bims-rimeca Biomed News
on RNA methylation in cancer
Issue of 2023–08–27
47 papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics
  1. ALKBH5 promotes hypopharyngeal squamous cell carcinoma apoptosis by targeting TLR2 in a YTHDF1/IGF2BP2-mediated manner.
  2. Intracellular Fusobacterium nucleatum infection increases METTL3-mediated m6A methylation to promote the metastasis of esophageal squamous cell carcinoma.
  3. METTL3-modified lncRNA-MALAT1 regulates the molecular axis of miR-124-3p/CDK4 involved in Ewing's sarcoma.
  4. m6A eraser FTO impairs gemcitabine resistance in pancreatic cancer through influencing NEDD4 mRNA stability by regulating the PTEN/PI3K/AKT pathway.
  5. METTL14 inhibits malignant progression of oral squamous cell carcinoma by targeting the autophagy-related gene RB1CC1 in an m6A-IGF2BP2-dependent manner.
  6. MAPK13 stabilization via m6A mRNA modification limits anti-cancer efficacy of rapamycin.
  7. Exploring the role of m6A methylation regulators in glioblastoma multiforme and their impact on the tumor immune microenvironment.
  8. A cryptic pocket in METTL3-METTL14 regulates m6A conversion and sensing.
  9. The implications of N6-methyladenosine (m6A) modification in esophageal carcinoma.
  10. SUMOylation of the m6A reader YTHDF2 by PIAS1 promotes viral RNA decay to restrict EBV replication.
  11. Structure-Based Design of Inhibitors of the m6A-RNA Writer Enzyme METTL3.
  12. RBM15-Mediated m6A Modification of K17 Affects Keratinocytes Response to IL-17A Stimulation in Psoriasis.
  13. Comprehensive analysis of m6A subtype classification for immune microenvironment of pituitary adenomas.
  14. METTL3-deficiency Suppresses Neural Apoptosis to Induce Protective Effects in Cerebral I/R Injury via Inhibiting RNA m6A Modifications: A Pre-clinical and Pilot Study.
  15. The RNA m6A modification might participate in microglial activation during hypoxic-ischemic brain damage in neonatal mice.
  16. RNA N6‑methyladenosine methyltransferase WTAP promotes the differentiation of endothelial progenitor cells.
  17. In Search of a Function for the N6-Methyladenosine in Epitranscriptome, Autophagy and Neurodegenerative Diseases.
  18. N6-methyladenosine modification of REG1α facilitates colorectal cancer progression via β-catenin/MYC/LDHA axis mediated glycolytic reprogramming.
  19. Xanthine derivatives inhibit FTO in an L-ascorbic acid-dependent manner.
  20. P300/SP1 complex mediating elevated METTL1 regulates CDK14 mRNA stability via internal m7G modification in CRPC.
  21. The m6A modification-mediated OGDHL exerts a tumor suppressor role in ccRCC by downregulating FASN to inhibit lipid synthesis and ERK signaling.
  22. Analysis of mRNA m6A modification and mRNA expression profiles in middle ear cholesteatoma.
  23. METTL3 Promotes the Growth and Invasion of Melanoma Cells by Regulating the lncRNA SNHG3/miR-330-5p Axis.
  24. Role of METTL3-mediated m6A modification in osteogenic differentiation of periodontal ligament stem cells extracted from adult periodontal ligaments ex-vivo.
  25. Changes in m6A in Steatotic Liver Disease.
  26. miR-33a-3p regulates METTL3-mediated AREG stability and alters EMT to inhibit pancreatic cancer invasion and metastasis.
  27. CircASPH Enhances Exosomal STING to Facilitate M2 Macrophage Polarization in Colorectal Cancer.
  28. Loss of NSUN6 inhibits osteosarcoma progression by downregulating EEF1A2 expression and activation of Akt/mTOR signaling pathway via m5C methylation.
  29. Propofol suppresses OGD/R-induced ferroptosis in neurons by inhibiting the HIF-1α/YTHDF1/BECN1 axis.
  30. Pathogen-induced m6A dynamics affect plant immunity.
  31. The role of m6A-modified CircEPHB4 in glioma pathogenesis: Insights into cancer stemness metastasis.
  32. Epigenetic m6A RNA Modification Reader YTHDF1 in Merkel Cell Carcinoma.
  33. Cuproptosis- and m6A-Related lncRNAs for Prognosis of Hepatocellular Carcinoma.
  34. Pan-cancer analysis and experimental validation revealed the m6A methyltransferase KIAA1429 as a potential biomarker for diagnosis, prognosis, and immunotherapy.
  35. Acyl-CoA Medium-Chain Synthetase-3 (ACSM3) Is Regulated by Insulin Like Growth Factor 2 mRNA Binding Protein 3 (IGF2BP3) and Inhibits Proliferation, Motility and Stem Cell Properties of Breast Invasive Carcinoma.
  36. The multi-omics analyses of acsl1 reveal its translational significance as a tumor microenvironmental and prognostic biomarker in clear cell renal cell carcinoma.
  37. N6-Methyladenosine Modification and Prognostic Analysis of UBE2K in Hepatocellular Carcinoma: A Potential Target.
  38. DNA N6-methyladenine methylase N6AMT1 controls neuropathic pain through epigenetically modifying Kcnj16 in dorsal horn neurons.
  39. Characterization of m 6 A modifiers and RNA modifications in uterine fibroids.
  40. TRDMT1-mediated RNA C-5 methylation as a novel target in anticancer therapy.
  41. The 3' Non-Coding Sequence Negatively Regulates PD-L1 Expression, and Its Regulators Are Systematically Identified in Pan-Cancer.
  42. FOXM1 maintains fatty acid homoeostasis through the SET7-H3K4me1-FASN axis.
  43. Droplet-based high-throughput single microbe RNA sequencing by smRandom-seq.
  44. ZXDC enhances cervical cancer metastasis through IGF2BP3-mediated activation of RhoA/ROCK signaling.
  45. DARS2 overexpression is associated with PET/CT metabolic parameters and affects glycolytic activity in lung adenocarcinoma.
  46. Liver Metastases across Cancer Types Sharing Tumor Environment Immunotolerance Can Impede Immune Response Therapy and Immune Monitoring.
  47. SHP2 Contributes to Resistance of KRAS p.G12C-Driven Lung Cancer Cells to MRTX849 by Regulating β-catenin/c-MYC Axis.
  1. Cell Death Discov. 2023 Aug 23. 9(1): 308
    ALKBH5 promotes hypopharyngeal squamous cell carcinoma apoptosis by targeting TLR2 in a YTHDF1/IGF2BP2-mediated manner.
    Jing Ye, Yuting Wu, Yao Chen, Yiyue Ren, Xiaohua Jiang, Zhihuai Dong, Jingna Zhang, Mao Jin, Xiaozhen Chen, Zhanggui Wang, Mang Xiao.
      Hypopharyngeal squamous cell carcinoma (HPSCC) is one of the most aggressive cancers and is notorious for its extremely poor prognosis. However, very few molecular biological studies have been performed. As a novel method of epigenetic gene modulation, N6-methyladenosine (m6A) RNA modification occurs in HPSCC. The expression of the m6A demethylase AlkB homolog 5 (ALKBH5) is frequently downregulated in human HPSCC. Furthermore, we found that ALKBH5 impaired cell proliferation by regulating human Toll-like receptor 2 (TLR2) in an m6A-dependent manner in HPSCC cells. ALKBH5 decreased TLR2 m6A modification, which could be recognized by the m6A readers IGF2BP2 and YTHDF1. IGF2BP2 facilitates TLR2 mRNA stability, whereas YTHDF1 promotes TLR2 mRNA translation. The current work uncovered a critical function of ALKBH5 in TLR2 regulation and provides a novel role for m6A demethylation of mRNA in HPSCC. The inhibition of m6A modification of ALKBH5 in HPSCC deserves further clinical investigation.
    DOI:  https://doi.org/10.1038/s41420-023-01589-6
  2. J Adv Res. 2023 Aug 22. pii: S2090-1232(23)00229-1. [Epub ahead of print]
    Intracellular Fusobacterium nucleatum infection increases METTL3-mediated m6A methylation to promote the metastasis of esophageal squamous cell carcinoma.
    Songhe Guo, Fangfang Chen, Linfang Li, Dou Shuheng, Qifan Li, Yuying Huang, Zijun Li, Wanli Liu, Ge Zhang.
       INTRODUCTION: The tumor-associated microbiota plays a vital role in cancer development. Accumulating evidence shows that Fusobacterium nucleatum (Fn) participates in the progression of multiple tumor types. However, the underlying mechanisms remain unclear.
    OBJECTIVES: This study examined the expression of methyltransferase-like protein 3 (METTL3) during Fn infection and elucidated the function and pathway of Fn-induced m6A methylation in esophageal squamous cell carcinoma (ESCC).
    METHODS: The abundance of Fn in patient tissues was determined by qPCR. Western blot, qRT-PCR, and immunohistochemistry were performed to measure METTL3 expression in cells and tissues. METTL3 function was evaluated in vitro by colony formation and cell migration assays. MeRIP-qPCR was performed to determine the relationship between METTL3 and c-Myc. In addition, the half-lives of genes that are downstream of METTL3 were determined with RNA stability assays.
    RESULTS: Fn was enriched in hepatocellular carcinoma (HCC), breast cancer (BRCA), ESCC, and colorectal cancer (CRC) tumor tissues. METTL3 expression was positively associated with Fn abundance in ESCC tissues. Fn could survive and proliferation as well as increase METTL3 expression in ESCC, HCC, CRC, and BRCA cells. Moreover, METTL3 overexpression promoted ESCC cells proliferation, migration in vivo and in vitro. Mechanistically, Intracellular Fn infection increases METTL3 transcription. METTL3 promoted c-Myc mRNA methylation in the 3'-untranslated Region (3'-UTR) and enhanced its mRNA stability in a YTH N6-Methyladenosine RNA binding protein 1(YTHDF1)-dependent manner, which contributes to Fn induced ESCC proliferation and metastasis.
    CONCLUSIONS: This study indicates that intracellular Fn infection promotes ESCC development and metastasis, and eradicating Fn infection may be a promising strategy for treating ESCC.
    Keywords:  Fusobacterium nucleatum; METTL3; c-Myc; esophageal squamous cell carcinoma; m6A methylation
    DOI:  https://doi.org/10.1016/j.jare.2023.08.014
  3. Cell Mol Biol (Noisy-le-grand). 2023 Jun 30. 69(6): 193-197
    METTL3-modified lncRNA-MALAT1 regulates the molecular axis of miR-124-3p/CDK4 involved in Ewing's sarcoma.
    Fangzhou He, Tingting Ren, Xiaodong Tang.
      N6-methyladenosine (m6A) modifications are considered key mechanisms in cancer. As an m6A-modified lncRNA, MALAT1 is associated with tumor progression. In this study, the MALAT1/miR-124-3p/CDK4 axis was studied to discover METTL3's effects on Ewing's sarcoma (ES). For this purpose, clinical ES samples were collected and ES cells were cultured to detect gene expression. Then, the interlink between METTL3, MALAT1, miR-124-3p, and CDK4 was studied and confirmed, and m6A modification of MALAT1 was determined. Finally, the Transwell method was used to test migration and invasion. Results showed that ES samples expressed low miR-124-3p and high METTL3, MALAT1 and CDK4. METTL3 elevated MALAT1 expression by m6A modification. MALAT1 enhanced CDK4 expression by competing with miR-124-3p. In ES cells, METTL3 silencing repressed cell migration and invasion by inhibiting MALAT1. In conclusion, METTL3 promotes tumorigenesis of ES through the MALAT1/miR-124-3p/CDK4 axis.
    DOI:  https://doi.org/10.14715/cmb/2023.69.6.29
  4. J Exp Clin Cancer Res. 2023 Aug 22. 42(1): 217
    m6A eraser FTO impairs gemcitabine resistance in pancreatic cancer through influencing NEDD4 mRNA stability by regulating the PTEN/PI3K/AKT pathway.
    Kai Lin, Endi Zhou, Ting Shi, Siqing Zhang, Jinfan Zhang, Ziruo Zheng, Yuetian Pan, Wentao Gao, Yabin Yu.
       BACKGROUND: Gemcitabine resistance has brought great challenges to the treatment of pancreatic cancer. The N6-methyladenosine (m6A) mutation has been shown to have a significant regulatory role in chemosensitivity; however, it is not apparent whether gemcitabine resistance can be regulated by fat mass and obesity-associated protein (FTO).
    METHODS: Cells with established gemcitabine resistance and tissues from pancreatic cancer patients were used to evaluate FTO expression. The biological mechanisms of the effects of FTO on gemcitabine resistant cells were investigated using CCK-8, colony formation assay, flow cytometry, and inhibitory concentration 50. Immunoprecipitation/mass spectrometry, MeRIP-seq, RNA sequencing and RIP assays, RNA stability, luciferase reporter, and RNA pull down assays were employed to examine the mechanism of FTO affecting gemcitabine resistant pancreatic cancer cells.
    RESULTS: The results revealed that FTO was substantially expressed in cells and tissues that were resistant to gemcitabine. Functionally, the gemcitabine resistance of pancreatic cancer could be enhanced by FTO, while its depletion inhibited the growth of gemcitabine resistant tumor cells in vivo. Immunoprecipitation/mass spectrometry showed that the FTO protein can be bound to USP7 and deubiquitinated by USP7, leading to the upregulation of FTO. At the same time, FTO knockdown significantly decreased the expression level of NEDD4 in an m6A-dependent manner. RNA pull down and RNA immunoprecipitation verified YTHDF2 as the reader of NEDD4, which promoted the chemoresistance of gemcitabine resistant cells. FTO knockdown markedly increased the PTEN expression level in an NEDD4-dependent manner and influenced the chemosensitivity to gemcitabine through the PI3K/AKT pathway in pancreatic cancer cells.
    CONCLUSION: In conclusion, we found that gemcitabine resistance in pancreatic cancer can be influenced by FTO that demethylates NEDD4 RNA in a m6A-dependent manner, which then influences the PTEN expression level and thereby affects the PI3K/AKT pathway. We also identified that the FTO level can be upregulated by USP7.
    Keywords:  FTO; Gemcitabine resistance; N6-methyladenosine; Pancreatic carcinoma; Proliferation
    DOI:  https://doi.org/10.1186/s13046-023-02792-0
  5. Clin Sci (Lond). 2023 Aug 24. pii: CS20230219. [Epub ahead of print]
    METTL14 inhibits malignant progression of oral squamous cell carcinoma by targeting the autophagy-related gene RB1CC1 in an m6A-IGF2BP2-dependent manner.
    Jianfeng Liang, Hongshi Cai, Chen Hou, Fan Song, Yaoqi Jiang, Ziyi Wang, Danqi Qiu, Yue Zhu, Fang Wang, Dongsheng Yu, Jinsong Hou.
      N6-methyladenosine (m6A) plays crucial roles in tumorigenesis and autophagy. However, the underlying mechanisms mediated by m6A and autophagy in the malignant progression of oral squamous cell carcinoma (OSCC) remain unclear. In this study, we revealed that downregulated expression of METTL14 was correlated with advanced clinicopathological characteristics and poor prognosis in OSCC. METTL14 knockdown significantly inhibited autophagy and facilitated malignant progression in vitro, and promoted tumor growth and metastasis in vivo. A cell model of rapamycin-induced autophagy was established to identify RB1CC1 as a potential target gene involved in m6A-regulated autophagy in OSCC, through RNA sequencing and methylated RNA immunoprecipitation sequencing (meRIP-seq) analysis. Mechanistically, we confirmed that METTL14 posttranscriptionally enhanced RB1CC1 expression in an m6A-IGF2BP2-dependent manner, thereby affecting autophagy and progression in OSCC, through methylated RNA immunoprecipitation qRT-PCR (meRIP-qPCR), RNA stability assays, mutagenesis assays and dual-luciferase reporter. Collectively, our findings demonstrated that METTL14 serves as an OSCC suppressor by regulating the autophagy-related gene RB1CC1 through m6A modification, which may provide a new insight for the diagnosis and therapy of OSCC.
    Keywords:  autophagy; epigenetics; methyltransferase-like 14; oncogenesis; oral squamous cell carcinoma
    DOI:  https://doi.org/10.1042/CS20230219
  6. J Biol Chem. 2023 Aug 18. pii: S0021-9258(23)02203-2. [Epub ahead of print] 105175
    MAPK13 stabilization via m6A mRNA modification limits anti-cancer efficacy of rapamycin.
    Joohwan Kim, Yujin Chun, Cuauhtemoc B Ramirez, Lauren A Hoffner, Sunhee Jung, Ki-Hong Jang, Varvara I Rubtsova, Cholsoon Jang, Gina Lee.
      N6-adenosine methylation (m6A) is the most abundant mRNA modification that controls gene expression through diverse mechanisms. Accordingly, m6A-dependent regulation of oncogenes and tumor suppressors contributes to tumor development. However, the role of m6A-mediated gene regulation upon drug treatment or resistance is poorly understood. Here, we report that m6A modification of mitogen-activated protein kinase 13 (MAPK13) mRNA determines the sensitivity of cancer cells to the mechanistic target of rapamycin complex 1 (mTORC1)-targeting agent rapamycin. mTORC1 induces m6A modification of MAPK13 mRNA at its 3' untranslated region (3'UTR) through the methyltransferase-like 3 (METTL3)-METTL14-Wilms' tumor 1-associating protein (WTAP) methyltransferase complex, facilitating its mRNA degradation via an m6A reader protein YTH domain family protein 2 (YTHDF2). Rapamycin blunts this process and stabilizes MAPK13. On the other hand, genetic or pharmacological inhibition of MAPK13 enhances rapamycin's anti-cancer effects, which suggests that MAPK13 confers a pro-growth signal upon rapamycin treatment, thereby limiting rapamycin efficacy. Together, our data indicate that rapamycin-mediated MAPK13 mRNA stabilization underlies drug resistance, and it should be considered as a promising therapeutic target to sensitize cancer cells to rapamycin.
    Keywords:  MAPK13; RNA modification; RNA stability; Rapamycin; m(6)A; mTORC1; p38
    DOI:  https://doi.org/10.1016/j.jbc.2023.105175
  7. FASEB J. 2023 Sep;37(9): e23155
    Exploring the role of m6A methylation regulators in glioblastoma multiforme and their impact on the tumor immune microenvironment.
    Xinpeng Deng, Xiaoke Sun, Ziliang Hu, Yiwen Wu, Chenhui Zhou, Jie Sun, Xiang Gao, Yi Huang.
      Although the role of N6-Methyladenosine (m6A) methylation factors has been established in multiple cancer types, its involvement in glioblastoma multiforme (GBM) remains limited. This study aims to explore the involvement of m6A regulators in GBM and examine their association with the tumor immune microenvironment (TIME). A comprehensive set of 24 candidate m6A RNA regulators was procured. Consensus clustering was performed based on these regulators to identify distinct GBM clusters. PD-L1 and PD-1 levels, immune cell infiltration, and immune scores were evaluated between two clusters. Prognostic signatures and correlation analysis with TIME were analyzed using Lasso and Spearman's analysis. GBM tissue was collected to verify the correlations. Eighteen m6A regulators (WTAP, YTHDF2, HNRNPC, CAPRIN1, YTHDF3, METTL14, GNL3, ZCCHC4, HNRNPD, YTHDF1, RBM15, PCIF1, RBM27, KIAA1429, MSI2, FTO, ALKBH5, and METTL3), PD-L1, and PD-1 were significantly upregulated in GBM tissue. These regulators were divided into two distinct molecular subtypes (clusters 1 and 2). Cluster 2 exhibited a significant increase in immune score, monocytes, M1 macrophages, activated mast cells, and eosinophils. HNRNPC, YWHAG, and ALKBH5 were significantly associated with TIME and positively correlated with PD-L1. Immune cell invasiveness profiles dynamically changed with copy number changes of these three m6A regulators. Finally, YWHAG and ALKBH5 were found to be independent prognostic indicators of GBM through risk analysis and were experimentally verified with clinical samples. YWHAG and ALKBH5 may be used as prognostic markers for patients with GBM. m6A methylation regulators may play an important role in regulating PD-L1/PD-1 expression and immune infiltration, thus having a significant impact on GBM TIME.
    Keywords:  N6-methyladenosine methylation; PD-1; PD-L1; RNA methylation; glioblastoma multiforme; immune infiltrates; prognosis
    DOI:  https://doi.org/10.1096/fj.202301343
  8. Res Sq. 2023 Aug 08. pii: rs.3.rs-3150186. [Epub ahead of print]
    A cryptic pocket in METTL3-METTL14 regulates m6A conversion and sensing.
    Yogesh Gupta, Shan Qi.
      The nuclear METTL3-METTL14 enzyme complex transfers a methyl group from S-adenosyl-L-methionine (SAM) to the N 6 amino group of an adenosine (A) base in RNA to convert it to m 6 A and in ssDNA to 6mA. m 6 A marks are prevalent in eukaryotic mRNAs and lncRNAs and modulate their stability and fate in a context-dependent manner. The cytoplasmic METTL3 can act as a m6A reader to regulate mRNA translation. However, the precise mechanism that actuates the switch from m 6 A writer to reader/sensor is unclear. Here, we present a ~2.5Å crystal structure of the methyltransferase core of human METTL3-METTL14 in complex with the reaction product, N 6 -methyladenosine monophosphate (m 6 A), representing a state post-catalysis but before the release of m 6 A. m 6 A occupies a novel evolutionarily conserved cryptic pocket in METTL3-METTL14 located ~16Å away from the SAM pocket that frequently mutates in cancer. We propose a two-step model of swiveling of target A upon conversion to m 6 A and sensing its methylation status by the cryptic pocket, enabling it to actuate enzymes' switch from writer to an m 6 A-sensor. Cancer-associated mutations cannot distinguish methylated from unmethylated adenine and show impaired RNA binding, de-stacking, and defective m 6 A writing and sensing.
    DOI:  https://doi.org/10.21203/rs.3.rs-3150186/v1
  9. Mol Biol Rep. 2023 Aug 20.
    The implications of N6-methyladenosine (m6A) modification in esophageal carcinoma.
    Cheng He, Xiao Teng, Luming Wang, Miaoqi Ni, Linhai Zhu, Jiacong Liu, Wang Lv, Jian Hu.
      Esophageal carcinoma (EC) is always diagnosed at advanced stage and its the mortality rate remains high. The patients usually miss the best opportunity for treatment because of non-specific symptoms and the survival rates are low. N6-methyladenosine (m6A) the predominant modification in eukaryotic messenger RNA(mRNA), serves vital roles in numerous bioprocess. This chemical modification is dynamic, reversible and consists of three regulators: m6A methyltransferases (writers), demethylases (erasers) and m6A-binding proteins (readers). Recently, a growing number of evidences have indicated relationships between m6A and EC. Whereas, lacking of cognition about the molecular mechanism of m6A modification in esophageal carcinoma. We will focus on the biological function roles of m6A modification in the tumorigenesis and development of EC. Recent studies showed that immunotherapy had a positive impact on EC. The relationship between m6A and immunotherapy in EC deserves further research and discussion. We will also discuss the potential clinical applications regarding diagnosis, treatment and prognosis of m6A modification for EC and provide perspectives for further studies.
    Keywords:  Diagnosis; Erasers; Esophageal carcinoma; N6-methyladenosine (m6A); Readers; Therapy; Writers
    DOI:  https://doi.org/10.1007/s11033-023-08575-2
  10. bioRxiv. 2023 Aug 09. pii: 2023.08.08.552509. [Epub ahead of print]
    SUMOylation of the m6A reader YTHDF2 by PIAS1 promotes viral RNA decay to restrict EBV replication.
    Febri Gunawan Sugiokto, Farjana Saiada, Kun Zhang, Renfeng Li.
      YTHDF2 is a member of the YTH protein family that binds to N6-methyladenosine (m6A)-modified RNA, regulating RNA stability and restricting viral replication, including Epstein-Barr virus (EBV). PIAS1 is an E3 SUMO ligase known as an EBV restriction factor, but its role in YTHDF2 SUMOylation remains unclear. In this study, we investigated the functional regulation of YTHDF2 by PIAS1. We found that PIAS1 promotes the SUMOylation of YTHDF2 at three specific lysine residues (K281, K571, and K572). Importantly, PIAS1 enhances the antiviral activity of YTHDF2, and SUMOylation-deficient YTHDF2 shows reduced anti-EBV activity. Mechanistically, YTHDF2 lacking SUMOylation exhibits reduced binding to EBV transcripts, leading to increased viral mRNA stability. Furthermore, PIAS1 mediates SUMOylation of YTHDF2's paralogs, YTHDF1 and YTHDF3. These results collectively uncover a unique mechanism whereby YTHDF2 controls EBV replication through PIAS1-mediated SUMOylation, highlighting the significance of SUMOylation in regulating viral mRNA stability and EBV replication.
    Importance: N6-methyladenosine (m6A) RNA modification pathway plays important roles in diverse cellular processes and viral life cycle. Here, we investigated the relationship between PIAS1 and the m6A reader protein YTHDF2, which is involved in regulating RNA stability by binding to m6A-modified RNA. We found that both the N-terminal and C-terminal regions of YTHDF2 interact with PIAS1. We showed that PIAS1 promotes the SUMOylation of YTHDF2 at three specific lysine residues. We also demonstrated that PIAS1 enhances the anti-Epstein-Barr virus (EBV) activity of YTHDF2. We further revealed that PIAS1 mediates the SUMOylation of other YTHDF family members, namely YTHDF1 and YTHDF3, due to their similarities with YTHDF2. These findings together illuminate an important regulatory mechanism of YTHDF2 in controlling viral RNA decay and EBV replication through PIAS1-mediated SUMOylation.
    DOI:  https://doi.org/10.1101/2023.08.08.552509
  11. ACS Bio Med Chem Au. 2023 Aug 16. 3(4): 359-370
    Structure-Based Design of Inhibitors of the m6A-RNA Writer Enzyme METTL3.
    Rajiv Kumar Bedi, Danzhi Huang, Yaozong Li, Amedeo Caflisch.
      Methyltransferase-like 3 (METTL3) and METTL14 form a heterodimeric complex that catalyzes the most abundant internal mRNA modification, N6-methyladenosine (m6A). METTL3 is the catalytic subunit that binds the co-substrate S-adenosyl methionine (SAM), while METTL14 is involved in mRNA binding. The m6A modification provides post-transcriptional level control over gene expression as it affects almost all stages of the mRNA life cycle, including splicing, nuclear export, translation, and decay. There is increasing evidence for an oncogenic role of METTL3 in acute myeloid leukemia. Here, we use structural and dynamic details of the catalytic subunit METTL3 for developing small-molecule inhibitors that compete with SAM. Starting from a hit identified by high-throughput docking, protein crystallography and molecular dynamics simulations were employed to guide the optimization of inhibitory activity. The potency was successfully improved by 8000-fold as measured by a homogeneous time-resolved fluorescence assay. The optimized compound is selective against the off-targets RNA methyltransferases METTL1 and METTL16.
    DOI:  https://doi.org/10.1021/acsbiomedchemau.3c00023
  12. Ann Clin Lab Sci. 2023 Jul;53(4): 529-538
    RBM15-Mediated m6A Modification of K17 Affects Keratinocytes Response to IL-17A Stimulation in Psoriasis.
    Manni Fu, Bei Zhou, Nian Shi, Rongjing Dong, Yongjun Chen.
       OBJECTIVE: Psoriasis is characterized by excessive proliferation and abnormal differentiation of epidermal keratinocytes. This study aimed to reveal the function and mechanism of a N6-methyladenosine (m6A) methyltransferase RNA-binding motif protein 15 (RBM15) in IL-17A-induced keratinocytes.
    METHODS: A immortalized keratinocyte cell line HaCaT was used to undergo the IL-17A stimulation. The mRNA levels were detected by qRT-PCR, whereas the protein levels were measured by western blotting. The change of keratinocytes proliferation was determined using CCK8 and EdU assays, and the inflammation factors (IL-8 and TNF-α) in keratinocytes were analyzed by qRT-PCR. The m6A modification of Keratin 17 (K17) was confirmed by MeRIP and mRNA stability assays.
    RESULTS: The levels of RBM15 and K17 in skin samples from patients with psoriasis and IL-17A-induced keratinocytes were upregulated, and showed the positive correlation. Silencing RBM15 suppressed viability, proliferation, and inflammation of keratinocytes that were enhanced by IL-17A stimulation. Moreover, RBM15 knockdown reduced the stability of K17 mRNA via m6A modification method. Since K17 is modified by RBM15, its overexpression relieved the effects of RBM15 knockdown on keratinocytes under IL-17A stimulation.
    CONCLUSION: This study revealed that RBM15 knockdown suppressed proliferation and inflammation by mediating m6A modification of K17 to reduce K17 stability in IL-17A-induced keratinocytes. Our findings may provide novel idea for improving the treatment of psoriasis.
    Keywords:  IL-17A; K17; RBM15; keratinocyte; psoriasis
  13. Int Immunopharmacol. 2023 Aug 20. pii: S1567-5769(23)01109-8. [Epub ahead of print]124(Pt A): 110784
    Comprehensive analysis of m6A subtype classification for immune microenvironment of pituitary adenomas.
    Feng Yuan, Xiangming Cai, Yingshuai Wang, Chaonan Du, Zixiang Cong, Xinrui Zeng, Chao Tang, Chiyuan Ma.
       BACKGROUND: N6-methyladenosine (m6A) RNA methylation and tumor immune microenvironment (IME) have an essential role in tumor development. However, their relationships in pituitary adenomas (PAs) remains unclear.
    METHODS: PA datasets from the Gene Expression Omnibus (GEO) and European Bioinformatics Institute (EMBL-EBI) were used. We utilized hierarchical clustering algorithms based on the m6A regulator gene set to identify m6A subtypes. ESTIMATE and CIBERSORT algorithms were applied to explore the compositions of stromal and immune cells. A nomogram model was constructed for the prediction of m6A subtypes in PAs. Immunohistochemistry and multiplex immunofluorescence staining were used to analyze the expression level of m6A regulator YTHDF2 in relation to M2 macrophages and immune checkpoints in PAs.
    RESULTS: We concluded the IME landscape of m6A subtype classification and characterized two emerging m6A subtypes. Different IME between these two m6A subtypes were identified. Simultaneously, a polygenic nomogram model was constructed for predicting m6A subtype classification, with excellent predictive performance (training set, AUC = 0.984; validation set, AUC = 0.986). YTHDF2 was highly expressed in PAs and accompanied by upregulated M2 macrophages and expression of PD-L1.
    CONCLUSIONS: We proposed two novel m6A subtypes in PAs for the first time and constructed a reliable and clinically accessible nomogram model for them. Meanwhile, YTHDF2 was first identified as a promising biomarker for immunotherapy and potential molecular target in PAs.
    Keywords:  Classification; Immune Microenvironment (IME); Immunotherapy; N6-methyladenosine (m(6)A); Nomogram; Pituitary Adenomas (PAs)
    DOI:  https://doi.org/10.1016/j.intimp.2023.110784
  14. Neurochem Res. 2023 Aug 23.
    METTL3-deficiency Suppresses Neural Apoptosis to Induce Protective Effects in Cerebral I/R Injury via Inhibiting RNA m6A Modifications: A Pre-clinical and Pilot Study.
    Gang Huang, Yuda Qiu, Yafei Fan, Jianfeng Liu.
      N6-Methyladenosine (m6A) RNA methylation involves in regulating the initiation, progression and aggravation of cerebral ischemia-reperfusion (I/R) injury, however, the detailed functions and mechanisms by which m6A drives cerebral I/R injury are not fully understood. This study found that methyltransferase-like 3 (METTL3) m6A-dependently regulated cerebral I/R injury trough regulating a novel LncRNA ABHD11-AS1/miR-1301-3p/HIF1AN/HIF-1α axis. Specifically, the middle cerebral artery occlusion (MCAO)/reperfusion mice models and glucose deprivation (OGD)/reoxygenation (RX) astrocyte cell models were respectively established, and we verified that METTL3, ABHD11-AS1 and HIF1AN were upregulated, whereas miR-1301-3p and HIF-1α were downregulated in both MCAO/reperfusion mice tissues and OGD/RX astrocytes. Mechanical experiments confirmed that METTL3 m6A dependently increased stability and expression levels of ABHD11-AS1, and elevated ABHD11-AS1 sponged miR-1301-3p to upregulate HIF1AN, resulting in the downregulation of HIF-1α. Moreover, silencing of METTL3 rescued MCAO/reperfusion and OGD/RX-induced oxidative stress-associated cell apoptosis and cell cycle arrest in both mice brain tissues in vivo and the mouse primary astrocytes in vitro, which were abrogated by overexpressing ABHD11-AS1 and downregulating miR-1301-3p. Taken together, our study firstly reported a novel METTL3/m6A/ ABHD11-AS1/miR-1301-3p/HIF1AN/HIF-1α signaling cascade in regulating the progression of cerebral I/R injury, and future work will focus on investigating whether the above genes can be used as biomarkers for the treatment of cerebral I/R injury by performing clinical studies.
    Keywords:  Cell apoptosis; Cerebral I/R injury; LncRNA ABHD11-AS1; METTL3; N6-Methyladenosine; Oxidative stress; cell cycle arrest
    DOI:  https://doi.org/10.1007/s11064-023-04015-6
  15. Hum Genomics. 2023 Aug 25. 17(1): 78
    The RNA m6A modification might participate in microglial activation during hypoxic-ischemic brain damage in neonatal mice.
    Xiaojuan Su, Lingyi Huang, Shiping Li, Junjie Ying, Fengyan Zhao, Shaopu Wang, Qian Liu, Yi Qu, Dezhi Mu.
       BACKGROUND: The RNA m6A modification has been implicated in multiple neurological diseases as well as macrophage activation. However, whether it regulates microglial activation during hypoxic-ischemic brain damage (HIBD) in neonates remains unknown. Here, we aim to examine whether the m6A modification is involved in modulating microglial activation during HIBD. We employed an oxygen and glucose deprivation microglial model for in vitro studies and a neonatal mouse model of HIBD. The brain tissue was subjected to RNA-seq to screen for significant changes in the mRNA m6A regulator. Thereafter, we performed validation and bioinformatics analysis of the major m6A regulators.
    RESULTS: RNA-seq analysis revealed that, among 141 m6A regulators, 31 exhibited significant differential expression (FC (abs) ≥ 2) in HIBD mice. We then subjected the major m6A regulators Mettl3, Mettl14, Fto, Alkbh5, Ythdf1, and Ythdf2 to further validation, and the results showed that all were significantly downregulated in vitro and in vivo. GO analysis reveals that regulators are mainly involved in the regulation of cellular and metabolic processes. The KEGG results indicate the involvement of the signal transduction pathway.
    CONCLUSIONS: Our findings demonstrate that m6A modification of mRNA plays a crucial role in the regulation of microglial activation in HIBD, with m6A-associated regulators acting as key modulators of microglial activation.
    Keywords:  Hypoxia–ischemia brain damage; Microglia activation; RNA m6A regulators; RNA-sequencing; m6A modification
    DOI:  https://doi.org/10.1186/s40246-023-00527-y
  16. Exp Ther Med. 2023 Sep;26(3): 420
    RNA N6‑methyladenosine methyltransferase WTAP promotes the differentiation of endothelial progenitor cells.
    Longyun Wu, Lili Niu, Zhou Yang, Qiaoyun Xia, Jingyuan Xu, Xiaolan Lu.
      N6-methyladenosine (m6A) serves a critical role in regulating gene expression and has been associated with various diseases; however, its role in the differentiation of endothelial progenitor cells (EPCs) remains unclear. The present study used liquid chromatography with tandem mass spectrometry and immunofluorescence assays to quantify the levels of m6A in human peripheral blood-derived EPCs (HPB-EPCs) before and after differentiation into mature cells. The present study performed Cell Counting Kit 8, Transwell, and tube formation assays to determine the effects of overexpression and knockdown of Wilms' tumor 1-associated protein (WTAP) on HPB-EPCs. The results revealed that the level of m6A modification was significantly increased during HPB-EPCs differentiation, and WTAP exhibited the most significant alteration among the enzymes involved in m6A regulation. When WTAP was overexpressed in HPB-EPCs, cell proliferation, invasion, and the formation of tubes were improved, whereas WTAP knockdown yielded the opposite effects. In conclusion, the present study highlighted the involvement of m6A in regulating EPC differentiation, with WTAP acting as a promoter of EPC differentiation.
    Keywords:  N6-methyladenosine; Wilms' tumor 1-associated protein; cell differentiation; endothelial progenitor cell
    DOI:  https://doi.org/10.3892/etm.2023.12119
  17. Neurol Int. 2023 Aug 10. 15(3): 967-979
    In Search of a Function for the N6-Methyladenosine in Epitranscriptome, Autophagy and Neurodegenerative Diseases.
    Naoko Suga, Yuka Ikeda, Sayuri Yoshikawa, Kurumi Taniguchi, Haruka Sawamura, Satoru Matsuda.
      Changes in epitranscriptome with N6-methyladenine (m6A) modification could be involved in the development of multiple diseases, which might be a prevalent modification of messenger RNAs (mRNAs) in eukaryotes. The m6A modification might be performed through the action of methyltransferases, demethylases, and methylation-binding proteins. Importantly, the m6A methylation may be associated with various neurological disorders including Alzheimer's disease (AD), Parkinson's disease (PD), depression, aging-related diseases, and/or aging itself. In addition, the m6A methylation might functionally regulate the eukaryotic transcriptome by influencing the splicing, export, subcellular localization, translation, stability, and decay of mRNAs. Neurodegenerative diseases may possess a wide variety of phenotypes, depending on the neurons that degenerate on occasion. Interestingly, an increasing amount of evidence has indicated that m6A modification could modulate the expression of autophagy-related genes and promote autophagy in neuronal cells. Oxidative stresses such as reactive oxygen species (ROS) could stimulate the m6A RNA methylation, which may also be related to the regulation of autophagy and/or the development of neurodegenerative diseases. Both m6A modification and autophagy could also play critical roles in regulating the health condition of neurons. Therefore, a comprehensive understanding of the m6A and autophagy relationship in human diseases may benefit in developing therapeutic strategies in the future. This paper reviews advances in the understanding of the regulatory mechanisms of m6A modification in the occurrence and development of neurodegenerative diseases and/or aging, discussing the possible therapeutic procedures related to mechanisms of m6A RNA methylation and autophagy.
    Keywords:  Alzheimer’s disease; N6-methyladenine; RNA-binding protein; neurodegenerative disease; reactive oxygen species
    DOI:  https://doi.org/10.3390/neurolint15030062
  18. Cell Death Dis. 2023 Aug 25. 14(8): 557
    N6-methyladenosine modification of REG1α facilitates colorectal cancer progression via β-catenin/MYC/LDHA axis mediated glycolytic reprogramming.
    Mingxia Zhou, Jing He, Yingxia Li, Libin Jiang, Jiaxuan Ran, Chang Wang, Chenxi Ju, Dan Du, Xinyu Xu, Xuexin Wang, Hongle Li, Fucheng He, Hongtao Wen.
      Aerobic glycolysis has been considered as a hallmark of colorectal cancer (CRC). However, the potential functional regulators of glycolysis in CRC remains to be elucidated. In the current study, we found that Regenerating islet-derived protein 1-alpha (REG1α) was significantly increased in both CRC tissues and serum, and positively associated with CRC patients' lymph node metastasis, advanced tumor stage, and unfavorable prognosis. Ectopic expression of REG1α contributed to various tumorigenic properties, including cell proliferation, cell cycle, migration, invasion, and glycolysis. In contrast, REG1α deficiency in CRC cells attenuated malignant properties and glucose metabolism. Mechanically, REG1α promoted CRC proliferation and metastasis via β-catenin/MYC axis-mediated glycolysis upregulation. Moreover, the malignant behaviors governed by REG1α could be effectively abolished by silencing of Wnt/β-catenin/MYC axis or glycolysis process using specific inhibitors. Besides, REG1α expression was mediated by METTL3 in an m6A-dependent manner. Overall, our work defines a novel regulatory model of the METTL3/REG1α/β-catenin/MYC axis in CRC, which indicates that REG1α could function as a novel biomarker and a potential therapeutic target for patients with CRC.
    DOI:  https://doi.org/10.1038/s41419-023-06067-6
  19. Chem Commun (Camb). 2023 Aug 21.
    Xanthine derivatives inhibit FTO in an L-ascorbic acid-dependent manner.
    Kamui Tanaka, Akiyo Suda, Motonari Uesugi, Shiroh Futaki, Miki Imanishi.
      Xanthine derivatives were identified as inhibitors of the N6-methyladenosine (m6A) demethylase activity of fat-mass-and-obesity-associated protein (FTO) by activity-based high-throughput screening using the m6A-sensitive ribonuclease MazF. Pentoxifylline exhibited L-ascorbic acid concentration-dependent inhibitory activity against FTO, an unprecedented mode of inhibition, indicating that L-ascorbic acid is a promising key for designing FTO-specific inhibitors.
    DOI:  https://doi.org/10.1039/d3cc02484a
  20. J Exp Clin Cancer Res. 2023 Aug 21. 42(1): 215
    P300/SP1 complex mediating elevated METTL1 regulates CDK14 mRNA stability via internal m7G modification in CRPC.
    Mingpeng Zhang, Duo Kan, Boya Zhang, Xueqiao Chen, Chun Wang, Songmao Chen, Wenlong Gao, Zhao Yang, Yang Li, Yutong Chen, Shimiao Zhu, Simeng Wen, Yuanjie Niu, Zhiqun Shang.
       BACKGROUND: N7-methylguanosine (m7G) modification is, a more common epigenetic modification in addition to m6A modification, mainly found in mRNA capsids, mRNA interiors, transfer RNA (tRNA), pri-miRNA, and ribosomal RNA (rRNA). It has been found that m7G modifications play an important role in mRNA transcription, tRNA stability, rRNA processing maturation, and miRNA biosynthesis. However, the role of m7G modifications within mRNA and its "writer" methyltransferase 1(METTL1) in tumors, particularly prostate cancer (PCa), has not been revealed.
    METHODS: The differential expression level of METTL1 between hormone-sensitive prostate cancer (HSPC) and castrate-resistant prostate cancer (CRPC) was evaluated via RNA-seq and in vitro experiments. The effects of METTL1 on CRPC progression were investigated through in vitro and in vivo assays. The upstream molecular mechanism of METTL1 expression upregulation and the downstream mechanism of its action were explored via Chromatin Immunoprecipitation quantitative reverse transcription polymerase chain reaction (CHIP-qPCR), Co-immunoprecipitation (Co-IP), luciferase reporter assay, transcriptome-sequencing, m7G AlkAniline-Seq, and mRNA degradation experiments, etc. RESULTS AND CONCLUSION: Here, we found that METTL1 was elevated in CRPC and that patients with METTL1 elevation tended to have a poor prognosis. Functionally, the knockdown of METTL1 in CRPC cells significantly limited cell proliferation and invasive capacity. Mechanistically, we unveiled that P300 can form a complex with SP1 and bind to the promoter region of the METTL1 gene via SP1, thereby mediating METTL1 transcriptional upregulation in CRPC. Subsequently, our findings indicated that METTL1 leads to enhanced mRNA stability of CDK14 by adding m7G modifications inside its mRNA, ultimately promoting CRPC progression.
    Keywords:  CDK14; CRPC; METTL1; P300/SP1 complex; m7G
    DOI:  https://doi.org/10.1186/s13046-023-02777-z
  21. Cell Death Dis. 2023 Aug 25. 14(8): 560
    The m6A modification-mediated OGDHL exerts a tumor suppressor role in ccRCC by downregulating FASN to inhibit lipid synthesis and ERK signaling.
    Jian Shi, Daojia Miao, Qingyang Lv, Keshan Wang, Qi Wang, Huageng Liang, Hongmei Yang, Zhiyong Xiong, Xiaoping Zhang.
      Metabolic reprogramming is a hallmark of cancer, and the impact of lipid metabolism as a crucial aspect of metabolic reprogramming on clear cell renal cell carcinoma (ccRCC) progression has been established. However, the regulatory mechanisms underlying the relationship between metabolic abnormalities and ccRCC progression remain unclear. Therefore, this study aimed to identify key regulatory factors of metabolic reprogramming in ccRCC and provide potential therapeutic targets for ccRCC patients. Potential metabolic regulatory factors in ccRCC were screened using bioinformatics analysis. Public databases and patient samples were used to investigate the aberrant expression of Oxoglutarate dehydrogenase-like (OGDHL) in ccRCC. The function of OGDHL in ccRCC growth and metastasis was evaluated through in vitro and in vivo functional experiments. Mechanistic insights were obtained through luciferase reporter assays, chromatin immunoprecipitation, RNA methylation immunoprecipitation, and mutagenesis studies. OGDHL mRNA and protein levels were significantly downregulated in ccRCC tissues. Upregulation of OGDHL expression effectively inhibited ccRCC growth and metastasis both in vitro and in vivo. Furthermore, FTO-mediated OGDHL m6A demethylation suppressed its expression in ccRCC. Mechanistically, low levels of OGDHL promoted TFAP2A expression by inhibiting ubiquitination levels, which then bound to the FASN promoter region and transcriptionally activated FASN expression, thereby promoting lipid accumulation and ERK pathway activation. Our findings demonstrate the impact of OGDHL on ccRCC progression and highlight the role of the FTO/OGDHL/TFAP2A/FASN axis in regulating ccRCC lipid metabolism and progression, providing new targets for ccRCC therapy.
    DOI:  https://doi.org/10.1038/s41419-023-06090-7
  22. Front Genet. 2023 ;14 1188048
    Analysis of mRNA m6A modification and mRNA expression profiles in middle ear cholesteatoma.
    Shumin Xie, Li Jin, Jun He, Jinfeng Fu, Tuanfang Yin, Jihao Ren, Wei Liu.
      Introduction: Middle ear cholesteatoma is characterized by the hyperproliferation of keratinocytes. In recent decades, N6-methyladenosine (m6A) modification has been shown to play an essential role in the pathogenesis of many proliferative diseases. However, neither the m6A modification profile nor its potential role in the pathogenesis of middle ear cholesteatoma has currently been investigated. Therefore, this study aimed to explore m6A modification patterns in middle ear cholesteatoma. Materials and methods: An m6A mRNA epitranscriptomic microarray analysis was performed to analyze m6A modification patterns in middle ear cholesteatoma tissue (n = 5) and normal post-auricular skin samples (n = 5). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to predict the potential biological functions and signaling pathways underlying the pathogenesis of middle ear cholesteatoma. Subsequently, m6A modification levels were verified by methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) in middle ear cholesteatoma tissue and normal skin samples, respectively. Results: A total of 6,865 distinctive m6A-modified mRNAs were identified, including 4,620 hypermethylated and 2,245 hypomethylated mRNAs, as well as 9,162 differentially expressed mRNAs, including 4,891 upregulated and 4,271 downregulated mRNAs, in the middle ear cholesteatoma group relative to the normal skin group. An association analysis between methylation and gene expression demonstrated that expression of 1,926 hypermethylated mRNAs was upregulated, while expression of 2,187 hypomethylated mRNAs and 38 hypermethylated mRNAs was downregulated. Moreover, GO analysis suggested that differentially methylated mRNAs might influence cellular processes and biological behaviors, such as cell differentiation, biosynthetic processes, regulation of molecular functions, and keratinization. KEGG pathway analysis demonstrated that the hypermethylated transcripts were involved in 26 pathways, including the Hippo signaling pathway, the p53 signaling pathway, and the inflammatory mediator regulation of transient receptor potential (TRP) channels, while the hypomethylated transcripts were involved in 13 pathways, including bacterial invasion of epithelial cells, steroid biosynthesis, and the Hippo signaling pathway. Conclusion: Our study presents m6A modification patterns in middle ear cholesteatoma, which may exert regulatory roles in middle ear cholesteatoma. The present study provides directions for mRNA m6A modification-based research on the epigenetic etiology and pathogenesis of middle ear cholesteatoma.
    Keywords:  expression profile; m6A; mRNA; methylation profile; middle ear cholesteatoma
    DOI:  https://doi.org/10.3389/fgene.2023.1188048
  23. Cell Transplant. 2023 Jan-Dec;32:32 9636897231188300
    METTL3 Promotes the Growth and Invasion of Melanoma Cells by Regulating the lncRNA SNHG3/miR-330-5p Axis.
    Shaojun Chu, Yulong Li, Baojin Wu, Guo Rong, Qiang Hou, Qin Zhou, Dexiang Du, Yufei Li.
      Accumulating evidence indicates that m6A methyltransferase 3 (METTL3) plays a pivotal role in different malignancies including melanoma. However, the function and underlying mechanisms by which METTL3 contributes to the tumorigenesis of melanoma remain undocumented. The association of METTL3 and long noncoding RNA (lncRNA) small nucleolar RNA host gene 3 (SNHG3) with clinicopathological characteristics and prognosis in patients with melanoma was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting, and The Cancer Genome Atlas data sets. The role of METTL3 in melanoma cells was assessed by in vitro and in vivo experiments. The m6A dot blot, methylated RNA immunoprecipitation (MeRIP), and RT-qPCR were used to verify METTL3-mediated m6A modification of lncRNA SNHG3. The effect of METTL3 on lncRNA SNHG3 was determined by luciferase gene reporter assay, RT-qPCR, and Western blotting. We found that METTL3 was upregulated in melanoma tissue samples and associated with poor survival in patients with melanoma. Knockdown of METTL3 suppressed the growth and invasion of melanoma cells in vitro and in vivo, whereas restored expression of METTL3 promoted these effects. Mechanistic investigations showed that knockdown of METTL3 reduced SNHG3 m6A levels and its messenger ribonucleic acid (mRNA) expression levels. SNHG3 could act as a sponge of microRNA (miR)-330-5p to upregulate the expression of CCHC-type zinc finger nucleic acid binding protein (CNBP). SNHG3 overexpression reversed METTL3-knockdown-caused antitumor effects, miR-330-5p upregulation and CNBP downregulation. SNHG3 had a positive correlation with METTL3 expression but a negative correlation with miR-330-5p expression in melanoma tissue samples. In conclusion, our findings demonstrated that METTL3-mediated m6A modification of lncRNA SNHG3 promoted the growth and invasion of melanoma cells by regulating the miR-330-5p/CNBP axis.
    Keywords:  METTL3; SNHG3; m6A; melanoma; miR-330-5p
    DOI:  https://doi.org/10.1177/09636897231188300
  24. Cell Mol Biol (Noisy-le-grand). 2023 Jun 30. 69(6): 23-28
    Role of METTL3-mediated m6A modification in osteogenic differentiation of periodontal ligament stem cells extracted from adult periodontal ligaments ex-vivo.
    Chun Yao, Haiyan Xin, Si Liu, Peng She, Yanhong Ma, Min Hu, Yan Jin.
      Periodontal ligament stem cells (PDLSCs) are identified as candidate cells for the regeneration of periodontal and alveolar bone tissues. This research was to analyze the effect of methyltransferase-like 3 (METTL3)-mediated m6A modification on the osteogenic differentiation of PDLSCs extracted from adult periodontal ligaments (PDLs) ex-vivo. From June 2022 to October 2022, 27 patients undergoing orthodontic treatment in our hospital were selected as the research population, with 31 teeth extracted in total. PDLSCs were isolated from PDLs by tissue block culture, and the results were analyzed. Then PDLSCs were induced to differentiate into osteoblasts, and changes in METTL3 and m6A levels during differentiation were observed. Additionally, abnormal METTL3 expression vectors were constructed and transfected into PDLSCs to observe the influence of METTL3 on the biological behavior and osteogenic differentiation of PDLSCs. PDLSCs isolated from ex-vivo PDLs were predominantly spindle-shaped, with high CD73, CD90 and CD105 levels and low CD11b, CD34 and CD45 levels, showing the characteristics of stem cells. Spearman correlation coefficients identified a positive connection between Runx2, Sp7, Alp, Bglap, METTL3 and m6A levels and osteogenic differentiation incubation time (P<0.05). As METTL3 expression was increased, the proliferation capacity and osteogenic differentiation ability of PDLSCs were enhanced (P<0.05), and the content of m6A was increased (P<0.05). However, the activity and osteogenic differentiation ability of PDLSCs was decreased after silencing METTL3 (P<0.05). In conclusion, METTL3-mediated m6A modification promoted the osteogenic differentiation of PDLSCs extracted from adult PDLs ex vivo. This study offered a novel understanding of the mechanisms underlying osteogenic differentiation, and implied a possible method for accelerating bone formation.
    DOI:  https://doi.org/10.14715/cmb/2023.69.6.4
  25. Genes (Basel). 2023 Aug 19. pii: 1653. [Epub ahead of print]14(8):
    Changes in m6A in Steatotic Liver Disease.
    Belinda J Petri, Matthew C Cave, Carolyn M Klinge.
      Fatty liver disease is one of the major causes of morbidity and mortality worldwide. Fatty liver includes non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), now replaced by a consensus group as metabolic dysfunction-associated steatotic liver disease (MASLD). While excess nutrition and obesity are major contributors to fatty liver, the underlying mechanisms remain largely unknown and therapeutic interventions are limited. Reversible chemical modifications in RNA are newly recognized critical regulators controlling post-transcriptional gene expression. Among these modifications, N6-methyladenosine (m6A) is the most abundant and regulates transcript abundance in fatty liver disease. Modulation of m6A by readers, writers, and erasers (RWE) impacts mRNA processing, translation, nuclear export, localization, and degradation. While many studies focus on m6A RWE expression in human liver pathologies, limitations of technology and bioinformatic methods to detect m6A present challenges in understanding the epitranscriptomic mechanisms driving fatty liver disease progression. In this review, we summarize the RWE of m6A and current methods of detecting m6A in specific genes associated with fatty liver disease.
    Keywords:  NAFLD; NASH; RNA modifications; epitranscriptome; fatty liver; m6A
    DOI:  https://doi.org/10.3390/genes14081653
  26. Sci Rep. 2023 Aug 21. 13(1): 13587
    miR-33a-3p regulates METTL3-mediated AREG stability and alters EMT to inhibit pancreatic cancer invasion and metastasis.
    Xiaowen Su, Tiantian Lai, Yue Tao, Yong Zhang, Changyong Zhao, Junjing Zhou, Enhong Chen, Maoqun Zhu, Shuo Zhang, Bei Wang, Yong Mao, Hao Hu.
      Recent studies have shown that amphoteric regulatory protein (AREG), a member of the epidermal growth factor (EGF) family, is expressed in many cancers and is an independent prognostic indicator for patients with pancreatic cancer, but whether AREG is regulated at the epigenetic level to promote the development of pancreatic cancer (PC) has not been elucidated. Our results support the notion that AREG is overexpressed in pancreatic cancer tissues and cell lines. Functionally, the deletion of AREG impedes pancreatic cancer (PC) cell proliferation, migration, and invasion. In addition, we identified and validated that methyltransferase-like 3 (METTL3) induced the m6A modification on AREG and facilitated the stability of AREG mRNA after sequencing. Additionally, we obtained experimental evidence that miR-33a-3p targets and inhibits METTL3 from taking action, as predicted by using the miRDB and RNAinter. Remediation experiments showed that miR-33a-3p inhibits PC progression through METTL3. In summary, this research reveals that miR-33a-3p inhibits m6A-induced stabilization of AREG by targeting METTL3, which plays a key role in the aggressive progression of PC. AREG could be a potential target for PC treatment.
    DOI:  https://doi.org/10.1038/s41598-023-39506-7
  27. Inflamm Bowel Dis. 2023 Aug 25. pii: izad113. [Epub ahead of print]
    CircASPH Enhances Exosomal STING to Facilitate M2 Macrophage Polarization in Colorectal Cancer.
    Yuting Zhang, Jiakun Guo, Liyin Zhang, Ying Li, Kangliang Sheng, Yawei Zhang, Liu Liu, Wenbin Gong, Kun Guo.
      Exosomes are considered a mediator of communication within the tumor microenvironment (TME), which modulates cancer progression through transmitting cargos between cancer cells and other cancer-related cells in TME. Circular RNAs (circRNAs) have emerged to be regulators in colorectal cancer (CRC) progression, but most of them have not been discussed in CRC. This study aims to investigate the role of circRNA aspartate beta-hydroxylase (circASPH) in CRC progression and its correlation with exosome-mediated TME. At first, we determined that circASPH was upregulated in CRC samples and cell lines. Functionally, the circASPH deficiency suppressed the malignant processes of CRC cells and also inhibited in vivo tumor growth via enhancing antitumor immunity. Mechanically, circASPH facilitated macrophage M2 polarization by upregulating exosomal stimulator of interferon genes (STING). CircASPH interacted with insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) to stabilize IGF2BP2 protein, therefore enhancing the stability of m6A-modified STING mRNA. In turn, coculture of STING-overexpressed macrophages recovered the suppression of silenced circASPH on the malignancy of CRC cells both in vitro and in vivo. Our study demonstrated that circASPH enhances exosomal STING to facilitate M2 macrophage polarization, which further accelerates CRC progression. The findings support circASPH as a promising therapeutic target for CRC treatment.
    Keywords:  STING; circASPH; colorectal cancer; exosome; macrophage polarization
    DOI:  https://doi.org/10.1093/ibd/izad113
  28. Exp Ther Med. 2023 Sep;26(3): 457
    Loss of NSUN6 inhibits osteosarcoma progression by downregulating EEF1A2 expression and activation of Akt/mTOR signaling pathway via m5C methylation.
    Sang Hu, Min Yang, Kangwen Xiao, Zhiqiang Yang, Lin Cai, Yuanlong Xie, Linlong Wang, Renxiong Wei.
      As an important 5-methylcytidine (m5C) methyltransferase, NOP2/Sun RNA methyltransferase family member 6 (NSUN6) has been reported to play an important role in the progression of several diseases. However, the role of NSUN6 in the progression of osteosarcoma (OS) remains unclear. This study aimed to identify the role of NSUN6 in the progression of OS and clarify the potential molecular mechanism. The present study discovered that NSUN6 was upregulated in OS and a higher NSUN6 expression was a strong indicator for poorer prognosis of patients with OS. In addition, the loss of NSUN6 led to reduced proliferation, migration and invasion of OS cells. Through bioinformatics analysis, RNA immunoprecipitation (RIP) and methylated RIP assays, eukaryotic elongation factor 1 α-2 (EEF1A2) was identified and validated as a potential target of NSUN6 in OS. Mechanistically, the expression of EEF1A2 was significantly suppressed following NSUN6 knockdown due to reduced EEF1A2 mRNA stability in an m5C-dependent manner. Meanwhile, NSUN6 deficiency inhibited m5C-dependent activation of Akt/mTOR signaling pathway. In addition, genetic overexpression of EEF1A2 or pharmacological activation of the Akt signaling pathway counteracted the suppressive effects of NSUN6 deficiency on the proliferation, invasion and migration of OS cells. The current findings suggested that NSUN6 may serve as a potential therapeutic target for OS treatment.
    Keywords:  5-methylcytidine; Akt/mTOR; NOP2/Sun RNA methyltransferase family member 6; RNA methylation; eukaryotic elongation factor 1 α-2
    DOI:  https://doi.org/10.3892/etm.2023.12156
  29. Brain Inj. 2023 Aug 23. 1-9
    Propofol suppresses OGD/R-induced ferroptosis in neurons by inhibiting the HIF-1α/YTHDF1/BECN1 axis.
    Hongyan Ma, Dongxue Ye, Yuqing Liu, Pei Wu, Lu Yu, Libo Guo, Yang Gao, Ying Liu, Haiyan Yan, Jinghui Shi.
       BACKGROUND: Ischemia/reperfusion (I/R) is a pathological process that causes severe damage. Propofol is known to alleviate I/R-related injury; however, the exact function and underlying mechanisms are not fully understood.
    METHODS: Using an oxygen glucose deprivation/re-oxygenation (OGD/R) method, an in vitro I/R injury model was induced. The cell viability and the level of Fe2+, glutathione synthetase (GSH), and malondialdehyde (MDA) were evaluated using kits. Luciferase reporter gene assay, chromatin immunoprecipitation, and RNA immunoprecipitation (RIP) were used to verify the interaction between molecules. The m6A level of BECN1 mRNA was determined through methylated RIP.
    RESULTS: Propofol-treated OGD/R models showed reduced levels of Fe2+ and MDA, while the cell viability and the level of GSH increased. Propofol inhibited ferroptosis by down-regulating HIF-1α in OGD/R-treated HT22 cells. HIF-1α is bound to the promoter region of YTHDF1 to promote its transcription, and YTHDF1 promoted ferroptosis by stabilizing the mRNA of BECN1. The suppressive effect of propofol on OGD/R-induced ferroptosis was reversed by the overexpression of YTHDF1.
    CONCLUSIONS: Our study revealed that the HIF-1α/YTHDF1/BECN1 axis in OGD/R-treated HT22 cells promotes ferroptosis, and administration of propofol can inhibit this axis to avoid cell death. This study provides a novel insight for the neuroprotective function of propofol.
    Keywords:  HIF-1α; Ischemia/reperfusion; ferroptosis; oxygen glucose deprivation/re-oxygenation; propofol
    DOI:  https://doi.org/10.1080/02699052.2023.2237881
  30. Plant Cell. 2023 Aug 23. pii: koad224. [Epub ahead of print]
    Pathogen-induced m6A dynamics affect plant immunity.
    Wil Prall, Arsheed H Sheikh, Jeremie Bazin, Jean Bigeard, Marilia Almeida-Trapp, Martin Crespi, Heribert Hirt, Brian D Gregory.
      Post-transcriptional regulation of mRNA mediated by methylation at the N6 position of adenine (N6-methyladenosine (m6A)) has profound effects on transcriptome regulation in plants. Focused studies across eukaryotes offer glimpses into the processes governed by m6A throughout developmental and disease states. However, we lack an understanding of the dynamics and the regulatory potential of m6A during biotic stress in plants. Here, we provide a comprehensive look into the effects of m6A on both the short-term and long-term response to pathogen signaling in Arabidopsis (Arabidopsis thaliana). We demonstrate that m6A-deficient plants are more resistant to bacterial and fungal pathogen infections and have altered immune responses. Furthermore, m6A deposition is specifically coordinated on transcripts involved in defense and immunity prior to and proceeding the pathogen signal flagellin. Consequently, the dynamic modulation of m6A on specific stress-responsive transcripts is correlated with changes in abundance and cleavage of these transcripts. Overall, we show that the m6A methylome is regulated prior to and during simulated and active pathogen stress and functions in the coordination and balancing of normal growth and pathogen responses.
    DOI:  https://doi.org/10.1093/plcell/koad224
  31. Ann Clin Transl Neurol. 2023 Aug 23.
    The role of m6A-modified CircEPHB4 in glioma pathogenesis: Insights into cancer stemness metastasis.
    Yuxiang Liao, Xiaohui Qiu, Jingping Liu, Zhiping Zhang, Bo Liu, Chen Jin.
       OBJECTIVE: While existing literature has provided insights into involvement of circEPHB4, SOX2 in glioma, their precise molecular mechanisms and synergistic implications in glioma pathogenesis still dim. This study aims to investigate significance and underlying mechanism of m6A-modified circEPHB4 in regulating SOX2/PHLDB2 axis in gliomas.
    METHODS: The mRNA and protein expression were tested by qRT-PCR and Western blot, respectively. ChIP assay was performed to detect SOX2 enrichment on the PHLDB2 promoter. Cell sphere-forming assay to detect self-renewal ability, flow cytometry to determine positivity of CD133 expressions, Malignant behavior of glioma cells were detected by CCK-8, plate colony formation, scratch, and transwell assays. Glioma xenograft models were constructed to investigate effects of CircEPHB4 in tumor development in vivo.
    RESULTS: Methyltransferase MELLT3 upregulated m6A modification of CircEPHB4, and YTHDC1 promoted cytoplasmic localization of m6A-modified CircEPHB4. Overexpression of wild-type CircEPHB4 enhanced glioma cells' stemness, metastasis, and proliferation. Cytoplasmic CircEPHB4 increased SOX2 mRNA stability by binding to IGF2BP2, and the effects observed by SOX2 knockdown were reversed by CircEPHB4 in glioma cells. SOX2 promoted transcriptional expression of PHLDB2 by enriching the PHLDB2 promoter region. SOX2 reversed the inhibition of PHLDB2 knockdown on stemness of glioma, cell proliferation, and metastasis. In vivo experiments also revealed that CircEPHB4 upregulated PHLDB2 expression by stabilizing SOX2 mRNA, which promoted in vivo tumor growth and accelerated stemness of glioma cells and metastasis.
    CONCLUSION: This study reveals functional interaction and molecular mechanisms of m6A-modified circEPHB4 in regulating SOX2/PHLDB2 axis, highlighting their importance in glioma pathogenesis and potential as therapeutic targets.
    DOI:  https://doi.org/10.1002/acn3.51864
  32. Cancers (Basel). 2023 Aug 12. pii: 4075. [Epub ahead of print]15(16):
    Epigenetic m6A RNA Modification Reader YTHDF1 in Merkel Cell Carcinoma.
    Aayami Jaguri, Martin Steinhoff, Aamir Ahmad.
      Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine cutaneous malignancy that commonly affects older individuals with a high mortality rate [...].
    DOI:  https://doi.org/10.3390/cancers15164075
  33. Biology (Basel). 2023 Aug 08. pii: 1101. [Epub ahead of print]12(8):
    Cuproptosis- and m6A-Related lncRNAs for Prognosis of Hepatocellular Carcinoma.
    Yuezhi Zhu, Jen Kit Tan, Jo Aan Goon.
      Cuproptosis and N6-methyladenosine (m6A) have potential as prognostic predictors in cancer patients, but their roles in hepatocellular carcinoma (HCC) are unclear. This study aimed to screen a total of 375 HCC samples were retrieved from the TCGA database, and lncRNAs related to cuproptosis and m6A were obtained through correlation analysis. To construct a risk assessment model, univariate Cox regression analysis and LASSO Cox regression were employed. Analyze the regulatory effect of relevant risk assessment models on tumor mutation load (TMB) and immune microenvironment. A total of five lncRNAs (AC007405.3, AL031985.3, TMCC1-AS1, MIR210HG, TMEM220-AS1) with independent overall survival-related risk models were obtained by LASSO survival regression. TP53 and CTNNB1 were the three genes found to have the most mutations in high-risk group patients. The high-risk group with low TMB had the worst survival, whereas the low-risk group with high TMB had the best survival. KEGG pathway analysis revealed that the high-risk group was enriched with cell cycle, oocyte meiosis, cell senescence, and glycolysis/glucose production pathways. We constructed a reliable cuproptosis- and m6A-related lncRNA model for the prognosis of HCC. The model may provide new insights into managing HCC patients, but further research is needed to validate it.
    Keywords:  LncRNAs; N6-methyladenosine; RNA methylation; cuproptosis; hepatocellular carcinoma; prognosis
    DOI:  https://doi.org/10.3390/biology12081101
  34. Aging (Albany NY). 2023 Aug 21. 15
    Pan-cancer analysis and experimental validation revealed the m6A methyltransferase KIAA1429 as a potential biomarker for diagnosis, prognosis, and immunotherapy.
    Chao Ma, Qiming Zheng, Yepeng Wang, Guoxiang Li, Mengmeng Zhao, Zhigang Sun.
       BACKGROUND: KIAA1429, also known as VIRMA (vir-like m6A methyltransferase associated), plays a crucial role in tumorigenesis by modulating the level of m6A methylation. Previous studies have reported the prevalent overexpression of KIAA1429 in multiple cancers, related to a poor prognosis. Nevertheless, the precise role of KIAA1429 in tumor progression and its impact on the immune response remains unclear.
    METHODS: A differential analysis of KIAA1429 expression was performed across cancers using data from the Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. We evaluated the role of KIAA1429 in the diagnosis, prognosis, and immunotherapy of tumor patients using bioinformatics methods. In addition, we also analyzed the associations between KIAA1429 and DNA methylation, immunotherapy. RT-qPCR was used to study the expression levels of KIAA1429 mRNA in 11 cell lines.
    RESULTS: KIAA1429 is found to be overexpressed in 28 cancer types, but its expression is relatively low in patients with acute myeloid leukemia (LAML) and ovarian serous cystadenocarcinoma (OV). Moreover, KIAA1429 demonstrates a positive correlation with advanced stages of multiple cancers. Kaplan-Meier (KM) analysis suggested that patients with elevated KIAA1429 expression had shorter survival. Furthermore, KIAA1429 shows strong associations with DNA methylation, tumor-infiltrating immune cells (TIICs), and the tumor microenvironment (TME). RT-qPCR results indicated significantly higher expression of KIAA1429 in tumor cells compared to matched-normal cells.
    CONCLUSIONS: In summary, our work illustrates that KIAA1429 expression is positively connected with poor prognosis in multiple cancers. Moreover, KIAA1429 could serve as a diagnostic factor and a predictor of immune response for specific tumor types.
    Keywords:  KIAA1429; diagnosis; immune infiltration; pan-cancer; prognosis
    DOI:  https://doi.org/10.18632/aging.204968
  35. Ann Clin Lab Sci. 2023 Jul;53(4): 539-547
    Acyl-CoA Medium-Chain Synthetase-3 (ACSM3) Is Regulated by Insulin Like Growth Factor 2 mRNA Binding Protein 3 (IGF2BP3) and Inhibits Proliferation, Motility and Stem Cell Properties of Breast Invasive Carcinoma.
    Wenjing Zhu, Xiuxing Wei, Meijiao Fang.
       OBJECTIVE: Breast invasive carcinoma (BRCA) has a high degree of malignancy, is prone to lymph node metastasis, and has a poor prognosis. This study aimed to explore the role of Acyl-CoA Medium-Chain Synthetase-3 (ACSM3) in BRCA, which was found down-regulated in liver cancer and malignant melanoma.
    METHODS: The expression level of ACSM3 in patients with BRCA and its correlation with the overall survival rate was analyzed. The impacts of ACSM3 on BRCA cell proliferation, motility and stem cell properties were then evaluated. The association between insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) and ACSM3 was verified, the influences of IGF2BP3 on the regulation of ACSM3 on cells were determined.
    RESULTS: Down-regulated ACSM3 level was associated with poor overall survival. ACSM3 overexpression weakened BRCA cell proliferation, motility and stem cell properties. Importantly, IGF2BP3 destabilized ACSM3 and downregulated its expression level. IGF2BP3 overexpression reversed the impacts of ACSM3 overexpression on cells, indicating that ACSM3 was regulated by IGF2BP3 in BRCA cells.
    CONCLUSION: We found that ACSM3 was regulated by IGF2BP3 and attenuated BRCA proliferation, invasion and stem cell properties. The role of ACSM3 in BRCA was first revealed, which provides a novel target for treatment.
    Keywords:  ACSM3; IGF2BP3; breast invasive carcinoma; motility; stem cell property
  36. Diagn Pathol. 2023 Aug 22. 18(1): 96
    The multi-omics analyses of acsl1 reveal its translational significance as a tumor microenvironmental and prognostic biomarker in clear cell renal cell carcinoma.
    Yang Yang, Jiayu Liang, Junjie Zhao, Xinyuan Wang, Dechao Feng, Hang Xu, Yu Shen, Yaowen Zhang, Jindong Dai, Zhipeng Wang, Qiang Wei, Zhenhua Liu.
       BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the dominant subtype of kidney cancer. Dysregulation of long-chain acyl-CoA synthetase 1 (ACSL1) is strongly implicated in undesirable results in varieties of cancers. Nevertheless, the dysregulation and associated multi-omics characteristics of ACSL1 in ccRCC remain elusive.
    METHODS: We probed the mRNA and protein profiles of ACSL1 in RCC using data from the Cancer Genome Atlas, Gene Expression Omnibus, the Human Protein Atlas (HPA), and Clinical Proteomic Tumor Analysis Consortium (CPTAC) and verified them in our patient cohort and RCC cell lines. Correlations between ACSL1 expression and clinicopathological features, epigenetic modification and immune microenvironment characteristics were analyzed to reveal the multi-omics profile associated with ACSL1.
    RESULTS: ACSL1 was down-regulated in ccRCC tissues compared to adjacent normal tissues. Lower expression of ACSL1 was linked to unfavorable pathological parameters and prognosis. The dysregulation of ACSL1 was greatly ascribed to CpG island-associated methylation modification. The ACSL1 high-expression subgroup had enriched fatty acid metabolism-related pathways and high expression of ferroptosis-related genes. In contrast, the ACSL1 low-expression subgroup exhibited higher immune and microenvironment scores, elevated expression of immune checkpoints PDCD1, CTLA4, LAG3, and TIGIT, and higher TIDE scores. Using data from the GDSC database, we corroborated that down-regulation of ACSL1 was associated with higher sensitivity towards Erlotinib, Pazopanib, and PI3K-Akt-mTOR-targeted therapeutic strategies.
    CONCLUSION: Taken together, our findings point to ACSL1 as a biomarker for prognostic prediction of ccRCC, identifying the tumor microenvironment (TME) phenotype, and even contributing to treatment decision-making in ccRCC patients.
    Keywords:  ACSL1; Clear cell renal cell carcinoma; Immune Microenvironment; Methylation; m6A modification
    DOI:  https://doi.org/10.1186/s13000-023-01384-y
  37. Crit Rev Eukaryot Gene Expr. 2023 ;33(8): 41-55
    N6-Methyladenosine Modification and Prognostic Analysis of UBE2K in Hepatocellular Carcinoma: A Potential Target.
    Jinhui Guo, Jie Zhao.
      The treatment of hepatocellular carcinoma (HCC) is still relatively lacking, the prognosis is poor, and the potential mechanism of carcinogenesis has not been thoroughly studied. In this study, Ubiquitin-conjugating enzyme E2K (UBE2K) transcript levels in HCC patients were up-regulated in two databases, GEO and TCGA. External validation was performed using Western blot experiments. Compared to normal liver cells, UBE2K was upregulated in HCC cell lines. The survival curve and prognosis model revealed that the expression of UBE2K was of high prognostic value in patients with HCC. Transwell assay, wound healing assay and sphere formation assay were used to evaluate the effects of knockdown and overexpression of UBE2K on HCC cells. Overexpression of UBE2K promoted the invasion, migration and stemness of HCC cells, while knocking down UBE2K attenuated the invasion, migration and stemness of HCC cells. Then, through a series of functional analysis (GO and KEEG), it was found that UBE2K played an important role in mRNA processing. We speculate that UBE2K may be involved in HCC progression through its own N6-methyladenosine modification. We therefore used a global methylation inhibitor (3-deazaadenosine) to treat HCC cells and found a gradient increase in the mRNA level of UBE2K. Collectively, the results suggest that UBE2K may be a promising molecular target for the treatment of HCC.
    DOI:  https://doi.org/10.1615/CritRevEukaryotGeneExpr.2023048801
  38. Pain. 2023 Aug 25.
    DNA N6-methyladenine methylase N6AMT1 controls neuropathic pain through epigenetically modifying Kcnj16 in dorsal horn neurons.
    Hui-Min Zhou, Heng-Jun Xu, Run-Hang Sun, Ming Zhang, Xiao-Tong Li, Ya-Xuan Zhao, Kehui Yang, Runa Wei, Qiaoqiao Liu, Siyuan Li, Zhouya Xue, Ling-Yun Hao, Li Yang, Qi-Hui Wang, Hong-Jun Wang, Fang Gao, Jun-Li Cao, Zhiqiang Pan.
       ABSTRACT: Nerve injury-induced aberrant changes in gene expression in spinal dorsal horn neurons are critical for the genesis of neuropathic pain. N6-methyladenine (m6A) modification of DNA represents an additional layer of gene regulation. Here, we report that peripheral nerve injury significantly decreased the level of m6A-specific DNA methyltransferase 1 (N6amt1) in dorsal horn neurons. This decrease was attributed, at least partly, to a reduction in transcription factor Nr2f6. Rescuing the decrease in N6amt1 reversed the loss of m6A at the promoter for inwardly rectifying potassium channel subfamily J member 16 (Kcnj16), mitigating the nerve injury-induced upregulation of Kcnj16 expression in the dorsal horn and alleviating neuropathic pain hypersensitivities. Conversely, mimicking the downregulation of N6amt1 in naive mice erased DNA m6A at the Kcnj16 promoter, elevated Kcnj16 expression, and led to neuropathic pain-like behaviors. Therefore, decreased N6amt1 caused by NR2F6 is required for neuropathic pain, likely through its regulation of m6A-controlled KCNJ16 in dorsal horn neurons, suggesting that DNA m6A modification may be a potential new target for analgesic and treatment strategies.
    DOI:  https://doi.org/10.1097/j.pain.0000000000002986
  39. bioRxiv. 2023 Aug 09. pii: 2023.08.07.552278. [Epub ahead of print]
    Characterization of m 6 A modifiers and RNA modifications in uterine fibroids.
    Jitu W George, Rosa A Cancino, Jennifer L Griffin Miller, Fang Qiu, Qishan Lin, M Jordan Rowley, Varghese M Chennathukuzhi, John S Davis.
      Uterine leiomyoma or fibroids are the most common prevalent noncancerous tumors of the uterine muscle layer. Common symptoms associated with fibroids include pelvic pain, heavy menstrual bleeding, anemia, and pelvic pressure. These tumors are a leading cause of gynecological care but lack long-term therapy as the origin and development of fibroids are not well understood. Several next-generation sequencing technologies have been performed to identify the underlying genetic and epigenetic basis of fibroids. However, there remains a systemic gap in our understanding of molecular and biological process that define uterine fibroids. Recent epitranscriptomics studies have unraveled RNA modifications that are associated with all forms of RNA and are thought to influence both normal physiological functions and the progression of diseases. We quantified RNA expression profiles by analyzing publicly available RNA-seq data for 15 known epigenetic mediators to identify their expression profile in uterine fibroids compared to myometrium. To validate our findings, we performed RT-qPCR on a separate cohort of uterine fibroids targeting these modifiers confirming our RNA-seq data. We then examined protein profiles of key m 6 A modifiers in fibroids and their matched myometrium. In concordance with our RNA expression profiles, no significant differences were observed in these proteins in uterine fibroids compared to myometrium. To determine abundance of RNA modifications, mRNA and small RNA from fibroids and matched myometrium were analyzed by UHPLC MS/MS. In addition to the prevalent N6-methyladenosine (m 6 A), we identified 11 other known modifiers but did not identify any aberrant expression in fibroids. We then mined a previously published dataset and identified differential expression of m 6 A modifiers that were specific to fibroid genetic sub-type. Our analysis also identified m 6 A consensus motifs on genes previously identified to be dysregulated in uterine fibroids. Overall, using state-of-the-art mass spectrometry, RNA expression and protein profiles, we characterized and identified differentially expressed m 6 A modifiers in relation to driver mutations. Despite the use of several different approaches, we identified limited differential expression of RNA modifiers and associated modifications in uterine fibroids. However, considering the highly heterogenous genomic and cellular nature of fibroids, and the possible contribution of single molecule m 6 A modifications to fibroid pathology, there is a need for greater in-depth characterization of m 6 A marks and modifiers in a larger and varied patient cohort.
    DOI:  https://doi.org/10.1101/2023.08.07.552278
  40. Biochim Biophys Acta Rev Cancer. 2023 Aug 23. pii: S0304-419X(23)00113-0. [Epub ahead of print] 188964
    TRDMT1-mediated RNA C-5 methylation as a novel target in anticancer therapy.
    Anna Lewinska, Jagoda Adamczyk-Grochala, Maciej Wnuk.
      Affected landscape of RNA modifications is frequently observed in different cancer cells that can be associated with the development of cancer cell phenotypic traits such as sustained proliferation, migration and invasion, apoptosis resistance and metabolic reprograming. DNMT2/TRDMT1 5-methylcytosine methyltransferase, initially classified as DNA methyltransferase, can methylate both tRNA and mRNA promoting tRNA stability and proper protein synthesis, and orchestrating DNA damage response (DDR) and DNA stability, respectively. TRDMT1 is associated with cancer progression as its levels can be elevated and its mutations can be observed in a number of cancer types. TRDMT1 gene knockout (KO) can sensitize cancer cells of different origin to radiotherapy and chemotherapy. In the present review paper, based on literature data, the physiological and pathophysiological roles of TRDMT1 in different biological systems are described with the emphasis on human normal and cancer cells. Potential TRDMT1 substrates, inhibitors and regulatory mechanisms of catalytic activity and cellular localization are also presented and evaluated. TRDMT1 as a novel promising target in anticancer therapy is proposed and discussed.
    Keywords:  Anticancer strategy; Chemotherapy-induced senescence; Genetic instability; RNA C-5 methylation; TRDMT1/DNMT2
    DOI:  https://doi.org/10.1016/j.bbcan.2023.188964
  41. Genes (Basel). 2023 Aug 13. pii: 1620. [Epub ahead of print]14(8):
    The 3' Non-Coding Sequence Negatively Regulates PD-L1 Expression, and Its Regulators Are Systematically Identified in Pan-Cancer.
    Zike Chen, Hui Pi, Wen Zheng, Xiaohong Guo, Conglin Shi, Zhiyang Wang, Jie Zhang, Xuanhao Qu, Lehan Liu, Haoliang Shen, Yang Lu, Miaomiao Chen, Weibing Zhang, Rong Sun, Yihui Fan.
      The 3'-untranslated region (3'-UTR) of PD-L1 is significantly longer than the coding sequences (CDSs). However, its role and regulators have been little studied. We deleted whole 3'-UTR region by CRISPR-Cas9. Prognostic analysis was performed using online tools. Immune infiltration analysis was performed using the Timer and Xcell packages. Immunotherapy response prediction and Cox regression was performed using the R software. MicroRNA network analysis was conducted by the Cytoscape software. The level of PD-L1 was significantly and dramatically up-regulated in cells after deleting the 3'-UTR. Additionally, we discovered a panel of 43 RNA-binding proteins (RBPs) whose expression correlates with PD-L1 in the majority of cancer cell lines and tumor tissues. Among these RBPs, PARP14 is widely associated with immune checkpoints, the tumor microenvironment, and immune-infiltrating cells in various cancer types. We also identified 38 microRNAs whose individual expressions are associated with PD-L1 across different cancers. Notably, miR-3139, miR-4761, and miR-15a-5p showed significant associations with PD-L1 in most cancer types. Furthermore, we revealed 21 m6A regulators that strongly correlate with PD-L1. Importantly, by combining the identified RBP and m6A regulators, we established an immune signature consisting of RBMS1, QKI, ZC3HAV1, and RBM38. This signature can be used to predict the responsiveness of cancer patients to immune checkpoint blockade treatment. We demonstrated the critical role of the 3'-UTR in the regulation of PD-L1 and identified a significant number of potential PD-L1 regulators across various types of cancer. The biomarker signature generated from our findings shows promise in predicting patient prognosis. However, further biological investigation is necessary to explore the potential of these PD-L1 regulators.
    Keywords:  3′-untranslated region (3′-UTR); PD-L1; RNA-binding proteins (RBPs); m6A regulator; miRNAs
    DOI:  https://doi.org/10.3390/genes14081620
  42. Cell Death Discov. 2023 Aug 24. 9(1): 310
    FOXM1 maintains fatty acid homoeostasis through the SET7-H3K4me1-FASN axis.
    Xixi Li, Weijie Su, Honglin Wu, Jiakun Xu, Hongxing Tang, Xiangkun Chen, Zhanqi Yin, Changming Zhang, Jia Yang, Yibing Yang, Nu Zhang, Lixuan Yang.
      Reprogramming of metabolic genes and subsequent alterations in metabolic phenotypes occur widely in malignant tumours, including glioblastoma (GBM). FOXM1 is a potent transcription factor that plays an oncogenic role by regulating the expression of many genes. As a SET domain containing protein, SET7 is a protein lysine methyltransferase which monomethylates histone proteins and other proteins. The epigenetic modification of histones regulates gene expressions by epigenetically modifying promoters of DNAs and inter vening in tumor development. Activation of FASN increased de novo fatty acid (FA) synthesis, a hallmark of cancer cells. Here, we report that FOXM1 may directly promote the transcription of SET7 and activate SET7-H3K4me1-FASN axis, which results in the maintenance of de novo FA synthesis.
    DOI:  https://doi.org/10.1038/s41420-023-01540-9
  43. Nat Commun. 2023 Aug 23. 14(1): 5130
    Droplet-based high-throughput single microbe RNA sequencing by smRandom-seq.
    Ziye Xu, Yuting Wang, Kuanwei Sheng, Raoul Rosenthal, Nan Liu, Xiaoting Hua, Tianyu Zhang, Jiaye Chen, Mengdi Song, Yuexiao Lv, Shunji Zhang, Yingjuan Huang, Zhaolun Wang, Ting Cao, Yifei Shen, Yan Jiang, Yunsong Yu, Yu Chen, Guoji Guo, Peng Yin, David A Weitz, Yongcheng Wang.
      Bacteria colonize almost all parts of the human body and can differ significantly. However, the population level transcriptomics measurements can only describe the average bacteria population behaviors, ignoring the heterogeneity among bacteria. Here, we report a droplet-based high-throughput single-microbe RNA-seq assay (smRandom-seq), using random primers for in situ cDNA generation, droplets for single-microbe barcoding, and CRISPR-based rRNA depletion for mRNA enrichment. smRandom-seq showed a high species specificity (99%), a minor doublet rate (1.6%), a reduced rRNA percentage (32%), and a sensitive gene detection (a median of ~1000 genes per single E. coli). Furthermore, smRandom-seq successfully captured transcriptome changes of thousands of individual E. coli and discovered a few antibiotic resistant subpopulations displaying distinct gene expression patterns of SOS response and metabolic pathways in E. coli population upon antibiotic stress. smRandom-seq provides a high-throughput single-microbe transcriptome profiling tool that will facilitate future discoveries in microbial resistance, persistence, microbe-host interaction, and microbiome research.
    DOI:  https://doi.org/10.1038/s41467-023-40137-9
  44. iScience. 2023 Aug 18. 26(8): 107447
    ZXDC enhances cervical cancer metastasis through IGF2BP3-mediated activation of RhoA/ROCK signaling.
    Yifang Mao, Xingyu Jiang, Peng Guo, Ying Ouyang, Xiangfu Chen, Meng Xia, Lixin Wu, Zihao Tang, Tianyi Liang, Yue Li, Mian He.
      Metastasis in cervical cancer (CC) has a significant negative impact on patient survival, highlighting the urgent need for investigation in this area. In this study, we identified significant overexpression of zinc finger, X-linked, duplicated family member C (ZXDC) in CC tissue with metastasis, which correlates with poor outcomes for CC patients. We observed that overexpression of ZXDC promotes, while silencing of ZXDC inhibits the metastasis of CC cells both in vitro and in vivo. Additionally, our research demonstrated that ZXDC activated RhoA/ROCK signaling pathway, leading to enhanced cytoskeleton remodeling in CC cells. Besides, we found that IGF2BP3 plays an essential role in the activation of ZXDC on the RhoA/ROCK signaling pathway by stabilizing RhoA mRNA. These findings reveal a mechanism whereby ZXDC promotes the cervical cancer metastasis by targeting IGF2BP3/RhoA/ROCK pathway.
    Keywords:  Cancer; Molecular biology; Omics; Transcriptomics
    DOI:  https://doi.org/10.1016/j.isci.2023.107447
  45. J Transl Med. 2023 Aug 26. 21(1): 574
    DARS2 overexpression is associated with PET/CT metabolic parameters and affects glycolytic activity in lung adenocarcinoma.
    Xu-Sheng Liu, Ling-Ling Yuan, Yan Gao, Xing Ming, Yao-Hua Zhang, Yu Zhang, Zi-Yue Liu, Yi Yang, Zhi-Jun Pei.
       BACKGROUND: This study investigated the correlation between the expression of DARS2 and metabolic parameters of 18F-FDG PET/CT, and explored the potential mechanisms of DARS2 affecting the proliferation and glycolysis of lung adenocarcinoma (LUAD) cells.
    METHODS: This study used genomics and proteomics to analyze the difference in DARS2 expression between LUAD samples and control samples. An analysis of 62 patients with LUAD who underwent 18F-FDG PET/CT examinations before surgery was conducted retrospectively. The correlation between DARS2 expression and PET/CT metabolic parameters, including SUVmax, SUVmean, MTV, and TLG, was examined by Spearman correlation analysis. In addition, the molecular mechanism of interfering with DARS2 expression in inhibiting LUAD cell proliferation and glycolysis was analyzed through in vitro cell experiments.
    RESULTS: DARS2 expression was significantly higher in LUAD samples than in control samples (p < 0.001). DARS2 has high specificity (98.4%) and sensitivity (95.2%) in the diagnosis of LUAD. DARS2 expression was positively correlated with SUVmax, SUVmean, and TLG (p < 0.001). At the same time, the sensitivity and specificity of SUVmax in predicting DARS2 overexpression in LUAD were 88.9% and 65.9%, respectively. In vitro cell experiments have shown that interfering with DARS2 expression can inhibit the proliferation and migration of LUAD cells, promote cell apoptosis, and inhibit the glycolytic activity of tumor cells by inhibiting the expression of glycolytic related genes SLC2A1, GPI, ALDOA, and PGAM1.
    CONCLUSIONS: Overexpression of DARS2 is associated with metabolic parameters on 18F-FDG PET/CT, which can improve LUAD diagnosis accuracy. DARS2 may be a useful biomarker to diagnose, prognosis, and target treatment of LUAD patients.
    Keywords:  DARS2; Glycolysis; Lung adenocarcinoma; PET/CT; SUVmax
    DOI:  https://doi.org/10.1186/s12967-023-04454-3
  46. J Adv Res. 2023 Aug 22. pii: S2090-1232(23)00227-8. [Epub ahead of print]
    Liver Metastases across Cancer Types Sharing Tumor Environment Immunotolerance Can Impede Immune Response Therapy and Immune Monitoring.
    Yuzhen Gao, Shipeng Chen, Hao Wang, Chenghao Wu, Rui An, Guoli Li, Min Yang, Ying Zhou, Yundong Zhou, Xinyou Xie, Hong Yu, Jun Zhang.
       BACKGROUND: Hepatic immune tolerance might contribute to the development of therapeutic resistance to immunotherapy. However, addressing this issue is challenging since the efficacy of immunotherapy in the context of liver metastasis (LM) remains poorly studied. Here, we aimed to establish an LM common immune feature (LMCIF) score to quantify the characteristics of LM immunotolerance across cancer types for assisting clinical disease management.
    METHODS: Large-scale clinical data were collected to identify the prognosis of LM. Multi-omics datasets of metastatic cancers with LM special immune-related pathways (LMSIPs) from the Molecular Signatures Database (MSigDB)were used to obtain an LMCIF cluster. Based on differential expression genes (DEGs), a novel LMCIF score for the LMCIF cluster was constructed. In addition, multi-omics, and immunohistochemistry (IHC) data from the public and in-house cohorts were used to explore the features of LM, and LMCIF score.
    RESULTS: Patients with LM had a worse prognosis and significantly lower infiltration of immune cells than patients with metastasis to other organs when analyzed with combined clinical and RNA sequencing data. After extracting the LMCIF cluster from 373 samples by utilizing 29 LMSIPs and validating them in a microarray cohort, an LMCIF score was established to confirm the role of the immunosuppressive environment as a contributor to the poor prognosis of LM across cancer types. Moreover, this LMCIF score could be used to predict the immune response of cancer patients undergoing immunotherapy. Finally, we identified that the majority of the 31 LMCIF genes exhibited a negative correlation with TME cells in LM patients, one of them, KRT19, which possessed the strongest positive correlation with LMCIF score, was confirmed to have an immunosuppressive effect through IHC analysis.
    CONCLUSIONS: Our results suggest that LM across cancer types share similar immunological profiles that induce immunotolerance and escape from immune monitoring. The novel LMCIF score represents a common liver metastasis immune cluster for predicting immunotherapy response, the results of which might benefit clinical disease management.
    Keywords:  Immunotherapy; Immunotolerance; KRT19; Liver Metastasis; Pan-cancer
    DOI:  https://doi.org/10.1016/j.jare.2023.08.011
  47. Ann Clin Lab Sci. 2023 Jul;53(4): 630-640
    SHP2 Contributes to Resistance of KRAS p.G12C-Driven Lung Cancer Cells to MRTX849 by Regulating β-catenin/c-MYC Axis.
    Wenli Li, Ya Wu, Lili Dai, Wei Wang, Lei Zhao.
       OBJECTIVE: SHP2 has been promulgated to be involved in chemoresistance in a variety of cancers. However, its relationship with MRTX849-resistance in KRAS G12C mutant lung cancer has not been revealed.
    METHODS: Lung cancer cell lines resistant to MRTX849 were first constructed by repeated dosing over 10 months, and the parental and drug-resistant strains were evaluated for SHP2 expression at different time points (2, 4, 6, 8, 10 months). We further analyzed whether SHP2 knockdown affects the sensitivity of MRTX849-resistant cells to MRTX849, and overexpression of SHP2 in the parental cell line to assess its effect on MRTX849 resistance, mainly by CCK-8, clonogenic assay, TUNEL staining and Western blotting to assess cell viability, proliferation, apoptosis, as well as β-catenin/c-MYC pathway protein expression.
    RESULTS: SHP2 expression remained largely unchanged in the parental cell line, whereas they were gradually upregulated in a time-dependent manner in the resistant cell line. SHP2 knockdown enhanced the sensitivity of MRTX849-resistant cell lines to MRTX849 and encouraged the killing of lung cancer cells by MRTX849, as indicated by a more significant decrease in cell viability and proliferation after knockdown of SHP2 in the presence of MRTX849 compared with MRTX849 untreated, while apoptosis was more significantly enhanced. Additionally, SHP2 overexpression enhanced the resistance of MRTX849 to lung cancer cells. Eventually, we confirmed that the MRTX849-resistance effect of SHP2 on lung cancer cells was through the activation of the β-catenin/c-MYC pathway.
    CONCLUSION: SHP2 contributes to resistance of KRAS G12C-driven lung cancer cells to MRTX849 by regulating β-catenin/c-MYC axis.
    Keywords:  KRAS G12C; MRTX849; SHP2; lung cancer; resistance
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