bims-rimeca Biomed News
on RNA methylation in cancer
Issue of 2023–05–21
forty-six papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics
  1. METTL3 promotes pancreatic cancer proliferation and stemness by increasing stability of ID2 mRNA in a m6A-dependent manner.
  2. METTL3 suppresses pancreatic ductal adenocarcinoma progression through activating endogenous dsRNA-induced anti-tumor immunity.
  3. Hypericin Ameliorates Depression-like Behaviors via Neurotrophin Signaling Pathway Mediating m6A Epitranscriptome Modification.
  4. m6A eraser ALKBH5 mitigates the apoptosis of cardiomyocytes in ischemia reperfusion injury through m6A/SIRT1 axis.
  5. RNA demethylase ALKBH5 promotes colorectal cancer progression by posttranscriptional activation of RAB5A in an m6A-YTHDF2-dependent manner.
  6. Short-chain fatty acid-butyric acid ameliorates granulosa cells inflammation through regulating METTL3-mediated N6-methyladenosine modification of FOSL2 in polycystic ovarian syndrome.
  7. METTL1/WDR4-mediated tRNA m7G modification and mRNA translation control promote oncogenesis and doxorubicin resistance.
  8. Insights into N6-methyladenosine (m6A) modification of noncoding RNA in tumor microenvironment.
  9. The m6A methyltransferase METTL16 inhibits the proliferation of pancreatic adenocarcinoma cancer cells via the p21 signaling pathway.
  10. FTO represses NLRP3-mediated pyroptosis and alleviates myocardial ischemia-reperfusion injury via inhibiting CBL-mediated ubiquitination and degradation of β-catenin.
  11. METTL14 represses osteoclast formation to ameliorate osteoporosis via enhancing GPX4 mRNA stability.
  12. Novel insights into the METTL3-METTL14 complex in musculoskeletal diseases.
  13. N6-methyladenosine reader IGF2BP3 as a prognostic Biomarker contribute to malignant progression of glioma.
  14. Identification of a New m6A Regulator-Related Methylation Signature for Predicting the Prognosis and Immune Microenvironment of Patients with Pancreatic Cancer.
  15. CSNK1D-mediated phosphorylation of HNRNPA2B1 induces miR-25-3p/miR-93-5p maturation to promote prostate cancer cell proliferation and migration through m6A-dependent manner.
  16. ALKBH4 Stabilization Is Required for Arsenic-Induced 6mA DNA Methylation Inhibition, Keratinocyte Malignant Transformation, and Tumorigenicity.
  17. m6A modification-tuned sphingolipid metabolism regulates postnatal liver development in male mice.
  18. Novel targets for gastric cancer: The tumor microenvironment (TME), N6-methyladenosine (m6A), pyroptosis, autophagy, ferroptosis and cuproptosis.
  19. PRMT1 mediated methylation of cGAS suppresses anti-tumor immunity.
  20. The cancer-testis lncRNA LINC01977 promotes HCC progression by interacting with RBM39 to prevent Notch2 ubiquitination.
  21. Epigenetically silenced lncRNA SNAI3-AS1 promotes ferroptosis in glioma via perturbing the m6A-dependent recognition of Nrf2 mRNA mediated by SND1.
  22. An Integrated Dual-Functional Nanotool Capable of Studying Single-Cell Epigenetics and Programmable Gene Regulation.
  23. SINEUP non-coding RNA activity depends on specific N6-methyladenosine nucleotides.
  24. Epigenetic inactivation of the 5-methylcytosine RNA methyltransferase NSUN7 is associated with clinical outcome and therapeutic vulnerability in liver cancer.
  25. Preliminary study based on methylation and transcriptome gene sequencing of lncRNAs and immune infiltration in hypopharyngeal carcinoma.
  26. The RNA Demethylase ALKBH5 Maintains Endoplasmic Reticulum Homeostasis by Regulating UPR, Autophagy, and Mitochondrial Function.
  27. SOX18 meditates the resistance of Bmi1-expressing cells to cetuximab in HNSCC.
  28. Myc derived circRNA promotes triple-negative breast cancer progression via reprogramming fatty acid metabolism.
  29. Overexpression of VIRMA confers vulnerability to breast cancers via the m6A-dependent regulation of unfolded protein response.
  30. m6A Modification Mediates Exosomal LINC00657 to Trigger Breast Cancer Progression Via Inducing Macrophage M2 Polarization.
  31. The role of m6A mRNA modification in normal and malignant hematopoiesis.
  32. The Functional Role and Regulatory Mechanism of FTO m6A RNA Demethylase in Human Uterine Leiomyosarcoma.
  33. BRSK2 in pancreatic β cells promotes hyperinsulinemia-coupled insulin resistance and its genetic variants are associated with human type 2 diabetes.
  34. N1-Methyladenosine modification of mRNA regulates neuronal gene expression and oxygen glucose deprivation/reoxygenation induction.
  35. The interface targeting hnRNPA2B1 regulates the repression of transthyretin against human retinal microvascular endothelial cells in high glucose environment.
  36. Xiao Chai Hu Tang alleviates the pancreatic tumorigenesis via improving the mtDNA N6-Methyladenine modification mediated mitochondrial dysfunction in Syrian hamster model.
  37. CTLA4 mRNA is downregulated by miR-155 in regulatory T cells, and reduced blood CTLA4 levels are associated with poor prognosis in metastatic melanoma patients.
  38. TRAF6 Promotes PRMT5 Activity in a Ubiquitination-Dependent Manner.
  39. A novel methionine metabolism-related signature predicts prognosis and immunotherapy response in lung adenocarcinoma.
  40. IGF2BP3 promotes adult myocardial regeneration by stabilizing MMP3 mRNA through interaction with m6A modification.
  41. Mena as a key enhancer factor of EMT to promote metastasis of human tongue squamous cell carcinoma.
  42. Polyamine metabolism patterns characterized tumor microenvironment, prognosis, and response to immunotherapy in colorectal cancer.
  43. RNA stability controlled by m6A methylation contributes to X-to-autosome dosage compensation in mammals.
  44. Overexpression of RACGAP1 by E2F1 Promotes Neuroendocrine Differentiation of Prostate Cancer by Stabilizing EZH2 Expression.
  45. Combinatorial quantification of 5mC and 5hmC at individual CpG dyads and the transcriptome in single cells reveals modulators of DNA methylation maintenance fidelity.
  46. The Regulatory Network of METTL3 in the Nervous System: Diagnostic Biomarkers and Therapeutic Targets.
  1. Cancer Lett. 2023 May 15. pii: S0304-3835(23)00173-8. [Epub ahead of print] 216222
    METTL3 promotes pancreatic cancer proliferation and stemness by increasing stability of ID2 mRNA in a m6A-dependent manner.
    Jiaoshun Chen, Haoxiang Zhang, Chaoyang Xiu, Chenggang Gao, Shihong Wu, Jianwei Bai, Qiang Shen, Tao Yin.
      In eukaryotes, N6-methyladenosine (m6A) is the most prevalent epigenetic alteration. Methyltransferase-like 3 (METTL3) is a key player in the control of m6A, although its function in pancreatic cancer is incompletely understood. In this study, we examined the role that METTL3 plays in pancreatic cancer cell proliferation and stemness. We discovered that in pancreatic cancer cells, METTL3-mediated m6A alterations regulate ID2 as a downstream target. The stability of ID2 mRNA was decreased and m6A modification was effectively eliminated by METTL3 knockdown in pancreatic cancer cells. We also demonstrate that m6a-YTHDF2 is necessary for the METTL3-mediated stabilization of ID2 mRNA. Additionally, we show that ID2 controls the stemness molecules NANOG and SOX2 via the PI3K-AKT pathway to support pancreatic cancer growth and stemness maintenance. Our data suggest that METTL3 may post-transcriptionally upregulate ID2 expression in an m6A-YTHDF2-dependent manner to further promote the stabilization of ID2 mRNA, which may be a new target for pancreatic cancer treatment.
    Keywords:  METTL3; Pancreatic cancer; Transcription regulation; YTHDF2; m6A methylation
    DOI:  https://doi.org/10.1016/j.canlet.2023.216222
  2. Cell Oncol (Dordr). 2023 May 13.
    METTL3 suppresses pancreatic ductal adenocarcinoma progression through activating endogenous dsRNA-induced anti-tumor immunity.
    Lili Zhu, Botai Li, Rongkun Li, Lipeng Hu, Yanli Zhang, Zhigang Zhang, Shuheng Jiang, Xueli Zhang.
       PURPOSE: Although immunotherapy improves clinical outcomes in several types of malignancies, as an immunologically 'cold' tumor, pancreatic ductal adenocarcinoma (PDAC) is arrantly resistant to immunotherapy. However, the role of N6-methyladenosine (m6A) modification in the immune microenvironment of PDAC is still poorly understood.
    METHODS: The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets were used to identify differentially expressed m6A related enzymes. The biological role and mechanism of METTL3 in PDAC growth and metastasis were determined in vitro and in vivo. RNA-sequencing and bioinformatics analysis were used to identify signaling pathways involved in METTL3. Western blot, m6A dot blot assays, co-immunoprecipitation, immunofluorescence, and flow cytometry were used to explore the molecular mechanism.
    RESULTS: Here, we demonstrate that METTL3, the key regulator of m6A modification, is downregulated in PDAC, and negatively correlates with PDAC malignant features. Elevated METTL3 suppresses PDAC growth and overcomes resistance to immune checkpoint blockade. Mechanistically, METTL3 promotes the accumulation of endogenous double-stranded RNA (dsRNA) through protecting m6A-transcripts from further Adenosine-to-inosine (A-to-I) editing. The dsRNA stress activates RIG-I-like receptors (RLRs) to enhance anti-tumor immunity, finally suppressing PDAC progression.
    CONCLUSION: Our findings indicate that tumor cell-intrinsic m6A modification participates in the regulation of tumor immune landscape. Adjusting the m6A level may be an effective strategy to overcome the resistance to immunotherapy and increase responsiveness to immunotherapy in PDAC.
    Keywords:  A-to-I; Antiviral immunity; Immunotherapy; Pancreatic ductal adenocarcinoma; dsRNA; m6A
    DOI:  https://doi.org/10.1007/s13402-023-00829-2
  3. Molecules. 2023 May 03. pii: 3859. [Epub ahead of print]28(9):
    Hypericin Ameliorates Depression-like Behaviors via Neurotrophin Signaling Pathway Mediating m6A Epitranscriptome Modification.
    Chunguang Lei, Ningning Li, Jianhua Chen, Qingzhong Wang.
      Hypericin, one of the major antidepressant constituents of St. John's wort, was shown to exert antidepressant effects by affecting cerebral CYP enzymes, serotonin homeostasis, and neuroinflammatory signaling pathways. However, its exact mechanisms are unknown. Previous clinical studies reported that the mRNA modification N6-methyladenosine (m6A) interferes with the neurobiological mechanism in depressed patients, and it was also found that the antidepressant efficacy of tricyclic antidepressants (TCAs) is related to m6A modifications. Therefore, we hypothesize that the antidepressant effect of hypericin may relate to the m6A modification of epitranscriptomic regulation. We constructed a UCMS mouse depression model and found that hypericin ameliorated depressive-like behavior in UCMS mice. Molecular pharmacology experiments showed that hypericin treatment upregulated the expression of m6A-modifying enzymes METTL3 and WTAP in the hippocampi of UCMS mice. Next, we performed MeRIP-seq and RNA-seq to study m6A modifications and changes in mRNA expression on a genome-wide scale. The genome-wide m6A assay and MeRIP-qPCR results revealed that the m6A modifications of Akt3, Ntrk2, Braf, and Kidins220 mRNA were significantly altered in the hippocampi of UCMS mice after stress stimulation and were reversed by hypericin treatment. Transcriptome assays and qPCR results showed that the Camk4 and Arhgdig genes might be related to the antidepressant efficacy of hypericin. Further gene enrichment results showed that the differential genes were mainly involved in neurotrophic factor signaling pathways. In conclusion, our results show that hypericin upregulates m6A methyltransferase METTL3 and WTAP in the hippocampi of UCMS mice and stabilizes m6A modifications to exert antidepressant effects via the neurotrophin signaling pathway. This suggests that METTL3 and WTAP-mediated changes in m6A modifications may be a potential mechanism for the pathogenesis of depression and the efficacy of antidepressants, and that the neurotrophin signaling pathway plays a key role in this process.
    Keywords:  N6-methyladenosine modification; antidepressant; hypericin; neurotrophin signaling pathway
    DOI:  https://doi.org/10.3390/molecules28093859
  4. PeerJ. 2023 ;11 e15269
    m6A eraser ALKBH5 mitigates the apoptosis of cardiomyocytes in ischemia reperfusion injury through m6A/SIRT1 axis.
    Liangliang Liu, Zhen Liu.
      Recent studies have shown that the potential regulatory role of N6-methyladenine (m6A) modification may affect the occurrence and development of various cardiovascular diseases. However, the regulatory mechanism of m6A modification on myocardial ischemia reperfusion injury (MIRI) is rarely reported. A mouse model of myocardial ischemia reperfusion (I/R) was established by ligation and perfusion of the left anterior descending coronary artery, and a cellular model of hypoxia/reperfusion (H/R) was conducted in cardiomyocytes (CMs). We found that the protein expression of ALKBH5 in myocardial tissues and cells were decreased, accompanied by increased m6A modification level. Overexpression of ALKBH5 significantly inhibited H/R-induced oxidative stress and apoptosis in CMs. Mechanistically, there was an enriched m6A motif in the 3'-UTR of SIRT1 genome, and ALKBH5 overexpression promoted the stability of SIRT1 mRNA. Furthermore, results using overexpression or knockdown of SIRT1 confirmed the protective effect of SIRT1 on H/R induced CMs apoptosis. Together, our study reveals a critical role of ALKBH5-medicated m6A on CM apoptosis, supplying an important regulating effect of m6A methylation in ischemic heart disease.
    Keywords:  ALKBH5; Cardiomyocytes
    DOI:  https://doi.org/10.7717/peerj.15269
  5. Clin Transl Med. 2023 May;13(5): e1279
    RNA demethylase ALKBH5 promotes colorectal cancer progression by posttranscriptional activation of RAB5A in an m6A-YTHDF2-dependent manner.
    Dingcheng Shen, Jinxin Lin, Yumo Xie, Zhuokai Zhuang, Gaopo Xu, Shaoyong Peng, Guannan Tang, Liangliang Bai, Mingxuan Zhu, Yu Zhang, Ziying Huang, Puning Wang, Xiaoxia Liu, Meijin Huang, Yanxin Luo, Xiaolin Wang, Huichuan Yu.
       BACKGROUND: N6-methyladenosine (m6A) modification is an emerging epigenetic regulatory mechanism in tumourigenesis. Considering that AlkB homolog 5 (ALKBH5) is a well-described m6A demethylase in previous enzyme assays, we aimed to investigate the role of m6A methylation alteration conferred by disturbed ALKBH5 in colorectal cancer (CRC) development.
    METHODS: Expression of ALKBH5 and its correlation with clinicopathological characteristics of CRC were evaluated using the prospectively maintained institutional database. The molecular role and underlying mechanism of ALKBH5 in CRC were explored using in vitro and in vivo experiments with methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNA-seq, MeRIP-qPCR, RIP-qPCR and luciferase reporter assays.
    RESULTS: ALKBH5 expression was significantly upregulated in CRC tissues compared to the paired adjacent normal tissues, and higher expression of ALKBH5 was independently associated with worse overall survival in CRC patients. Functionally, ALKBH5 promoted the proliferative, migrative and invasive abilities of CRC cells in vitro and enhanced subcutaneous tumour growth in vivo. Mechanistically, RAB5A was identified as the downstream target of ALKBH5 in CRC development, and ALKBH5 posttranscriptionally activated RAB5A by m6A demethylation, which impeded the YTHDF2-mediated degradation of RAB5A mRNA. In addition, we demonstrated that dysregulation of the ALKBH5-RAB5A axis could affect the tumourigenicity of CRC.
    CONCLUSIONS: ALKBH5 facilitates the progression of CRC by augmenting the expression of RAB5A via an m6A-YTHDF2-dependent manner. Our findings suggested that ALKBH5-RAB5A axis might serve as valuable biomarkers and effective therapeutic targets for CRC.
    Keywords:  ALKBH5; N6-methyladenosine (m6A); RAB5A; colorectal cancer (CRC)
    DOI:  https://doi.org/10.1002/ctm2.1279
  6. Clin Epigenetics. 2023 05 13. 15(1): 86
    Short-chain fatty acid-butyric acid ameliorates granulosa cells inflammation through regulating METTL3-mediated N6-methyladenosine modification of FOSL2 in polycystic ovarian syndrome.
    Kailu Liu, Xi He, Jingyu Huang, Simin Yu, Meiting Cui, Mengya Gao, Li Liu, Yu Qian, Ying Xie, Miao Hui, Yanli Hong, Xiaowei Nie.
      Polycystic ovary syndrome (PCOS) is an endocrine and metabolic disorder characterized by chronic low-grade inflammation. Previous studies have demonstrated that the gut microbiome can affect the host tissue cells' mRNA N6-methyladenosine (m6A) modifications. This study aimed to understand the role of intestinal flora in ovarian cells inflammation by regulating mRNA m6A modification particularly the inflammatory state in PCOS. The gut microbiome composition of PCOS and Control groups was analyzed by 16S rRNA sequencing, and the short chain fatty acids were detected in patients' serum by mass spectrometry methods. The level of butyric acid was found to be decreased in the serum of the obese PCOS group (FAT) compared to other groups, and this was correlated with increased Streptococcaceae and decreased Rikenellaceae based on the Spearman's rank test. Additionally, we identified FOSL2 as a potential METTL3 target using RNA-seq and MeRIP-seq methodologies. Cellular experiments demonstrated that the addition of butyric acid led to a decrease in FOSL2 m6A methylation levels and mRNA expression by suppressing the expression of METTL3, an m6A methyltransferase. Additionally, NLRP3 protein expression and the expression of inflammatory cytokines (IL-6 and TNF-α) were downregulated in KGN cells. Butyric acid supplementation in obese PCOS mice improved ovarian function and decreased the expression of local inflammatory factors in the ovary. Taken together, the correlation between the gut microbiome and PCOS may unveil crucial mechanisms for the role of specific gut microbiota in the pathogenesis of PCOS. Furthermore, butyric acid may present new prospects for future PCOS treatments.
    Keywords:  Butyric acid; Gut microbiome; Inflammation; PCOS; m6A modification
    DOI:  https://doi.org/10.1186/s13148-023-01487-9
  7. Oncogene. 2023 Apr 25.
    METTL1/WDR4-mediated tRNA m7G modification and mRNA translation control promote oncogenesis and doxorubicin resistance.
    Zhaoyu Wang, Peng Yu, Yutong Zou, Jieyi Ma, Hui Han, Wei Wei, Chunlong Yang, Siyi Zheng, Siyao Guo, Juan Wang, Lianlian Liu, Shuibin Lin.
      Osteosarcoma is the most common bone tumor that leads to high mortality in adolescents and children. The tRNA N7-methylguanosine methyltransferase METTL1 is located in chromosome 12q14.1, a region that is frequently amplified in osteosarcoma patients, while its functions and underlying mechanisms in regulation of osteosarcoma remain unknown. Herein we show that METTL1 and WDR4 are overexpressed in osteosarcoma and associated with poor patient prognosis. Knockdown of METTL1 or WDR4 causes decreased tRNA m7G modification level and impairs osteosarcoma progression in vitro and in vivo. Conversely, METTL1/WDR4 overexpression promotes osteosarcoma proliferation, migration and invasion capacities. tRNA methylation and mRNA translation profiling indicate that METTL1/WDR4 modified tRNAs enhance translation of mRNAs with more m7G tRNA-decoded codons, including extracellular matrix (ECM) remodeling effectors, which facilitates osteosarcoma progression and chemoresistance to doxorubicin. Our study demonstrates METTL1/WDR4 mediated tRNA m7G modification plays crucial oncogenic functions to enhance osteosarcoma progression and chemoresistance to doxorubicin via alteration of oncogenic mRNA translation, suggesting METTL1 inhibition combined with chemotherapy is a promising strategy for treatment of osteosarcoma patients.
    DOI:  https://doi.org/10.1038/s41388-023-02695-6
  8. Aging (Albany NY). 2023 May 12. 15
    Insights into N6-methyladenosine (m6A) modification of noncoding RNA in tumor microenvironment.
    YanJun Zhang, Lijuan Zhan, Jing Li, Xue Jiang, Li Yin.
      N6-methyladenosine (m6A) is the most abundant RNA modification in eukaryotes, and it participates in the regulation of pathophysiological processes in various diseases, including malignant tumors, by regulating the expression and function of both coding and non-coding RNAs (ncRNAs). More and more studies demonstrated that m6A modification regulates the production, stability, and degradation of ncRNAs and that ncRNAs also regulate the expression of m6A-related proteins. Tumor microenvironment (TME) refers to the internal and external environment of tumor cells, which is composed of numerous tumor stromal cells, immune cells, immune factors, and inflammatory factors that are closely related to tumors occurrence and development. Recent studies have suggested that crosstalk between m6A modifications and ncRNAs plays an important role in the biological regulation of TME. In this review, we summarized and analyzed the effects of m6A modification-associated ncRNAs on TME from various perspectives, including tumor proliferation, angiogenesis, invasion and metastasis, and immune escape. Herein, we showed that m6A-related ncRNAs can not only be expected to become detection markers of tumor tissue samples, but can also be wrapped into exosomes and secreted into body fluids, thus exhibiting potential as markers for liquid biopsy. This review provides a deeper understanding of the relationship between m6A-related ncRNAs and TME, which is of great significance to the development of a new strategy for precise tumor therapy.
    Keywords:  biomarker; exosome; m6A; ncRNA; targeted therapy; tumor metastasis; tumor microenvironment
    DOI:  https://doi.org/10.18632/aging.204679
  9. Front Oncol. 2023 ;13 1138238
    The m6A methyltransferase METTL16 inhibits the proliferation of pancreatic adenocarcinoma cancer cells via the p21 signaling pathway.
    Fuming Xie, Yao Zheng, Wen Fu, Bojing Chi, Xianxing Wang, Junfeng Zhang, Jianyou Gu, Jingyang Yin, Qiang Zhou, Shixiang Guo, Lei Cai, Jiali Yang, Songsong Liu, Huaizhi Wang.
       Background: Many studies have reported that N6-methyladenosine (m6A) modification plays a critical role in the epigenetic regulation of organisms and especially in the pathogenesis of malignant diseases. However, m6A research has mainly focused on methyltransferase activity mediated by METTL3, and few studies have focused on METTL16. The aim of this study was to investigate the mechanism of METTL16, which mediates m6A modification, and its role in pancreatic adenocarcinoma (PDAC) cell proliferation.
    Methods: Clinicopathologic and survival data were retrospectively collected from 175 PDAC patients from multiple clinical centers to detect the expression of METTL16. CCK-8, cell cycle, EdU and xenograft mouse model experiments were used to evaluate the proliferation effect of METTL16. Potential downstream pathways and mechanisms were explored via RNA sequencing, m6A sequencing, and bioinformatic analyses. Regulatory mechanisms were studied through methyltransferase inhibition, RIP, MeRIP‒qPCR assays.
    Results: We found that METTL16 expression was markedly downregulated in PDAC, and multivariate Cox regression analyses revealed that METTL16 was a protective factor for PDAC patients. We also demonstrated that METTL16 overexpression inhibited PDAC cell proliferation. Furthermore, we identified a METTL16-p21 signaling axis, with downregulation of METTL16 resulting in inhibition of CDKN1A (p21). Additionally, METTL16 silencing and overexpression experiments highlighted m6A modification alterations in PDAC.
    Conclusions: METTL16 plays a tumor-suppressive role and suppresses PDAC cell proliferation through the p21 pathway by mediating m6A modification. METTL16 may be a novel marker of PDAC carcinogenesis and target for the treatment of PDAC.
    Keywords:  METTL16; cell proliferation; m6A; p21; pancreatic adenocarcinoma
    DOI:  https://doi.org/10.3389/fonc.2023.1138238
  10. FASEB J. 2023 Jun;37(6): e22964
    FTO represses NLRP3-mediated pyroptosis and alleviates myocardial ischemia-reperfusion injury via inhibiting CBL-mediated ubiquitination and degradation of β-catenin.
    Fei Sun, Cheng An, Can Liu, Ying Hu, Yue Su, Zhixiang Guo, Hong Che, Shenglin Ge.
      Cardiac ischemia/reperfusion (I/R) injury is a complicated pathological event, which has close association with pyroptosis. This study uncovered the regulatory mechanisms of fat mass and obesity-associated protein (FTO) in NLRP3-mediated pyroptosis during cardiac I/R injury. H9c2 cells were stimulated with oxygen-glucose deprivation/reoxygenation (OGD/R). Cell viability and pyroptosis were detected by CCK-8 and flow cytometry. Western blotting or RT-qPCR was performed to analyze target molecule expression. NLRP3 and Caspase-1 expression was observed by immunofluorescence staining. IL-18 and IL-1β production was detected by ELISA. The total m6A and m6A level of CBL was determined by dot blot assay and methylated RNA immunoprecipitation-qPCR, respectively. The interaction between IGF2BP3 and CBL mRNA was confirmed by RNA pull-down and RIP assays. The protein interaction between CBL and β-catenin and β-catenin ubiquitination were evaluated by Co-IP. Myocardial I/R model was established in rats. We determined infarct size by TTC staining and pathological changes by H&E staining. LDH, CK-MB, LVFS, and LVEF were also assessed. FTO and β-catenin were down-regulated, while CBL was up-regulated by OGD/R stimulation. FTO/β-catenin overexpression or CBL silencing restrained OGD/R-induced NLRP3 inflammasome-mediated pyroptosis. CBL repressed β-catenin expression via ubiquitination and degradation. FTO reduced the mRNA stability of CBL by inhibiting m6A modification. CBL-mediated ubiquitination and degradation of β-catenin were involved in FTO-induced pyroptosis inhibition during myocardial I/R injury. FTO inhibits NLRP3-mediated pyroptosis to attenuate myocardial I/R injury via repressing CBL-induced ubiquitination degradation of β-catenin.
    Keywords:  CBL; FTO; NLRP3-mediated pyroptosis; myocardial I/R injury; β-catenin
    DOI:  https://doi.org/10.1096/fj.202201793RR
  11. Environ Toxicol. 2023 May 17.
    METTL14 represses osteoclast formation to ameliorate osteoporosis via enhancing GPX4 mRNA stability.
    Mingsi Deng, Jia Luo, Heng Cao, Yong Li, Liangjian Chen, Gengyan Liu.
      Excessive bone resorption by osteoclasts results in the development of multiple bone disorders including osteoporosis. This study aimed to explore the biological function of methyltransferase-like14 (METTL14) in osteoclast formation, as well as its related mechanisms. Expression levels of METTL14, GPX4 and osteoclast-related proteins TRAP, NFATc1, c-Fos were detected by qRT-PCR and Western blotting. The osteoporosis model was established in mice by bilateral ovariectomy (OVX). Bone histomorphology was determined by micro-CT and H&E staining. NFATc1 expression in bone tissues was determined by immunohistochemical staining. Proliferation of primary bone marrow macrophages cells (BMMs) was assessed by MTT assay. Osteoclast formation was observed by TRAP staining. The regulatory mechanism was evaluated by RNA methylation quantification assay, MeRIP-qPCR, dual luciferase reporter assay, and RIP, respectively. METTL14 was down-regulated in the serum samples of postmenopausal osteoporotic women, which was positively associated with bone mineral density (BMD). Osteoclast formation was promoted in OVX-treated METTL14+/- mice as compared with wild-type littermates. Conversely, METTL14 overexpression repressed RANKL-induced osteoclast differentiation of BMMs. Mechanistically, METTL14-mediated m6A modification post-transcriptionally stabilized glutathione peroxidase 4 (GPX4), with the assistance of Hu-Antigen R (HuR). Finally, GPX4 depletion-mediated osteoclast formation in BMMs could be counteracted by METTL14 or HuR overexpression. Collectively, METTL14 inhibits osteoclastogenesis and bone resorption via enhancing GPX4 stability through an m6A-HuR dependent mechanism. Therefore, targeting METTL14 might be a novel promising treatment strategy for osteoporosis.
    Keywords:  GPX4; METTL14; m6A modification; osteoclast formation; osteoporosis
    DOI:  https://doi.org/10.1002/tox.23829
  12. Cell Death Discov. 2023 May 18. 9(1): 170
    Novel insights into the METTL3-METTL14 complex in musculoskeletal diseases.
    Yeqiu Xu, Yuanzhuang Zhang, Yinzhou Luo, Guanzhen Qiu, Jie Lu, Ming He, Yong Wang.
      N6-methyladenosine (m6A) modification, catalyzed by methyltransferase complexes (MTCs), plays many roles in multifaceted biological activities. As the most important subunit of MTCs, the METTL3-METTL14 complex is reported to be the initial factor that catalyzes the methylation of adenosines. Recently, accumulating evidence has indicated that the METTL3-METTL14 complex plays a key role in musculoskeletal diseases in an m6A-dependent or -independent manner. Although the functions of m6A modifications in a variety of musculoskeletal diseases have been widely recognized, the critical role of the METTL3-METTL14 complex in certain musculoskeletal disorders, such as osteoporosis, osteoarthritis, rheumatoid arthritis and osteosarcoma, has not been systematically revealed. In the current review, the structure, mechanisms and functions of the METTL3-METTL14 complex and the mechanisms and functions of its downstream pathways in the aforementioned musculoskeletal diseases are categorized and summarized.
    DOI:  https://doi.org/10.1038/s41420-023-01435-9
  13. Transl Cancer Res. 2023 Apr 28. 12(4): 992-1005
    N6-methyladenosine reader IGF2BP3 as a prognostic Biomarker contribute to malignant progression of glioma.
    Xin Zheng, Shenggang Li, Ju Yu, Chungang Dai, Suji Yan, Gang Chen, Chao Sun.
       Background: Glioblastoma (GBM) is a highly aggressive cancer having a dismal prognosis. N6-methyladenosine (m6A) is closely related to GBM progression. The significance of m6A modifications depends on the m6A readers, whose functions in glioma progression are largely unknown. This study sought to investigate the expression of the m6A related gene in glioma and its effect on the malignant progression of glioma.
    Methods: The expression differences between low-grade gliomas (LGGs) and high-grade gliomas (HGGs), and among 19 m6A-related genes were analyzed by The Cancer Genome Atlas (TCGA). Survival probability was analyzed in terms of the high or low expression of insulin growth factor-2 binding protein 3 (IGF2BP3) in the TCGA data set. The clinicopathological data of 40 patients with glioma were analyzed retrospectively, and the expression of IGF2BP3 in the tumor tissues was analyzed by immunohistochemistry (IHC). Lentiviral vectors harboring short-hairpin RNA (shRNA) were used to knock down IGF2BP3 in the glioma cell lines U87 and U251, and the results were verified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot. The effects of IGF2BP3 on the proliferation, invasion, and tumorigenicity of the glioma cells were verified by Cell Counting Kit-8 (CCK-8), transwell invasion, and subcutaneous tumorigenesis experiments in nude mice. The cell cycle phases were measured by flow cytometry.
    Results: The sequencing of TCGA data identified IGF2BP3 as the most significantly altered m6A-related gene. Patients with high IGF2BP3 expression had a significantly reduced survival probability (P<0.001) compared to those with low IGF2BP3 expression. IGF2BP3 was more upregulated in the HGGs than the LGGs. The downregulation of IGF2BP3 inhibited the proliferation, migration, and invasiveness of the glioma cells, and xenograft tumor growth in the mice. According to TCGA data, IGF2BP3 was closely related to cell cycle regulators, such as cyclin-dependent kinase 1 (CDK1) and cell-division cycle protein 20 homologue (CDC20). Further, the knockdown of IGF2BP3 affected the expression of CDK1 and the cell cycle process.
    Conclusions: IGF2BP3 expression in glioma is positively correlated with tumor grade and enhanced glioma cell proliferation, invasion, and tumorigenicity. IGF2BP3 knockdown decreased the expression of CDK1 and the cell cycle process. The current study showed that IGF2BP3 may serve as a biomarker of prognosis and a therapeutic target in glioma.
    Keywords:  N6-methyladenosine (m6A); glioma; insulin growth factor-2 binding protein 3 (IGF2BP3); invasion; proliferation
    DOI:  https://doi.org/10.21037/tcr-23-449
  14. Mediators Inflamm. 2023 ;2023 5565054
    Identification of a New m6A Regulator-Related Methylation Signature for Predicting the Prognosis and Immune Microenvironment of Patients with Pancreatic Cancer.
    Tianle Zou, Dan Shi, Weiwei Wang, Guoyong Chen, Xianbin Zhang, Yu Tian, Peng Gong.
      Pancreatic cancer (PC) is a malignant tumor of the digestive system that has a bad prognosis. N6-methyladenosine (m6A) is involved in a wide variety of biological activities due to the fact that it is the most common form of mRNA modification in mammals. Numerous research has accumulated evidence suggesting that a malfunction in the regulation of m6A RNA modification is associated with various illnesses, including cancers. However, its implications in PC remain poorly characterized. The methylation data, level 3 RNA sequencing data, and clinical information of PC patients were all retrieved from the TCGA datasets. Genes associated with m6A RNA methylation were compiled from the existing body of research and made available for download from the m6Avar database. The LASSO Cox regression method was used to construct a 4-gene methylation signature, which was then used to classify all PC patients included in the TCGA dataset into either a low- or high-risk group. In this study, based on the set criteria of |cor| > 0.4 and p value < 0.05. A total of 3507 gene methylation were identified to be regulated by m6A regulators. Based on the univariate Cox regression analysis and identified 3507 gene methylation, 858 gene methylation was significantly associated with the patient's prognosis. The multivariate Cox regression analysis identified four gene methylation (PCSK6, HSP90AA1, TPM3, and TTLL6) to construct a prognosis model. Survival assays indicated that the patients in the high-risk group tend to have a worse prognosis. ROC curves showed that our prognosis signature had a good prediction ability on patient survival. Immune assays suggested a different immune infiltration pattern in patients with high- and low-risk scores. Moreover, we found that two immune-related genes, CTLA4 and TIGIT, were downregulated in high-risk patients. We generated a unique methylation signature that is related to m6A regulators and is capable of accurately predicting the prognosis for patients with PC. The findings might prove useful for therapeutic customization and the process of making medical decisions.
    DOI:  https://doi.org/10.1155/2023/5565054
  15. Cell Mol Life Sci. 2023 May 19. 80(6): 156
    CSNK1D-mediated phosphorylation of HNRNPA2B1 induces miR-25-3p/miR-93-5p maturation to promote prostate cancer cell proliferation and migration through m6A-dependent manner.
    Feng Qi, Wenyi Shen, Xiyi Wei, Yifei Cheng, Fan Xu, Yuxiao Zheng, Lu Li, Chao Qin, Xiao Li.
      It has been reported that heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) is highly expressed in prostate cancer (PCa) and associated with poor prognosis of patients with PCa. Nevertheless, the specific mechanism underlying HNRNPA2B1 functions in PCa remains not clear. In our study, we proved that HNRNPA2B1 promoted the progression of PCa through in vitro and in vivo experiments. Further, we found that HNRNPA2B1 induced the maturation of miR-25-3p/miR-93-5p by recognizing primary miR-25/93 (pri-miR-25/93) through N6-methyladenosine (m6A)-dependent manner. In addition, both miR-93-5p and miR-25-3p were proven as tumor promoters in PCa. Interestingly, by mass spectrometry analysis and mechanical experiments, we found that casein kinase 1 delta (CSNK1D) could mediate the phosphorylation of HNRNPA2B1 to enhance its stability. Moreover, we further proved that miR-93-5p targeted BMP and activin membrane-bound inhibitor (BAMBI) mRNA to reduce its expression, thereby activating transforming growth factor β (TGF-β) pathway. At the same time, miR-25-3p targeted forkhead box O3 (FOXO3) to inactivate FOXO pathway. These results collectively indicated that CSNK1D stabilized HNRNPA2B1 facilitates the processing of miR-25-3p/miR-93-5p to regulate TGF-β and FOXO pathways, resulting in PCa progression. Our findings supported that HNRNPA2B1 might be a promising target for PCa treatment.
    Keywords:  CSNK1D; HNRNPA2B1; Prostate cancer; m6A
    DOI:  https://doi.org/10.1007/s00018-023-04798-5
  16. Water (Basel). 2022 Nov 02. pii: 3595. [Epub ahead of print]14(22):
    ALKBH4 Stabilization Is Required for Arsenic-Induced 6mA DNA Methylation Inhibition, Keratinocyte Malignant Transformation, and Tumorigenicity.
    Yan-Hong Cui, Emma Wilkinson, Jack Peterson, Yu-Ying He.
      Inorganic arsenic is one of the well-known human skin carcinogens. However, the molecular mechanism by which arsenic promotes carcinogenesis remains unclear. Previous studies have established that epigenetic changes, including changes in DNA methylation, are among the critical mechanisms that drive carcinogenesis. N6-methyladenine (6mA) methylation on DNA is a widespread epigenetic modification that was initially found on bacterial and phage DNA. Only recently has 6mA been identified in mammalian genomes. However, the function of 6mA in gene expression and cancer development is not well understood. Here, we show that chronic low doses of arsenic induce malignant transformation and tumorigenesis in keratinocytes and lead to the upregulation of ALKBH4 and downregulation of 6mA on DNA. We found that reduced 6mA levels in response to low levels of arsenic were mediated by the upregulation of the 6mA DNA demethylase ALKBH4. Moreover, we found that arsenic increased ALKBH4 protein levels and that ALKBH4 deletion impaired arsenic-induced tumorigenicity in vitro and in mice. Mechanistically, we found that arsenic promoted ALKBH4 protein stability through reduced autophagy. Together, our findings reveal that the DNA 6mA demethylaseALKBH4 promotes arsenic tumorigenicity and establishes ALKBH4 as a promising target for arsenic-induced tumorigenesis.
    Keywords:  6mA DNA methylation; ALKBH4; arsenic; autophagy; epigenetics; skin cancer
    DOI:  https://doi.org/10.3390/w14223595
  17. Nat Metab. 2023 May 15.
    m6A modification-tuned sphingolipid metabolism regulates postnatal liver development in male mice.
    Shiguan Wang, Shanze Chen, Jianfeng Sun, Pan Han, Bowen Xu, Xinying Li, Youquan Zhong, Zaichao Xu, Peng Zhang, Ping Mi, Cuijuan Zhang, Lixiang Li, Haiyan Zhang, Yuchen Xia, Shiyang Li, Mathias Heikenwalder, Detian Yuan.
      Different organs undergo distinct transcriptional, epigenetic and physiological alterations that guarantee their functional maturation after birth. However, the roles of epitranscriptomic machineries in these processes have remained elusive. Here we demonstrate that expression of RNA methyltransferase enzymes Mettl3 and Mettl14 gradually declines during postnatal liver development in male mice. Liver-specific Mettl3 deficiency causes hepatocyte hypertrophy, liver injury and growth retardation. Transcriptomic and N6-methyl-adenosine (m6A) profiling identify the neutral sphingomyelinase, Smpd3, as a target of Mettl3. Decreased decay of Smpd3 transcripts due to Mettl3 deficiency results in sphingolipid metabolism rewiring, characterized by toxic ceramide accumulation and leading to mitochondrial damage and elevated endoplasmic reticulum stress. Pharmacological Smpd3 inhibition, Smpd3 knockdown or Sgms1 overexpression that counteracts Smpd3 can ameliorate the abnormality of Mettl3-deficent liver. Our findings demonstrate that Mettl3-N6-methyl-adenosine fine-tunes sphingolipid metabolism, highlighting the pivotal role of an epitranscriptomic machinery in coordination of organ growth and the timing of functional maturation during postnatal liver development.
    DOI:  https://doi.org/10.1038/s42255-023-00808-9
  18. Biomed Pharmacother. 2023 May 15. pii: S0753-3322(23)00673-X. [Epub ahead of print]163 114883
    Novel targets for gastric cancer: The tumor microenvironment (TME), N6-methyladenosine (m6A), pyroptosis, autophagy, ferroptosis and cuproptosis.
    Peizheng Yang, Wanting Yang, Zhong Wei, Yan Li, Yinfeng Yang, Jinghui Wang.
      Gastric cancer (GC) is a fatal illness, and its mortality rate is very high all over the world. At present, it is a serious health problem for any country. It is a multifactorial disease due to the rising drug resistance and the increasing global cancer burden, the treatment of GC still faces many obstacles and problems. In recent years, research on GC is being carried out continuously, and we hope to address the new targets of GC treatment through this review. At the same time, we also hope to discover new ways to fight GC and create more gospel for clinical patients. First, we discuss the descriptive tumor microenvironment (TME), N6-methyladenosine (m6A), pyroptosis, autophagy, ferroptosis, and cuproptosis. Finally, we expounded on the new or potential targets of GC treatment.
    Keywords:  Autophagy; Cuproptosis; Ferroptosis; Gastric cancer (GC); N6-methyladenosine (m6A); Pyroptosis; The tumor microenvironment (TME)
    DOI:  https://doi.org/10.1016/j.biopha.2023.114883
  19. Nat Commun. 2023 May 17. 14(1): 2806
    PRMT1 mediated methylation of cGAS suppresses anti-tumor immunity.
    Jing Liu, Xia Bu, Chen Chu, Xiaoming Dai, John M Asara, Piotr Sicinski, Gordon J Freeman, Wenyi Wei.
      Activation of the cGAS/STING innate immunity pathway is essential and effective for anti-tumor immunotherapy. However, it remains largely elusive how tumor-intrinsic cGAS signaling is suppressed to facilitate tumorigenesis by escaping immune surveillance. Here, we report that the protein arginine methyltransferase, PRMT1, methylates cGAS at the conserved Arg133 residue, which prevents cGAS dimerization and suppresses the cGAS/STING signaling in cancer cells. Notably, genetic or pharmaceutical ablation of PRMT1 leads to activation of cGAS/STING-dependent DNA sensing signaling, and robustly elevates the transcription of type I and II interferon response genes. As such, PRMT1 inhibition elevates tumor-infiltrating lymphocytes in a cGAS-dependent manner, and promotes tumoral PD-L1 expression. Thus, combination therapy of PRMT1 inhibitor with anti-PD-1 antibody augments the anti-tumor therapeutic efficacy in vivo. Our study therefore defines the PRMT1/cGAS/PD-L1 regulatory axis as a critical factor in determining immune surveillance efficacy, which serves as a promising therapeutic target for boosting tumor immunity.
    DOI:  https://doi.org/10.1038/s41467-023-38443-3
  20. Cell Death Discov. 2023 May 18. 9(1): 169
    The cancer-testis lncRNA LINC01977 promotes HCC progression by interacting with RBM39 to prevent Notch2 ubiquitination.
    Anliang Xia, Qi Yue, Mingming Zhu, Jianbo Xu, Siyuan Liu, Yue Wu, Zhangding Wang, Zhu Xu, Hongda An, Qiang Wang, Shouyu Wang, Beicheng Sun.
      Cancer-testis genes are involved in the occurrence and development of cancer, but the role of cancer-testis-associated lncRNAs (CT-lncRNAs) in hepatocellular carcinoma (HCC) remains to be explored. Here, we discovered a novel CT-lncRNA, LINC01977, based on the Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) databases. LINC01977 was exclusively expressed in testes and highly expressed in HCC. High LINC01977 levels correlated with poorer overall survival (OS) in individuals with HCC. Functional assays showed that LINC01977 promoted HCC growth and metastasis in vitro and in vivo. Mechanistically, LINC01977 directly bound to RBM39 to promote the further entry of Notch2 into the nucleus, thereby preventing the ubiquitination and degradation of Notch2. Furthermore, the RNA binding protein IGF2BP2, one of the m6A modification readers, enhanced the stability of LINC01977, resulting in its high level in HCC. Therefore, the data suggest that LINC01977 interacts with RBM39 and promotes the progression of HCC by inhibiting Notch2 ubiquitination and degradation, indicating that LINC01977 may be a potential biomarker and therapeutic target for HCC patients.
    DOI:  https://doi.org/10.1038/s41420-023-01459-1
  21. J Exp Clin Cancer Res. 2023 May 19. 42(1): 127
    Epigenetically silenced lncRNA SNAI3-AS1 promotes ferroptosis in glioma via perturbing the m6A-dependent recognition of Nrf2 mRNA mediated by SND1.
    Jianglin Zheng, Qing Zhang, Zhen Zhao, Yue Qiu, Yujie Zhou, Zhipeng Wu, Cheng Jiang, Xuan Wang, Xiaobing Jiang.
       BACKGROUND: Ferroptosis has been linked to tumor progression and resistance to antineoplastic therapy. Long noncoding RNA (lncRNA) exerts a regulatory role in various biological processes of tumor cells, while the function and molecular mechanism of lncRNA in ferroptosis are yet to be clarified in glioma.
    METHODS: Both gain-of-function and loss-of-function experiments were employed to investigate the effects of SNAI3-AS1 on the tumorigenesis and ferroptosis susceptibility of glioma in vitro and in vivo. Bioinformatics analysis, Bisulfite sequencing PCR, RNA pull-down, RIP, MeRIP and dual-luciferase reporter assay were performed to explore the low expression mechanism of SNAI3-AS1 and the downstream mechanism of SNAI3-AS1 in ferroptosis susceptibility of glioma.
    RESULTS: We found that ferroptosis inducer erastin downregulates SNAI3-AS1 expression in glioma by increasing the DNA methylation level of SNAI3-AS1 promoter. SNAI3-AS1 functions as a tumor suppressor in glioma. Importantly, SNAI3-AS1 enhances the anti-tumor activity of erastin by promoting ferroptosis both in vitro and in vivo. Mechanistically, SNAI3-AS1 competitively binds to SND1 and perturbs the m6A-dependent recognition of Nrf2 mRNA 3'UTR by SND1, thereby reducing the mRNA stability of Nrf2. Rescue experiments confirmed that SND1 overexpression and silence can rescue the gain- and loss-of-function ferroptotic phenotypes of SNAI3-AS1, respectively.
    CONCLUSIONS: Our findings elucidate the effect and detailed mechanism of SNAI3-AS1/SND1/Nrf2 signalling axis in ferroptosis, and provide a theoretical support for inducing ferroptosis to improve glioma treatment.
    Keywords:  Ferroptosis; Glioma; LncRNA; Nrf2; m6A
    DOI:  https://doi.org/10.1186/s13046-023-02684-3
  22. Angew Chem Int Ed Engl. 2023 May 15. e202302930
    An Integrated Dual-Functional Nanotool Capable of Studying Single-Cell Epigenetics and Programmable Gene Regulation.
    Xiao-Mei Shi, Yi-Tong Xu, Bing Wang, Zheng Li, Si-Yuan Yu, Hang Dong, Wei-Wei Zhao, Dechen Jiang, Hong-Yuan Chen, Jing-Juan Xu.
      Single-cell epigenetics is envisioned to decipher manifold epigenetic phenomena and to contribute to our accurate knowledge about basic epigenetic mechanisms. Engineered nanopipette technology has gained momentum in single-cell study, whereas solutions to epigenetic study remain unachieved. This study addresses the challenge by exploring N6-methyladenine (m6A)-bearing deoxyribozyme (DNAzyme) confined within nanopipette for profiling representative m6A modifying enzyme of fat mass and obesity-associated protein (FTO). Electroosmotic intracellular extraction of FTO could remove the m6A and cause DNAzyme cleavage, leading to the altered ionic current signal. Because the cleavage can release a DNA sequence, we simultaneously program it as an antisense strand against FTO-mRNA, intracellular injection of which has been shown to induce early stage apoptosis. This nanotool thus features the dual functions of studying single-cell epigenetics and programmable gene regulation.
    Keywords:  Electrochemistry; epigenetics; gene regulation; nanopipette; single cell
    DOI:  https://doi.org/10.1002/anie.202302930
  23. Mol Ther Nucleic Acids. 2023 Jun 13. 32 402-414
    SINEUP non-coding RNA activity depends on specific N6-methyladenosine nucleotides.
    Bianca Pierattini, Sabrina D'Agostino, Carlotta Bon, Omar Peruzzo, Andrej Alendar, Azzurra Codino, Gloria Ros, Francesca Persichetti, Remo Sanges, Piero Carninci, Claudio Santoro, Stefano Espinoza, Paola Valentini, Luca Pandolfini, Stefano Gustincich.
      SINEUPs are natural and synthetic antisense long non-coding RNAs (lncRNAs) selectively enhancing target mRNAs translation by increasing their association with polysomes. This activity requires two RNA domains: an embedded inverted SINEB2 element acting as effector domain, and an antisense region, the binding domain, conferring target selectivity. SINEUP technology presents several advantages to treat genetic (haploinsufficiencies) and complex diseases restoring the physiological activity of diseased genes and of compensatory pathways. To streamline these applications to the clinic, a better understanding of the mechanism of action is needed. Here we show that natural mouse SINEUP AS Uchl1 and synthetic human miniSINEUP-DJ-1 are N6-methyladenosine (m6A) modified by METTL3 enzyme. Then, we map m6A-modified sites along SINEUP sequence with Nanopore direct RNA sequencing and a reverse transcription assay. We report that m6A removal from SINEUP RNA causes the depletion of endogenous target mRNA from actively translating polysomes, without altering SINEUP enrichment in ribosomal subunit-associated fractions. These results prove that SINEUP activity requires an m6A-dependent step to enhance translation of target mRNAs, providing a new mechanism for m6A translation regulation and strengthening our knowledge of SINEUP-specific mode of action. Altogether these new findings pave the way to a more effective therapeutic application of this well-defined class of lncRNAs.
    Keywords:  MT: Non-coding RNAs; N6-methyladenosine; Nanopore direct RNA sequencing; RNA therapeutics; SINEUP; lncRNA; m6A; translation
    DOI:  https://doi.org/10.1016/j.omtn.2023.04.002
  24. Mol Cancer. 2023 05 12. 22(1): 83
    Epigenetic inactivation of the 5-methylcytosine RNA methyltransferase NSUN7 is associated with clinical outcome and therapeutic vulnerability in liver cancer.
    Vanessa Ortiz-Barahona, Marta Soler, Veronica Davalos, Carlos A García-Prieto, Maxime Janin, Fernando Setien, Irene Fernández-Rebollo, Joan J Bech-Serra, Carolina De La Torre, Sonia Guil, Alberto Villanueva, Pei-Hong Zhang, Li Yang, Marco Guarnacci, Ulrike Schumann, Thomas Preiss, Ugne Balaseviciute, Robert Montal, Josep M Llovet, Manel Esteller.
       BACKGROUND: RNA modifications are important regulators of transcript activity and an increasingly emerging body of data suggests that the epitranscriptome and its associated enzymes are altered in human tumors.
    METHODS: Combining data mining and conventional experimental procedures, NSUN7 methylation and expression status was assessed in liver cancer cell lines and primary tumors. Loss-of-function and transfection-mediated recovery experiments coupled with RNA bisulfite sequencing and proteomics determined the activity of NSUN7 in downstream targets and drug sensitivity.
    RESULTS: In this study, the initial screening for genetic and epigenetic defects of 5-methylcytosine RNA methyltransferases in transformed cell lines, identified that the NOL1/NOP2/Sun domain family member 7 (NSUN7) undergoes promoter CpG island hypermethylation-associated with transcriptional silencing in a cancer-specific manner. NSUN7 epigenetic inactivation was common in liver malignant cells and we coupled bisulfite conversion of cellular RNA with next-generation sequencing (bsRNA-seq) to find the RNA targets of this poorly characterized putative RNA methyltransferase. Using knock-out and restoration-of-function models, we observed that the mRNA of the coiled-coil domain containing 9B (CCDC9B) gene required NSUN7-mediated methylation for transcript stability. Most importantly, proteomic analyses determined that CCDC9B loss impaired protein levels of its partner, the MYC-regulator Influenza Virus NS1A Binding Protein (IVNS1ABP), creating sensitivity to bromodomain inhibitors in liver cancer cells exhibiting NSUN7 epigenetic silencing. The DNA methylation-associated loss of NSUN7 was also observed in primary liver tumors where it was associated with poor overall survival. Interestingly, NSUN7 unmethylated status was enriched in the immune active subclass of liver tumors.
    CONCLUSION: The 5-methylcytosine RNA methyltransferase NSUN7 undergoes epigenetic inactivation in liver cancer that prevents correct mRNA methylation. Furthermore, NSUN7 DNA methylation-associated silencing is associated with clinical outcome and distinct therapeutic vulnerability.
    Keywords:  5-methylcytosine RNA methyltransferase; DNA methylation; Epigenetics; Liver cancer; NSUN7; RNA modifications
    DOI:  https://doi.org/10.1186/s12943-023-01785-z
  25. Front Oncol. 2023 ;13 1117622
    Preliminary study based on methylation and transcriptome gene sequencing of lncRNAs and immune infiltration in hypopharyngeal carcinoma.
    Kainan Wu, Fen Chang, Wenming Li, Dongmin Wei, Shengda Cao, Yulin Xie, Ce Li, Dapeng Lei.
       Background: Hypopharyngeal squamous cell cancer (HSCC) is one of the most malignant tumors of the head and neck. It is not easy to detect in the early stage due to its hidden location; thus, lymph node metastasis is highly likely at diagnosis, leading to a poor prognosis. It is believed that epigenetic modification is related to cancer invasion and metastasis. However, the role of m6A-related lncRNA in the tumor microenvironment (TME) of HSCC remains unclear.
    Methods: The whole transcriptome and methylation sequencing of 5 pairs of HSCC tissues and adjacent tissues were performed to identify the methylation and transcriptome profiles of lncRNAs. The biological significance of lncRNAs differentially expressing the m6A peak was analyzed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. By constructing an m6A lncRNA-microRNA network, the mechanism of m6A lncRNAs in HSCC was analyzed. The relative expression levels of selected lncRNAs were examined by quantitative polymerase chain reaction. The CIBERSORT algorithm was used to evaluate the relative proportion of immune cell infiltration in HSCC and paracancerous tissues.
    Results: Based on an in-depth analysis of the sequencing results, 14413 differentially expressed lncRNAs were revealed, including 7329 up-regulated and 7084 down-regulated lncRNAs. Additionally, 4542 up-methylated and 2253 down-methylated lncRNAs were detected. We demonstrated methylation patterns and gene expression profiles of lncRNAs of HSCC transcriptome. In the intersection analysis of lncRNAs and methylated lncRNAs, 51 lncRNAs with up-regulated transcriptome and methylation and 40 lncRNAs with down-regulated transcriptome and methylation were screened, and significantly differentiated lncRNAs were further studied. In the immune cell infiltration analysis, B cell memory was significantly elevated in cancer tissue, while γδT cell amount was significantly decreased.
    Conclusion: m6A modification of lncRNAs might be involved in HSCC pathogenesis. Infiltration of immune cells in HSCC might provide a new direction for its treatment. This study provides new insights for exploring the possible HSCC pathogenesis and searching for new potential therapeutic targets.
    Keywords:  MeRIP-seq; hypopharyngeal carcinoma; immune cell infiltration; lncRNAs; miRNAs; transcriptome sequencing
    DOI:  https://doi.org/10.3389/fonc.2023.1117622
  26. Cells. 2023 04 29. pii: 1283. [Epub ahead of print]12(9):
    The RNA Demethylase ALKBH5 Maintains Endoplasmic Reticulum Homeostasis by Regulating UPR, Autophagy, and Mitochondrial Function.
    Panneerdoss Subbarayalu, Pooja Yadav, Santosh Timilsina, Daisy Medina, Kunal Baxi, Robert Hromas, Ratna K Vadlamudi, Yidong Chen, Patrick Sung, Manjeet K Rao.
      Eukaryotic cells maintain cellular fitness by employing well-coordinated and evolutionarily conserved processes that negotiate stress induced by internal or external environments. These processes include the unfolded protein response, autophagy, endoplasmic reticulum-associated degradation (ERAD) of unfolded proteins and altered mitochondrial functions that together constitute the ER stress response. Here, we show that the RNA demethylase ALKBH5 regulates the crosstalk among these processes to maintain normal ER function. We demonstrate that ALKBH5 regulates ER homeostasis by controlling the expression of ER lipid raft associated 1 (ERLIN1), which binds to the activated inositol 1, 4, 5,-triphosphate receptor and facilitates its degradation via ERAD to maintain the calcium flux between the ER and mitochondria. Using functional studies and electron microscopy, we show that ALKBH5-ERLIN-IP3R-dependent calcium signaling modulates the activity of AMP kinase, and consequently, mitochondrial biogenesis. Thus, these findings reveal that ALKBH5 serves an important role in maintaining ER homeostasis and cellular fitness.
    Keywords:  ALKBH5; ER homeostasis; ERLIN1; autophagy; m6A; mitochondria; unfolded protein response
    DOI:  https://doi.org/10.3390/cells12091283
  27. Oral Dis. 2023 May 15.
    SOX18 meditates the resistance of Bmi1-expressing cells to cetuximab in HNSCC.
    Caihua Zhang, Zhi Chen, Nailin Gao, Gan Xiong, Peng Chen, Hui Li, Demeng Chen, Qianting He, Liang Peng.
       OBJECTIVE: Head and neck squamous cell carcinoma (HNSCC) is the most common type of malignancy in the head and neck region worldwide. The therapeutic strategies for HNSCC remain unsatisfying and limited. Here, we found a population of resistant Bmi1-expressing cells in the presence of cetuximab treatment and reported a novel role of SRY-box transcription factor 18 (SOX18), a member of the SOX family, in promoting HNSCC resistance to cetuximab. This study aimed to investigate the regulatory mechanism of Sox18 in Bmi1-positive cells and to search for better therapeutic targets.
    METHODS: We successfully obtained Bmi1CreER , RosatdTomato , and RosaDTA mice and identified Bmi1-expressing cells through lineage tracing. SOX18 expression in HNSCC and normal tissues was analyzed by immunohistochemistry, colocalization of Sox18, and Bmi1-expressing cells was analyzed by immunofluorescence, and SOX18 expression in SCC9 cell lines was quantified by western blotting and quantitative real-time PCR. The investigation of the mechanism of SOX18-mediated cetuximab resistance in Bmi1-positive cells was based on the analysis of single-cell RNA-seq data obtained from the Gene Expression Omnibus (GEO) database. Western blotting was performed to verify the results obtained from the single-cell RNA-seq analysis.
    RESULTS: In our study, we demonstrated that Bmi1-expressing cells were resistant to cetuximab treatment and that depletion of Bmi1-expressing cells improved cetuximab efficacy in HNSCC. We then discovered that Sox18 mediated the stem cell-like properties of Bmi1-expressing cells and promoted cellular cetuximab resistance through an oxidative phosphorylation pathway. There was a significant downregulation of key genes in the oxidative phosphorylation pathway in Sox18 knockout cell lines.
    CONCLUSIONS: Taken together, the findings of our study suggest that Sox18 mediates the resistance of Bmi1-expressing cells to cetuximab in HNSCC via the oxidative phosphorylation pathway.
    Keywords:  HNSCC; Sox18; cancer stem cells; cetuximab; oxidative phosphorylation
    DOI:  https://doi.org/10.1111/odi.14596
  28. Discov Oncol. 2023 May 12. 14(1): 67
    Myc derived circRNA promotes triple-negative breast cancer progression via reprogramming fatty acid metabolism.
    Shengting Wang, Yufang Wang, Yue Wang, Qian Li, Kaixuan Zeng, Xiaoming Li, Xinghua Feng.
      Myc is a well-known proto-oncogene that is frequently amplified and activated in breast cancer, especially in triple-negative breast cancer (TNBC). However, the role of circular RNA (circRNA) generated by Myc remains unclear. Herein, we found that circMyc (hsa_circ_0085533) was remarkably upregulated in TNBC tissues and cell lines, which was attributed to gene amplification. Genetic knockdown of circMyc mediated by lentiviral vector significantly inhibited TNBC cell proliferation and invasion. Importantly, circMyc increased cellular triglycerides, cholesterols and lipid droplet contents. CircMyc was detected in both cytoplasm and nucleus, cytoplasmic circMyc could directly bind to HuR protein, facilitating the binding of HuR to SREBP1 mRNA, resulting in increasing SREBP1 mRNA stability. Nuclear circMyc bound to Myc protein, facilitating the occupation of Myc on SREBP1 promoter, leading to increasing SREBP1 transcription. As a result, the elevated SREBP1 increased the expression of its downstream lipogenic enzymes, enhancing lipogenesis and TNBC progression. Moreover, the orthotopic xenograft model showed that depletion of circMyc markedly inhibited lipogenesis and reduced tumor size. Clinically, high circMyc was closely related to larger tumor volume, later clinical stage and lymph node metastasis, functioning as an adverse prognostic factor. Collectively, our findings characterize a novel Myc-derived circRNA controlling TNBC tumorigenesis via regulation of metabolic reprogramming, implying a promising therapeutic target.
    Keywords:  CircRNA; Metabolic reprogramming; Triple-negative breast cancer
    DOI:  https://doi.org/10.1007/s12672-023-00679-2
  29. Cell Mol Life Sci. 2023 May 19. 80(6): 157
    Overexpression of VIRMA confers vulnerability to breast cancers via the m6A-dependent regulation of unfolded protein response.
    Quintin Lee, Renhua Song, Dang Anh Vu Phan, Natalia Pinello, Jessica Tieng, Anni Su, James M Halstead, Alex C H Wong, Michelle van Geldermalsen, Bob S-L Lee, Bowen Rong, Kristina M Cook, Mark Larance, Renjing Liu, Fei Lan, Jessamy C Tiffen, Justin J-L Wong.
      Virilizer-like m6A methyltransferase-associated protein (VIRMA) maintains the stability of the m6A writer complex. Although VIRMA is critical for RNA m6A deposition, the impact of aberrant VIRMA expression in human diseases remains unclear. We show that VIRMA is amplified and overexpressed in 15-20% of breast cancers. Of the two known VIRMA isoforms, the nuclear-enriched full-length but not the cytoplasmic-localised N-terminal VIRMA promotes m6A-dependent breast tumourigenesis in vitro and in vivo. Mechanistically, we reveal that VIRMA overexpression upregulates the m6A-modified long non-coding RNA, NEAT1, which contributes to breast cancer cell growth. We also show that VIRMA overexpression enriches m6A on transcripts that regulate the unfolded protein response (UPR) pathway but does not promote their translation to activate the UPR under optimal growth conditions. Under stressful conditions that are often present in tumour microenvironments, VIRMA-overexpressing cells display enhanced UPR and increased susceptibility to death. Our study identifies oncogenic VIRMA overexpression as a vulnerability that may be exploited for cancer therapy.
    Keywords:  Breast cancer; Endoplasmic reticulum stress; Messenger RNA; N6-methyladenosine (m6a); Unfolded protein response; VIRMA
    DOI:  https://doi.org/10.1007/s00018-023-04799-4
  30. Clin Breast Cancer. 2023 May 15. pii: S1526-8209(23)00092-7. [Epub ahead of print]
    m6A Modification Mediates Exosomal LINC00657 to Trigger Breast Cancer Progression Via Inducing Macrophage M2 Polarization.
    Jiafeng Chen, Yuxin Zhou, Minhua Wu, Yijie Yuan, Weizhu Wu.
       BACKGROUND: Exosome-mediated transfer of long noncoding RNAs (lncRNAs) is critical for the cell-cell crosstalk in the tumor microenvironment. Nevertheless, the role of breast cancer (BC) cell-derived exosomal lncRNA in macrophage polarization during the development of BC remains unclear.
    METHODS: The key lncRNAs carried by BC cell-derived exosomes were identified by RNA-seq. CCK-8, flow cytometry, and transwell assay were conducted to analyze the role of LINC00657 in BC cells. In addition, immunofluorescence, qRT-PCR, western blot, and MeRIP-PCR were used to evaluate the function and underlying mechanism of exosomal LINC00657 in macrophage polarization.
    RESULTS: LINC00657 was distinctly upregulated in BC-derived exosomes and it was associated with increased m6A methylation modification levels. In addition, the depletion of LINC00657 significantly diminished the proliferative activity, migration and invasion potential of BC cells, and it also accelerated cell apoptosis. Exosomal LINC00657 from MDA-MB-231 cells could facilitate macrophage M2 activation, thus stimulating BC development in turn. Furthermore, LINC00657 activated the TGF-β signaling pathway by sequestering miR-92b-3p in macrophages.
    CONCLUSION: Exosomal LINC00657 secreted by BC cells could induce macrophage M2 activation, and these macrophages preferentially contributed to the malignant phenotype of BC cells. These results improve our understanding of BC and suggest a new therapeutic strategy for patients with BC.
    Keywords:  BC; Exosomes; TAM; TME; lncRNAs
    DOI:  https://doi.org/10.1016/j.clbc.2023.04.007
  31. J Leukoc Biol. 2023 May 17. pii: qiad061. [Epub ahead of print]
    The role of m6A mRNA modification in normal and malignant hematopoiesis.
    Zhangjing Ma, Rio Sugimura, Kathy O Lui.
      Hematopoiesis is a highly orchestrated biological process sustaining the supply of leukocytes involved in the maintenance of immunity, O2 and CO2 exchange, and wound healing throughout the life-time of an animal including human. During early hematopoietic cell development, several waves of hematopoiesis require the precise regulation of hematopoietic ontogeny as well as the maintenance of hematopoietic stem and progenitor cells (HSPCs) in the hematopoietic tissues, such as the fetal liver and bone marrow (BM). Recently, emerging evidence has suggested the critical role of m6A mRNA modification, an epigenetic modification dynamically regulated by its effector proteins, in the generation and maintenance of hematopoietic cells during embryogenesis. In the adulthood, m6A has also been demonstrated to be involved in the functional maintenance of HSPCs in the BM and umbilical cord blood, as well as the progression of malignant hematopoiesis. In this review, we focus on recent progress in identifying the biological functions of m6A mRNA modification, its regulators and downstream gene targets during normal and pathological hematopoiesis. We propose that targeting m6A mRNA modification could offer novel insights into therapeutic development against abnormal and malignant hematopoietic cell development in the future.
    Keywords:  hematopoiesis; leukemia; m6A; mRNA modification
    DOI:  https://doi.org/10.1093/jleuko/qiad061
  32. Int J Mol Sci. 2023 Apr 27. pii: 7957. [Epub ahead of print]24(9):
    The Functional Role and Regulatory Mechanism of FTO m6A RNA Demethylase in Human Uterine Leiomyosarcoma.
    Qiwei Yang, Ayman Al-Hendy.
      Uterine leiomyosarcoma (uLMS) is the most frequent subtype of uterine sarcoma that presents a poor prognosis and high rates of recurrence and metastasis. The origin and molecular mechanism underlying and driving its clinical and biological behavior remain largely unknown. Recently, we and others have revealed the role of microRNAs, DNA methylation, and histone modifications in contributing to the pathogenesis of uLMS. However, the connection between reversible m6A RNA methylation and uLMS pathogenesis remains unclear. In this study, we assessed the role and mechanism of FTO m6A RNA demethylase in the pathogenesis of uLMS. Immunohistochemistry analysis revealed that the levels of RNA demethylases FTO and ALKBH5 were aberrantly upregulated in uLMS tissues compared to adjacent myometrium with a significant change by histochemical scoring assessment (p < 0.01). Furthermore, the inhibition of FTO demethylase with its small, potent inhibitor (Dac51) significantly decreased the uLMS proliferation dose-dependently via cell cycle arrest. Notably, RNA-seq analysis revealed that the inhibition of FTO with Dac51 exhibited a significant decrease in cell-cycle-related genes, including several CDK members, and a significant increase in the expression of CDKN1A, which correlated with a Dac51-exerted inhibitory effect on cell proliferation. Moreover, Dac51 treatment allowed the rewiring of several critical pathways, including TNFα signaling, KRAS signaling, inflammation response, G2M checkpoint, and C-Myc signaling, among others, leading to the suppression of the uLMS phenotype. Moreover, transcription factor (TF) analyses suggested that epitranscriptional alterations by Dac51 may alter the cell cycle-related gene expression via TF-driven pathways and epigenetic networks in uLMS cells. This intersection of RNA methylation and other epigenetic controls and pathways provides a framework to better understand uterine diseases, particularly uLMS pathogenesis with a dysregulation of RNA methylation machinery. Therefore, targeting the vulnerable epitranscriptome may provide an additional regulatory layer for a promising and novel strategy for treating patients with this aggressive uterine cancer.
    Keywords:  DAC51; RNA methylation; apoptosis; cell cycle; demethylase; histone modifications; miRNAs; transcription factors; transcriptome analysis; uterine leiomyosarcoma
    DOI:  https://doi.org/10.3390/ijms24097957
  33. J Mol Cell Biol. 2023 May 15. pii: mjad033. [Epub ahead of print]
    BRSK2 in pancreatic β cells promotes hyperinsulinemia-coupled insulin resistance and its genetic variants are associated with human type 2 diabetes.
    Rufeng Xu, Kaiyuan Wang, Zhengjian Yao, Yan Zhang, Li Jin, Jing Pang, Yuncai Zhou, Kai Wang, Dechen Liu, Yaqin Zhang, Peng Sun, Fuqiang Wang, Xiaoai Chang, Tengli Liu, Shusen Wang, Yalin Zhang, Shuyong Lin, Cheng Hu, Yunxia Zhu, Xiao Han.
      Brain-specific serine/threonine-protein kinase 2 (BRSK2) plays critical roles in insulin secretion and β-cell biology. Whether BRSK2 is associated with human type 2 diabetes mellitus (T2DM) is never appreciated. Here, we report that BRSK2 genetic variants are closely related to worsening glucose metabolism due to hyperinsulinemia and insulin resistance in Chinese population. The BRSK2 protein levels are significantly accumulated in β cells from T2DM patients and high-fat-diet (HFD)-fed mice due to enhanced protein stability. Mice with inducible loss-of-function Brsk2 (βKO) are metabolic normal with high potential of insulin secretion under chow-diet condition. Moreover, βKO mice prevent from HFD-induced hyperinsulinemia, obesity, insulin resistance, and glucose intolerance. Conversely, gain-of-function Brsk2 in mature β cells reversibly triggers hyperglycemia due to β-cell hypersecretion-coupled insulin resistance. Mechanistically, BRSK2 senses lipid signals and induces basal insulin secretion in a kinase-dependent manner. The enhanced basal insulin secretion drives insulin resistance and β-cell exhaustion, and thus the onset of T2DM in mice with HFD feeding or β-cell gain-of-function BRSK2. These findings reveal that BRSK2 links hyperinsulinemia to systematic insulin resistance via interplaying between β cells and insulin-sensitive tissues in human genetic variant population or under nutrient-overload conditions.
    Keywords:  BRSK2; genetic variant; hyperinsulinemia; insulin resistance; type 2 diabetes mellitus; β-cell hypersecretion
    DOI:  https://doi.org/10.1093/jmcb/mjad033
  34. Cell Death Discov. 2023 May 12. 9(1): 159
    N1-Methyladenosine modification of mRNA regulates neuronal gene expression and oxygen glucose deprivation/reoxygenation induction.
    Zhangyang Qi, Chi Zhang, Huan Jian, Mengfan Hou, Yongfu Lou, Yi Kang, Wei Wang, Yigang Lv, Shenghui Shang, Chaoyu Wang, Xueying Li, Shiqing Feng, Hengxing Zhou.
      N1-Methyladenosine (m1A) is an abundant modification of transcripts, plays important roles in regulating mRNA structure and translation efficiency, and is dynamically regulated under stress. However, the characteristics and functions of mRNA m1A modification in primary neurons and oxygen glucose deprivation/reoxygenation (OGD/R) induced remain unclear. We first constructed a mouse cortical neuron OGD/R model and then used methylated RNA immunoprecipitation (MeRIP) and sequencing technology to demonstrate that m1A modification is abundant in neuron mRNAs and dynamically regulated during OGD/R induction. Our study suggests that Trmt10c, Alkbh3, and Ythdf3 may be m1A-regulating enzymes in neurons during OGD/R induction. The level and pattern of m1A modification change significantly during OGD/R induction, and differential methylation is closely associated with the nervous system. Our findings show that m1A peaks in cortical neurons aggregate at both the 5' and 3' untranslated regions. m1A modification can regulate gene expression, and peaks in different regions have different effects on gene expression. By analysing m1A-seq and RNA-seq data, we show a positive correlation between differentially methylated m1A peaks and gene expression. The correlation was verified by using qRT-PCR and MeRIP-RT-PCR. Moreover, we selected human tissue samples from Parkinson's disease (PD) and Alzheimer's disease (AD) patients from the Gene Expression Comprehensive (GEO) database to analyse the selected differentially expressed genes (DEGs) and differential methylation modification regulatory enzymes, respectively, and found similar differential expression results. We highlight the potential relationship between m1A modification and neuronal apoptosis following OGD/R induction. Furthermore, by mapping mouse cortical neurons and OGD/R-induced modification characteristics, we reveal the important role of m1A modification in OGD/R and gene expression regulation, providing new ideas for research on neurological damage.
    DOI:  https://doi.org/10.1038/s41420-023-01458-2
  35. Diabet Med. 2023 Apr 25. e15125
    The interface targeting hnRNPA2B1 regulates the repression of transthyretin against human retinal microvascular endothelial cells in high glucose environment.
    Yikun Tian, Lu Ye, Jun Shao, Yu Xin.
       BACKGROUND: The interaction between transthyretin (TTR) and heterogeneous nuclear ribonucleoprotein (hnRNP)A2B1 is involved in the neovascularization of human retinal microvascular endothelial cells (hRECs) under hyperglycemic conditions. However, whether the TTR-hnRNPA2B1 interface can be altered, and how this protein-protein interaction and associated downstream pathways are regulated is unclear.
    METHODS: We performed homologous sequential analysis and binding energy assays using Discovery Studio and designed substitution targeting three fragments of the interface (fragment 1: aa 34-39, -RKAADD-; fragment 2, aa 61-68, -EEEFVEGI-; and fragment 3, aa 96-102, -TANDSGP-) to disrupt or stabilize the TTR-hnRNPA2B1 complex and were subjected to Co-immunoprecipitation analysis. To investigate the effect of TTR-hnRNPA2B1 interface alterations on the physiological properties of hRECs, we performed CCK-8, EdU, migration, wound healing, and tube formation assays. To study the downstream genes, we performed qRT-PCR and western blot.
    RESULTS: Nineteen TTR substitutions were recombinantly expressed in soluble form, results indicated that reducing the binding energy stabilized the TTR-hnRNPA2B1, while increasing the binding energy had the opposite effect. The native TTR significantly prohibited the proliferation, DNA synthesis, migration, and tube formation capacities of hRECs, while fragment 1 always reduced these effects. However, the I68R and D99R substitutions in fragments 2 and 3, respectively, increased the inhibitory effect of TTR. Furthermore, our qRT-PCR and western blot results showed that the expression and protein levels of STAT-4, miR-223-3p, and FBXW7 were also regulated by the alteration of the TTR-hnRNPA2B1 interface.
    CONCLUSION: This work suggests that the formation of the TTR-hnRNPA2B1 complex plays vital roles in hyperglycemia, and modification of this interface regulates the TTR-mediated inhibition of hREC neovascularization via the STAT-4/miR-223-3p/FBXW7 pathway. This mechanism could have important implications for diabetic retinopathy treatment.
    Keywords:  Transthyretin (TTR); binding interface; heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1); human retinal microvascular endothelial cells (hRECs); hyperglycemia
    DOI:  https://doi.org/10.1111/dme.15125
  36. Phytomedicine. 2023 Apr 26. pii: S0944-7113(23)00201-5. [Epub ahead of print]116 154840
    Xiao Chai Hu Tang alleviates the pancreatic tumorigenesis via improving the mtDNA N6-Methyladenine modification mediated mitochondrial dysfunction in Syrian hamster model.
    Jun Cai, Wenyuan Shen, Guixian Zhang, Xia Li, Hongsheng Shen, Wenchang Li, Cheng Tan, Ting Zhang, Mengrou Shi, Zibo Yang, Yuan Li, Hongbin Liu, Xiumei Zhao.
       BACKGROUND: Pancreatic intraepithelial neoplasia (PanIN) is the most common precursor lesion of pancreatic ductal adenocarcinoma (PDAC), which is a highly malignant tumor and lack of effective treatment. Although Xiao Chai Hu Tang (XCHT) has a good therapeutic effect on pancreatic cancer patients with advanced stage, the effect and mechanism of XCHT remains unclear in pancreatic tumorigenesis.
    PURPOSE: To assess the therapeutic effects of XCHT on the malignant transformation from PanIN to PDAC and to reveal its mechanisms of pancreatic tumorigenesis.
    METHODS: Syrian golden hamster were induced by N-Nitrosobis (2-oxopropyl) amine (BOP) to establish the pancreatic tumorigenesis model. The morphological changes of pancreatic tissue were observed by H&E and Masson staining; the Gene ontology (GO) analysis the transcriptional profiling changes; the mitochondrial ATP generation, mitochondrial redox status, mitochondrial DNA (mtDNA) N6-methyladenine (6mA) level and relative mtDNA genes expressions were examined. In addition, immunofluorescence detect the cell localization of 6mA in human pancreatic cancer PANC1 cell. Using the TCGA database, the prognostic effect of mtDNA 6mA demethylation ALKBH1 expression on pancreatic cancer patients was analyzed.
    RESULTS: We confirmed the mtDNA 6mA levels were gradually increased with the mitochondrial dysfunction in PanINs progression. XCHT showed the effect to inhibit the occurrence and development of pancreatic cancer in Syrian hamster pancreatic tumorigenesis model. In addition, the lack of ALKBH1 mediated mtDNA 6mA increase, mtDNA coded genes down-expression and abnormal redox status were rescued by XCHT.
    CONCLUSIONS: ALKBH1/mtDNA 6mA mediated mitochondrial dysfunction to induce the occurrence and progression of pancreatic cancer. XCHT can improve ALKBH1 expression and mtDNA 6mA level, regulate the oxidative stress and expression of mtDNA coded genes. This study investigated a new molecular mechanism of pancreatic tumorigenesis, and revealed the therapeutic efficacy of XCHT in pancreatic tumorigenesis for the first time.
    Keywords:  ALKBH1; Mitochondrial dysfunction; N6-Methyladenine modification; Pancreatic tumorigenesis; Xiao Chai Hu Tang; mtDNA
    DOI:  https://doi.org/10.1016/j.phymed.2023.154840
  37. Front Immunol. 2023 ;14 1173035
    CTLA4 mRNA is downregulated by miR-155 in regulatory T cells, and reduced blood CTLA4 levels are associated with poor prognosis in metastatic melanoma patients.
    Prasanna Kumar Vaddi, Douglas Grant Osborne, Andrew Nicklawsky, Nazanin K Williams, Dinoop Ravindran Menon, Derek Smith, Jonathan Mayer, Anna Reid, Joanne Domenico, Giang Huong Nguyen, William A Robinson, Melanie Ziman, Dexiang Gao, Zili Zhai, Mayumi Fujita.
      Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is an immune checkpoint expressed in regulatory T (Treg) cells and activated T lymphocytes. Despite its potential as a treatment strategy for melanoma, CTLA-4 inhibition has limited efficacy. Using data from The Cancer Genome Atlas (TCGA) melanoma database and another dataset, we found that decreased CTLA4 mRNA was associated with a poorer prognosis in metastatic melanoma. To investigate further, we measured blood CTLA4 mRNA in 273 whole-blood samples from an Australian cohort and found that it was lower in metastatic melanoma than in healthy controls and associated with worse patient survival. We confirmed these findings using Cox proportional hazards model analysis and another cohort from the US. Fractionated blood analysis revealed that Treg cells were responsible for the downregulated CTLA4 in metastatic melanoma patients, which was confirmed by further analysis of published data showing downregulated CTLA-4 surface protein expression in Treg cells of metastatic melanoma compared to healthy donors. Mechanistically, we found that secretomes from human metastatic melanoma cells downregulate CTLA4 mRNA at the post-transcriptional level through miR-155 while upregulating FOXP3 expression in human Treg cells. Functionally, we demonstrated that CTLA4 expression inhibits the proliferation and suppressive function of human Treg cells. Finally, miR-155 was found to be upregulated in Treg cells from metastatic melanoma patients compared to healthy donors. Our study provides new insights into the underlying mechanisms of reduced CTLA4 expression observed in melanoma patients, demonstrating that post-transcriptional silencing of CTLA4 by miRNA-155 in Treg cells may play a critical role. Since CTLA-4 expression is downregulated in non-responder melanoma patients to anti-PD-1 immunotherapy, targeting miRNA-155 or other factors involved in regulating CTLA4 expression in Treg cells without affecting T cells could be a potential strategy to improve the efficacy of immunotherapy in melanoma. Further research is needed to understand the molecular mechanisms regulating CTLA4 expression in Treg cells and identify potential therapeutic targets for enhancing immune-based therapies.
    Keywords:  CTLA-4; biomarker; melanoma; miRNA-155; regulatory T cells
    DOI:  https://doi.org/10.3389/fimmu.2023.1173035
  38. Cancers (Basel). 2023 Apr 27. pii: 2501. [Epub ahead of print]15(9):
    TRAF6 Promotes PRMT5 Activity in a Ubiquitination-Dependent Manner.
    Liu Liu, Shasha Yin, Wenjian Gan.
      Protein arginine methyltransferase 5 (PRMT5) is the primary enzyme generating symmetric dimethylarginine (sDMA) on numerous substrates, through which it regulates many cellular processes, such as transcription and DNA repair. Aberrant expression and activation of PRMT5 is frequently observed in various human cancers and associated with poor prognosis and survival. However, the regulatory mechanisms of PRMT5 remain poorly understood. Here, we report that TRAF6 serves as an upstream E3 ubiquitin ligase to promote PRMT5 ubiquitination and activation. We find that TRAF6 catalyzes K63-linked ubiquitination of PRMT5 and interacts with PRMT5 in a TRAF6-binding-motif-dependent manner. Moreover, we identify six lysine residues located at the N-terminus as the primarily ubiquitinated sites. Disruption of TRAF6-mediated ubiquitination decreases PRMT5 methyltransferase activity towards H4R3 in part by impairing PRMT5 interaction with its co-factor MEP50. As a result, mutating the TRAF6-binding motifs or the six lysine residues significantly suppresses cell proliferation and tumor growth. Lastly, we show that TRAF6 inhibitor enhances cellular sensitivity to PRMT5 inhibitor. Therefore, our study reveals a critical regulatory mechanism of PRMT5 in cancers.
    Keywords:  PRMT5; TRAF6; arginine methylation; cancer; cell proliferation; ubiquitination
    DOI:  https://doi.org/10.3390/cancers15092501
  39. Aging (Albany NY). 2023 May 02. 15
    A novel methionine metabolism-related signature predicts prognosis and immunotherapy response in lung adenocarcinoma.
    Qing-Hua Chang, Yuan-Cui Zhang, Dong-Ying Zhang, Ting Mao, Ran Chang, Nan Wang, Yun Ye, Zi-Jun Xu.
      Recent research revealed methionine metabolism as a key mediator of tumor initiation and immune evasion. However, the relationship between methionine metabolism and tumor microenvironment (TME) in lung adenocarcinoma (LUAD) remains unknown. Here, we comprehensively analyzed the genomic alterations, expression patterns, and prognostic values of 68 methionine-related regulators (MRGs) in LUAD. We found that most MRGs were highly prognostic based on 30 datasets including 5024 LUAD patients. Three distinct MRG modification patterns were identified, which showed significant differences in clinical outcomes and TME characteristics: The C2 subtype was characterized by higher immune score, while the C3 subtype had more malignant cells and worse survival. We developed a MethScore to measure the level of methionine metabolism in LUAD. MethScore was positively correlated with T-cell dysfunction and tumor-associated macrophages (TAMs), indicating a dysfunctional TME phenotype in the high MethScore group. In addition, two immunotherapy cohorts confirmed that patients with a lower MethScore exhibited significant clinical benefits. Our study highlights the important role of methionine metabolism in modeling the TME. Evaluating methionine modification patterns will enhance our understanding of TME characteristics and can guide more effective immunotherapy strategies.
    Keywords:  immunotherapy; lung adenocarcinoma; methionine metabolism; molecular subtype; tumor microenvironment
    DOI:  https://doi.org/10.18632/aging.204687
  40. Cell Death Discov. 2023 May 15. 9(1): 164
    IGF2BP3 promotes adult myocardial regeneration by stabilizing MMP3 mRNA through interaction with m6A modification.
    Simeng Li, Siman Shen, Hao Xu, Shuyun Cai, Xiaodong Yuan, Changsen Wang, Xiaojun Zhang, Suyun Chen, Jianning Chen, De-Li Shi, Liangqing Zhang.
      Myocardial infarction that causes damage to heart muscle can lead to heart failure. The identification of molecular mechanisms promoting myocardial regeneration represents a promising strategy to improve cardiac function. Here we show that IGF2BP3 plays an important role in regulating adult cardiomyocyte proliferation and regeneration in a mouse model of myocardial infarction. IGF2BP3 expression progressively decreases during postnatal development and becomes undetectable in the adult heart. However, it becomes upregulated after cardiac injury. Both gain- and loss-of-function analyses indicate that IGF2BP3 regulates cardiomyocyte proliferation in vitro and in vivo. In particular, IGF2BP3 promotes cardiac regeneration and improves cardiac function after myocardial infarction. Mechanistically, we demonstrate that IGF2BP3 binds to and stabilizes MMP3 mRNA through interaction with N6-methyladenosine modification. The expression of MMP3 protein is also progressively downregulated during postnatal development. Functional analyses indicate that MMP3 acts downstream of IGF2BP3 to regulate cardiomyocyte proliferation. These results suggest that IGF2BP3-mediated post-transcriptional regulation of extracellular matrix and tissue remodeling contributes to cardiomyocyte regeneration. They should help to define therapeutic strategy for ameliorating myocardial infarction by inducing cell proliferation and heart repair.
    DOI:  https://doi.org/10.1038/s41420-023-01457-3
  41. Oral Dis. 2023 May 19.
    Mena as a key enhancer factor of EMT to promote metastasis of human tongue squamous cell carcinoma.
    Hao Cui, Xiang Liang, Yifei Zhu, Sadam Ahmed Elayah, Hong Qi, Linyang Xie, Zhichen Guo, Fang Siya, Yu Ming, Gong Yuxin, Junbo Tu, Sijia Na.
       OBJECTIVE: The aim of this study was to investigate the effect of mammalian-enabled (Mena) on tongue squamous cell carcinoma (TSCC) metastasis and its mechanism.
    MATERIALS AND METHODS: Immunochemistry was performed to investigate the Mena and tumor-related markers expression, and its clinicopathological characteristics in 46 TSCC specimens. TSCC cell SCC9 and Cal27 untransfected or stable transfected with Mena overexpression and small interfering RNA were used to determine the role of Mena in cell proliferation, cell migration, invasion and metastasis, and EMT-related markers in vitro, and the effect of Mena on TSCC growth and metastasis through tumor-bearing and tumor metastasis immunodeficient mice models in vivo.
    RESULTS: Immunochemistry showed that the expression of Mena was significantly correlated with lymphatic metastasis and TNM stage, E-cadherin, Vimentin, and MMP2. Mena did not affect cell proliferation and colony formation in vitro, and tumor growth in vivo. However, it promoted cell migration and invasion in vitro, and TSCC metastasis in vivo.
    CONCLUSIONS: Mena expression is associated with lymphatic metastasis and tumor stage and promotes TSCC invasion and metastasis by inducing the EMT process. Thus, Mena may be a biomarker for prognosis and targeted therapy in TSCC patients.
    Keywords:  Mena; epithelial-mesenchymal transition; metastasis; tongue squamous cell carcinoma
    DOI:  https://doi.org/10.1111/odi.14616
  42. Cancer Cell Int. 2023 May 18. 23(1): 96
    Polyamine metabolism patterns characterized tumor microenvironment, prognosis, and response to immunotherapy in colorectal cancer.
    Enkui Zhang, Chengsheng Ding, Shuchun Li, Batuer Aikemu, Xueliang Zhou, Xiaodong Fan, Jing Sun, Xiao Yang, Minhua Zheng.
       BACKGROUND: Changes in Polyamine metabolism (PAM) have been shown to establish a suppressive tumor microenvironment (TME) and substantially influence the progression of cancer in the recent studies. However, newly emerging data have still been unable to fully illuminate the specific effects of PAM in human cancers. Here, we analyzed the expression profiles and clinical relevance of PAM genes in colorectal cancer (CRC).
    METHODS: Based on unsupervised consensus clustering and principal component analysis (PCA) algorithm, we designed a scoring model to evaluate the prognosis of CRC patients and characterize the TME immune profiles, with related independent immunohistochemical validation cohort. Through comparative profiling of cell communities defined by single cell sequencing data, we identified the distinct characteristics of polyamine metabolism in the TME of CRC.
    RESULTS: Three PAM patterns with distinct prognosis and TME features were recognized from 1224 CRC samples. Moreover, CRC patients could be divided into high- and low-PAMscore subgroups by PCA-based scoring system. High PAMscore subgroup were associated to more advanced stage, higher infiltration level of immunosuppressive cells, and unfavorable prognosis. These results were also validated in CRC samples from other public CRC datasets and our own cohort, which suggested PAM genes were ideal biomarkers for predicting CRC prognosis. Notably, PAMscore also corelated with microsatellite instability-high (MSI-H) status, higher tumor mutational burden (TMB), and increased immune checkpoint gene expression, implying a potential role of PAM genes in regulating response to immunotherapy. To further confirm above results, we demonstrated a high-resolution landscape of TME and cell-cell communication network in different PAM patterns using single cell sequencing data and found that polyamine metabolism affected the communication between cancer cells and several immune cells such as T cells, B cells and myeloid cells.
    CONCLUSION: In total, our findings highlighted the significance of polyamine metabolism in shaping the TME and predicting the prognosis of CRC patients, providing novel strategies for immunotherapy and the targeting polyamine metabolites.
    Keywords:  Colorectal cancer; Immunotherapy; Polyamine metabolism; Prediction; Tumor microenvironment
    DOI:  https://doi.org/10.1186/s12935-023-02892-z
  43. Nat Struct Mol Biol. 2023 May 18.
    RNA stability controlled by m6A methylation contributes to X-to-autosome dosage compensation in mammals.
    Cornelia Rücklé, Nadine Körtel, M Felicia Basilicata, Anke Busch, You Zhou, Peter Hoch-Kraft, Kerstin Tretow, Fridolin Kielisch, Marco Bertin, Mihika Pradhan, Michael Musheev, Susann Schweiger, Christof Niehrs, Oliver Rausch, Kathi Zarnack, Claudia Isabelle Keller Valsecchi, Julian König.
      In mammals, X-chromosomal genes are expressed from a single copy since males (XY) possess a single X chromosome, while females (XX) undergo X inactivation. To compensate for this reduction in dosage compared with two active copies of autosomes, it has been proposed that genes from the active X chromosome exhibit dosage compensation. However, the existence and mechanisms of X-to-autosome dosage compensation are still under debate. Here we show that X-chromosomal transcripts have fewer m6A modifications and are more stable than their autosomal counterparts. Acute depletion of m6A selectively stabilizes autosomal transcripts, resulting in perturbed dosage compensation in mouse embryonic stem cells. We propose that higher stability of X-chromosomal transcripts is directed by lower levels of m6A, indicating that mammalian dosage compensation is partly regulated by epitranscriptomic RNA modifications.
    DOI:  https://doi.org/10.1038/s41594-023-00997-7
  44. Aging Dis. 2023 Mar 01.
    Overexpression of RACGAP1 by E2F1 Promotes Neuroendocrine Differentiation of Prostate Cancer by Stabilizing EZH2 Expression.
    Zhengshuai Song, Qi Cao, Bin Guo, Ye Zhao, Xuechao Li, Ning Lou, Chenxi Zhu, Gang Luo, Song Peng, Guohao Li, Ke Chen, Yong Wang, Hailong Ruan, Yonglian Guo.
      Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer. It is characterized by the loss of androgen receptor (AR) signaling in neuroendocrine transdifferentiation, and finally, resistance to AR-targeted therapy. With the application of a new generation of potent AR inhibitors, the incidence of NEPC is gradually increasing. The molecular mechanism of neuroendocrine differentiation (NED) after androgen deprivation therapy (ADT) remains largely unclear. In this study, using NEPC-related genome sequencing database analyses, we screened RACGAP1, a common differentially expressed gene. We investigated RACGAP1 expression in clinical prostate cancer specimens by IHC. Regulated pathways were examined by Western blotting, qRT-PCR, luciferase reporter, chromatin immunoprecipitation, and immunoprecipitation assays. The corresponding function of RACGAP1 in prostate cancer was analyzed by CCK-8 and Transwell assays. The changes of neuroendocrine markers and AR expression in C4-2-R and C4-2B-R cells were detected in vitro. We confirmed that RACGAP1 contributed to NE transdifferentiation of prostate cancer. Patients with high tumor RACGAP1 expression had shorter relapse-free survival time. The expression of RACGAP1 was induced by E2F1. RACGAP1 promoted neuroendocrine transdifferentiation of prostate cancer by stabilizing EZH2 expression in the ubiquitin-proteasome pathway. Moreover, overexpression of RACGAP1 promoted enzalutamide resistance of castration-resistant prostate cancer (CRPC) cells. Our results showed that the upregulation of RACGAP1 by E2F1 increased EZH2 expression, which drove NEPC progression. This study explored the molecular mechanism of NED and may provide novel methods and ideas for targeted therapy of NEPC.
    DOI:  https://doi.org/10.14336/AD.2023.0202
  45. bioRxiv. 2023 May 06. pii: 2023.05.06.539708. [Epub ahead of print]
    Combinatorial quantification of 5mC and 5hmC at individual CpG dyads and the transcriptome in single cells reveals modulators of DNA methylation maintenance fidelity.
    Alex Chialastri, Saumya Sarkar, Elizabeth E Schauer, Shyl Lamba, Siddharth S Dey.
      Transmission of 5-methylcytosine (5mC) from one cell generation to the next plays a key role in regulating cellular identity in mammalian development and diseases. While recent work has shown that the activity of DNMT1, the protein responsible for the stable inheritance of 5mC from mother to daughter cells, is imprecise; it remains unclear how the fidelity of DNMT1 is tuned in different genomic and cell state contexts. Here we describe Dyad-seq, a method that combines enzymatic detection of modified cytosines with nucleobase conversion techniques to quantify the genome-wide methylation status of cytosines at the resolution of individual CpG dinucleotides. We find that the fidelity of DNMT1-mediated maintenance methylation is directly related to the local density of DNA methylation, and for genomic regions that are lowly methylated, histone modifications can dramatically alter the maintenance methylation activity. Further, to gain deeper insights into the methylation and demethylation turnover dynamics, we extended Dyad-seq to quantify all combinations of 5mC and 5-hydroxymethylcytosine (5hmC) at individual CpG dyads to show that TET proteins preferentially hydroxymethylate only one of the two 5mC sites in a symmetrically methylated CpG dyad rather than sequentially convert both 5mC to 5hmC. To understand how cell state transitions impact DNMT1-mediated maintenance methylation, we scaled the method down and combined it with the measurement of mRNA to simultaneously quantify genome-wide methylation levels, maintenance methylation fidelity and the transcriptome from the same cell (scDyad&T-seq). Applying scDyad&T-seq to mouse embryonic stem cells transitioning from serum to 2i conditions, we observe dramatic and heterogenous demethylation and the emergence of transcriptionally distinct subpopulations that are closely linked to the cell-to-cell variability in loss of DNMT1-mediated maintenance methylation activity, with regions of the genome that escape 5mC reprogramming retaining high levels of maintenance methylation fidelity. Overall, our results demonstrate that while distinct cell states can substantially impact the genome-wide activity of the DNA methylation maintenance machinery, locally there exists an intrinsic relationship between DNA methylation density, histone modifications and DNMT1-mediated maintenance methylation fidelity that is independent of cell state.
    DOI:  https://doi.org/10.1101/2023.05.06.539708
  46. Biomolecules. 2023 Apr 11. pii: 664. [Epub ahead of print]13(4):
    The Regulatory Network of METTL3 in the Nervous System: Diagnostic Biomarkers and Therapeutic Targets.
    Xiaojuan Su, Yi Qu, Dezhi Mu.
      Methyltransferase-like 3 (METTL3) is a typical component of N6-methyladenosine writers that exhibits methyltransferase activity and deposits methyl groups on RNA. Currently, accumulating studies have demonstrated the involvement of METTL3 in the regulation of neuro-physiological and pathological events. However, no reviews have comprehensively summarized and analyzed the roles and mechanisms of METTL3 in these events. Herein, we are focused on reviewing the roles of METTL3 in regulating normal neurophysiological (Neurogenesis, Synaptic Plasticity and Glial Plasticity, Neurodevelopment, Learning and Memory,) and neuropathological (Autism Spectrum Disorder, Major Depressive Disorder, Neurodegenerative disorders, Brain Tumors, Brain Injuries, and Other Brain Disorders) events. Our review found that although the down-regulated levels of METTL3 function through different roles and mechanisms in the nervous system, it primarily inactivates neuro-physiological events and triggers or worsens neuropathological events. In addition, our review suggests that METTL3 could be used as a diagnostic biomarker and therapeutic target in the nervous system. Collectively, our review has provided an up-to-date research outline of METTL3 in the nervous system. In addition, the regulatory network for METTL3 in the nervous system has been mapped, which could provide directions for future research, biomarkers for clinical diagnosis, and targets for disease treatment. Furthermore, this review has provided a comprehensive view, which could improve our understanding of METTL3 functions in the nervous system.
    Keywords:  METTL3; diagnostic biomarkers; neurobiological events; neurological disorders; regulatory network; therapeutic targets
    DOI:  https://doi.org/10.3390/biom13040664
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