a ceRNA to sponge miR-218-5p and subsequently upregulated SKAP1 expression in CRC cells. SKAP1 overexpression reversed the suppressive effect of THUMPD3-AS1 knockdown on proliferation, migration and invision of CRC cells.
    CONCLUSIONS: THUMPD3-AS1 promotes CRC cell growth and aggressiveness by regulating the miR-218-5p/SKAP1 axis.
    Keywords:  Colorectal cancer; SKAP1; THUMPD3-AS1; miR-218-5p
    DOI:  https://doi.org/10.1007/s12013-022-01074-4
  44. Phytother Res. 2022 May 11.
    Modification of lysine deacetylation regulates curcumol-induced necroptosis through autophagy in hepatic stellate cells.
    Sumin Sun, Zhanghao Li, Sheng Huan, Jun Kai, Siwei Xia, Ying Su, Shufan Ji, Anping Chen, Shijun Wang, Xuefen Xu, Jiangjuan Shao, Feng Zhang, Biyun Zhang, Zili Zhang, Shizhong Zheng.
      The excessive deposition of extracellular matrix (ECM) is the main characteristic of liver fibrosis, and hepatic stellate cells (HSCs) are the main source of ECM. The removal of activated HSCs has a reversal effect on liver fibrosis. Western blot and MTT analysis indicated that curcumol could relieve hepatic fibrosis by promoting HSCs receptor-interacting protein kinase 1/3 (RIP1/RIP3)-dependent necroptosis. Importantly, autophagy flow was monitored by constructing the mRFP-GFP-LC3 plasmid, and it was found that curcumol cleared activated HSCs in a necroptosis manner that was dependent on autophagy. Our study suggested that the activation of necrosome formed by RIP1 and RIP3 depended on Atg5, and that autophagosomes were also necessary for curcumol-induced necroptosis. Furthermore, microscale thermophoresis and co-immunoprecipitation assay results proved that curcumol could target Sirt1 to regulate autophagy by reducing the acetylation level of Atg5. The HSCs-specific silencing of Sirt1 exacerbated CCl4 -induced liver fibrosis in mice. The deacetylation of Atg5 not only accelerated the accumulation of autophagosomes but also enhanced the interaction between Atg5 and RIP1/RIP3 to induce necroptosis. Overall, our study indicated that curcumol could activate Sirt1 to promote Atg5 deacetylation and enhanced its protein-protein interaction function, thereby inducing autophagy and promoting the necroptosis of HSCs to reduce liver fibrosis.
    Keywords:  Sirt1; autophagy; curcumol; hepatic stellate cell; liver fibrosis; necroptosis
    DOI:  https://doi.org/10.1002/ptr.7483
  45. PLoS One. 2022 ;17(5): e0268217
    Survival of HT29 cancer cells is influenced by hepatocyte growth factor receptor inhibition through modulation of self-DNA-triggered TLR9-dependent autophagy response.
    Bettina Bohusné Barta, Ágnes Simon, Lőrinc Nagy, Titanilla Dankó, Regina Eszter Raffay, Gábor Petővári, Viktória Zsiros, Anna Sebestyén, Ferenc Sipos, Györgyi Műzes.
      HGFR activation drives the malignant progression of colorectal cancer, and its inhibition displays anti-autophagic activity. The interrelated role of HGFR inhibition and TLR9/autophagy signaling in HT29 cancer cells subjected to modified self-DNA treatments has not been clarified. We analyzed this complex interplay with cell metabolism and proliferation measurements, TLR9, HGFR and autophagy inhibitory assays and WES Simple Western blot-based autophagy flux measurements, gene expression analyses, immunocytochemistry, and transmission electron microscopy. The overexpression of MyD88 and caspase-3 was associated with enhanced HT29 cell proliferation, suggesting that incubation with self-DNAs could suppress the apoptosis-induced compensatory cell proliferation. HGFR inhibition blocked the proliferation-reducing effect of genomic and hypermethylated, but not that of fragmented DNA. Lowest cell proliferation was achieved with the concomitant use of genomic DNA, HGFR inhibitor, and chloroquine, when the proliferation stimulating effect of STAT3 overexpression could be outweighed by the inhibitory effect of LC3B, indicating the putative involvement of HGFR-mTOR-ULK1 molecular cascade in HGFR inhibitor-mediated autophagy. The most intense cell proliferation was caused by the co-administration of hypermethylated DNA, TLR9 and HGFR inhibitors, when decreased expression of both canonical and non-canonical HGFR signaling pathways and autophagy-related genes was present. The observed ultrastructural changes also support the context-dependent role of HGFR inhibition and autophagy on cell survival and proliferation. Further investigation of the influence of the studied signaling pathways and cellular processes can provide a basis for novel, individualized anti-cancer therapies.
    DOI:  https://doi.org/10.1371/journal.pone.0268217
  46. Appl Biochem Biotechnol. 2022 May 11.
    MicroRNA-19a-3p Acts as an Oncogene in Gastric Cancer and Exerts the Effect by Targeting SMOC2.
    Hui Xu, Guochun Lu, Shengkun Zhou, Fu Fang.
      MicroRNAs are reported to be involved in tumor development. This study aims to investigate the biological functions and molecular mechanisms of microRNA-19a-3p in gastric cancer cells. TCGA-based expression analysis and qRT-PCR assay illustrated that microRNA-19a-3p was overexpressed in gastric cancer. MTT and Transwell assays indicated that microRNA-19a-3p could strengthen the proliferation, migration, and invasion of gastric cancer cells. SMOC2 was bioinformatically predicted as the target of microRNA-19a-3p, followed by identified using a dual-luciferase assay. The effects of microRNA-19a-3p/SMOC2 regulatory axis on gastric cancer cells were examined by MTT and Transwell assays as well. Concludingly, this study demonstrated that microRNA-19a-3p could promote the aggressive cell phenotypes of gastric cancer cells by targeting SMOC2.
    Keywords:  Cell proliferation rate; Gastric cancer (GC); Invasion; MicroRNA-19a-3p; Migration; SMOC2
    DOI:  https://doi.org/10.1007/s12010-022-03944-2
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