bims-rimeca Biomed News
on RNA methylation in cancer
Issue of 2023–01–15
forty-five papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics
  1. FTO demethylates m6A modifications in CDKAL1 mRNA and promotes gastric cancer chemoresistance by altering mitochondrial dynamics.
  2. METTL3 alleviates D-gal-induced renal tubular epithelial cellular senescence via promoting miR-181a maturation.
  3. ALKBH5 alleviates hypoxia postconditioning injury in D-galactose-induced senescent cardiomyocytes by regulating STAT3.
  4. N6-Methyladenosine RNA Modifications Regulate the Response to Platinum through Nicotinamide N-methyltransferase.
  5. NPTX2 Promotes EMT in Cutaneous Squamous Cell Carcinoma via Mettl3-Mediated m6A Methylation of Snail.
  6. ALKBH5 activates FAK signaling through m6A demethylation in ITGB1 mRNA and enhances tumor-associated lymphangiogenesis and lymph node metastasis in ovarian cancer.
  7. Novel Insights into The Roles of N6-methyladenosine (m6A) Modification and Autophagy in Human Diseases.
  8. ALKBH5 prevents hepatocellular carcinoma progression by post-transcriptional inhibition of PAQR4 in an m6A dependent manner.
  9. The RNA m6A reader YTHDF1 is required for acute myeloid leukemia progression.
  10. Stabilization of IGF2BP1 by USP10 promotes breast cancer metastasis via CPT1A in an m6A-dependent manner.
  11. RBM10 recruits METTL3 to induce N6-methyladenosine-MALAT1-dependent modification, inhibiting the invasion and migration of NSCLC.
  12. Constructing and validating of m6a-related genes prognostic signature for stomach adenocarcinoma and immune infiltration: Potential biomarkers for predicting the overall survival.
  13. METTL3-Mediated lncSNHG7 m6A Modification in the Osteogenic/Odontogenic Differentiation of Human Dental Stem Cells.
  14. The N6-methyladenosine METTL3 regulates tumorigenesis and glycolysis by mediating m6A methylation of the tumor suppressor LATS1 in breast cancer.
  15. Crosstalk between 5-methylcytosine and N6-methyladenosine machinery defines disease progression, therapeutic response and pharmacogenomic landscape in hepatocellular carcinoma.
  16. METTL16 drives leukemogenesis and leukemia stem cell self-renewal by reprogramming BCAA metabolism.
  17. Prognostic Values of METTL3 and Its Roles in Tumor Immune Microenvironment in Pan-Cancer.
  18. Upregulation of LRRC8A by m5C modification-mediated mRNA stability suppresses apoptosis and facilitates tumorigenesis in cervical cancer.
  19. Epitranscriptomic N6-Methyladenosine Profile of SARS-CoV-2-Infected Human Lung Epithelial Cells.
  20. STRIP2 motivates non-small cell lung cancer progression by modulating the TMBIM6 stability through IGF2BP3 dependent.
  21. RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia.
  22. The regulation of N6-methyladenosine modification in PD-L1-induced anti-tumor immunity.
  23. N6-Methyladenosine and Reader Protein YTHDF2 Enhance the Innate Immune Response by Mediating DUSP1 mRNA Degradation and Activating Mitogen-Activated Protein Kinases during Bacterial and Viral Infections.
  24. Emerging Mutual Regulatory Roles between m6A Modification and microRNAs.
  25. Integrative analysis implicates the significance of m6A in the liver fibrosis of biliary atresia by regulating THY1.
  26. Synthesis of 2'-modified N6-methyladenosine phosphoramidites and their incorporation into siRNA.
  27. A potassium-chloride co-transporter promotes tumor progression and castration resistance of prostate cancer through m6A reader YTHDC1.
  28. Bone Marrow Mesenchymal Stem Cells Reversed Ovarian Aging-related m6A RNA Methylation Modification Profile in Aged Granulosa Cells.
  29. Helicobacter pylori-induced NAT10 stabilizes MDM2 mRNA via RNA acetylation to facilitate gastric cancer progression.
  30. Enhancer RNA promotes resistance to radiotherapy in bone-metastatic prostate cancer by m6A modification.
  31. A New Bacterial Adenosine-Derived Nucleoside as an Example of RNA Modification Damage.
  32. Quantification of substoichiometric modification reveals global tsRNA hypomodification, preferences for angiogenin-mediated tRNA cleavage, and idiosyncratic epitranscriptomes of human neuronal cell-lines.
  33. Inhibition of IGF2BP1 attenuates renal injury and inflammation by alleviating m6A modifications and E2F1/MIF pathway.
  34. Characterization of ligamentum flavum hypertrophy based on m6A RNA methylation modification and the immune microenvironment.
  35. Analysis of M6A associated lncRNAs in prognosis and immune response of NSCLC patients.
  36. Impact of m6A demethylase (ALKBH5, FTO) genetic polymorphism and expression levels on the development of pulmonary tuberculosis.
  37. P. gingivalis infection upregulates PD-L1 expression on dendritic cells, suppresses CD8+ T cell responses, and aggravates oral cancer.
  38. Shaping the landscape of N6-methyladenosine RNA methylation in Arabidopsis.
  39. CTP synthase 2 predicts inferior survival and mediates DNA damage response via interacting with BRCA1 in chronic lymphocytic leukemia.
  40. ABRO1 arrests postnatal cardiomyocyte proliferation and myocardial repair by blocking N6-methyladenosine methylation and expression of PSPH.
  41. Targeting TRMT5 suppresses hepatocellular carcinoma progression via inhibiting the HIF-1α pathways.
  42. Targeted systematic evolution of an RNA platform neutralizing DNMT1 function and controlling DNA methylation.
  43. The miRNA125a-5p and miRNA125b-1-5p cluster induces cell invasion by down-regulating DDB2-reduced epithelial-to-mesenchymal transition (EMT) in colorectal cancer.
  44. Identification of 2-methylthio-methylenethio-N6-(cis-4-hydroxyisopentenyl)-adenosine (msms2io6A37) as a novel modification at adenosine 37 of tRNAs from Salmonella typhimurium.
  45. The novel m6A writer METTL5 as prognostic biomarker probably associating with the regulation of immune microenvironment in kidney cancer.
  1. Clin Exp Pharmacol Physiol. 2023 Jan 11.
    FTO demethylates m6A modifications in CDKAL1 mRNA and promotes gastric cancer chemoresistance by altering mitochondrial dynamics.
    Na Liu, Chang Liu, Zixuan Wang, Longqing Wang, Jiang Wang, Jing Kong.
      N6-methyladenosine (m6A) modification is the most common mRNA modification that is considered a new layer of mRNA epigenetic regulation. Demethylase fat mass and obesity-associated protein (FTO) are important in the dynamic regulation of m6A, but their role in gastric cancer (GC) is not fully understood. This study revealed that FTO and CDKAL1 were upregulated in GC cells and tissue. CDKAL1 is the downstream target of FTO-mediated m6A modification, with FTO promoting GC cell proliferation through CDKAL1 and inducing mitochondrial fusion, eventually causing GC chemoresistance. In conclusion, FTO contributes to the increasing resistance of GC cells to 5-fluorouracil (5-Fu) by upregulating CDKAL1 and inducing mitochondrial fusion.
    Keywords:  CDKAL1; FTO; Gastric cancer; chemoresistance; mitochondrial
    DOI:  https://doi.org/10.1111/1440-1681.13748
  2. Mech Ageing Dev. 2023 Jan 03. pii: S0047-6374(22)00156-7. [Epub ahead of print]210 111774
    METTL3 alleviates D-gal-induced renal tubular epithelial cellular senescence via promoting miR-181a maturation.
    Yu Zhang, Xinran Ni, Lu Wei, Yue Yu, Bei Zhu, Yun Bai, Xiaohua Pei, Fei Gao, Lulu Guo, Zhenzhu Yong, Weihong Zhao.
      Methyltransferase-like protein 3 (METTL3) mediated N6-Methyladenosine (m6A) modification has been implicated in many physiological and pathological processes. However, its function and mechanism in kidney aging are not entirely clear. Here, we investigated changes in m6A levels of aging kidneys and the role of METTL3 in senescent renal tubular epithelial cells and its potential mechanisms. First, we used the naturally aged mouse model and the D-galactose (D-gal)-induced aged mouse model. Dot blot and m6A RNA methylation quantification showed significantly decreased m6A levels in both models. In addition, we observed that METTL3 was down-regulated in D-gal-induced senescent human renal tubular epithelial cell line (HK-2). METTL3 reduction was associated with senescence-related phenotypes of HK-2 cells. We also found that miR-181a-5p attenuated HK-2 senescence by targeting the NF-κB pathway. Moreover, METTL3 was able to promote the maturation of miR-181a-5p and then inhibited the expression of NF-κB and IL-1α. Taken together, we demonstrate that the METTL3/miR-181a-5p/NF-κB axis counteracts HK-2 senescence. Our results suggest that METTL3 may be a novel biomarker and a potential therapy target for kidney aging.
    Keywords:  Cell senescence; Kidney aging; METTL3; m(6)A modification; miR-181a
    DOI:  https://doi.org/10.1016/j.mad.2022.111774
  3. Shock. 2023 Jan 09.
    ALKBH5 alleviates hypoxia postconditioning injury in D-galactose-induced senescent cardiomyocytes by regulating STAT3.
    Xuan Zhang, Feixiang Li, Ji Ma, Xiaobei Zhang, Yiqing Yin.
       ABSTRACT: Ischemic postconditioning (I/Post) reduces ischemia/reperfusion (I/R) injury by activating endogenous cardioprotection mechanisms, such as the JAK/STAT3 and PI3K/Akt pathways, which offer a traditional approach to myocardial protection. According to the previous study, cardioprotection by I/Post may lost in aged mice, and in our previous research, hypoxic postconditioning (H/Post) lacked a protective effect in senescent cardiomyocytes, which was associated with low expression of long noncoding RNA (lncRNA) H19. The N6-methyladenosine (m6A) modification is a dynamic and reversible process that has been confirmed plays a role in cardiovascular diseases. However, the mechanisms of m6A modification in myocardial I/Post remains to be explored. Neonatal cardiomyocytes were isolated from 2-day-old Sprague Dawley rats and senescence was induced by D-galactose, followed by stimulation of hypoxia-reoxygenation (H/R) and H/Post. Hypoxic injury was evaluated by cell viability and the Bcl-2/Bax protein ratio. Total m6A levels were measured using a colorimetric m6A RNA Methylation Quantification Kit and the m6A modified and differentially expressed mRNA was determined by methylated RNA immunoprecipitation (MeRIP). We found that H/Post increased m6A methylation and decreased RNA mA demethylase alkB homolog 5 (ALKBH5) expression in aged cardiomyocytes. Furthermore, ALKBH5 knockdown exacerbated injury following H/Post in senescent cardiomyocytes. Additionally, ALKBH5 regulated STAT3 expression by mediating its m6A modification and lncRNA H19/miR-124-3p. ALKBH5 also alleviated the H/Post injury induced by the low expression of STAT3 in senescent cardiomyocytes.
    DOI:  https://doi.org/10.1097/SHK.0000000000002031
  4. Mol Cancer Ther. 2023 Jan 09. pii: MCT-22-0278. [Epub ahead of print]
    N6-Methyladenosine RNA Modifications Regulate the Response to Platinum through Nicotinamide N-methyltransferase.
    Hao Huang, Guangyuan Zhao, Horacio Cardenas, Andres Felipe Valdivia, Yinu Wang, Daniela Matei.
      Development of resistance to platinum (Pt) in ovarian cancer (OC) remains a major clinical challenge. Here we focused on identifying epitranscriptomic modifications linked to Pt resistance. FTO is a N6-methyladenosine (m6A) RNA demethylase recently described by us as a tumor suppressor in OC. We hypothesized that FTO induced removal of m6A marks regulates the cellular response of OC cells to Pt and is linked to the development of resistance. To study the involvement of FTO in the cellular response to Pt, we used OC cells in which FTO was knocked down (KD) via shRNA or overexpressed and Pt-resistant (Pt-R) models derived through repeated cycles of exposure to Pt. We found that FTO was significantly downregulated in Pt-R vs. sensitive OC cells. Forced expression of FTO, but not of mutant FTO, increased sensitivity to Pt in vitro and in vivo (p<0.05). Increased numbers of -H2AX foci, measuring DNA double strand breaks, and increased apoptosis were observed after exposure to Pt in FTO overexpressing vs. control cells. Through integrated RNA-sequencing and MeRIP-sequencing, we identified and validated the enzyme nicotinamide N-methyltransferase (NNMT), as a new FTO target linked to Pt response. NNMT was upregulated and demethylated in FTO overexpressing cells. Treatment with an NNMT inhibitor or NNMT knockdown restored sensitivity to Pt in FTO overexpressing cells. Our results support a new function for FTO-dependent m6A RNA modifications in regulating the response to Pt through NNMT, a newly identified RNA methylated gene target.
    DOI:  https://doi.org/10.1158/1535-7163.MCT-22-0278
  5. J Invest Dermatol. 2023 Jan 10. pii: S0022-202X(23)00009-X. [Epub ahead of print]
    NPTX2 Promotes EMT in Cutaneous Squamous Cell Carcinoma via Mettl3-Mediated m6A Methylation of Snail.
    Tong Kang, Cheng Zhang, Hao Lei, Ruiting Luo, Meng Liu, Shengbang Wang, Xiu Zhang, Qiqi Duan, Shengxiang Xiao, Yan Zheng.
      Cutaneous squamous cell carcinoma (cSCC) is the second most common type of skin cancer. Neuronal pentraxin 2 (NPTX2), a member of the neuronal pentraxin family, is reported to play inconsistent roles in different cancers. The role and mechanism of NPTX2 in cSCC remains unclear. In this study, we found that NPTX2 was overexpressed in both skin lesions and cell lines of cSCC. In vitro studies demonstrated that NPTX2 facilitated cell proliferation, migration, invasion, colony formation, and epithelial-mesenchymal translation (EMT) in A431 and SCL-1 cells. NPTX2 interacted with Methyltransferase-like 3 (Mettl3), increased Mettl3 expression, and improved N6-methyladenosine (m6A) modification in cSCC cell lines. Mechanistically, NPTX2 facilitated EMT by promoting Mettl3-mediated m6A of Snail. Mettl3 knockdown and m6A inhibition reversed the impacts of NPTX2 overexpression on cSCC cells. In vivo studies verified the role of NPTX2 as an oncogene in cSCC. Therefore, NPTX2 may be a potential therapeutic target for cSCC.
    Keywords:  EMT; Mettl3; NPTX2; cSCC; m(6)A
    DOI:  https://doi.org/10.1016/j.jid.2022.12.015
  6. Theranostics. 2023 ;13(2): 833-848
    ALKBH5 activates FAK signaling through m6A demethylation in ITGB1 mRNA and enhances tumor-associated lymphangiogenesis and lymph node metastasis in ovarian cancer.
    Rui Sun, Lin Yuan, Yi Jiang, Yicong Wan, Xiaolling Ma, Jing Yang, Guodong Sun, Shulin Zhou, Hui Wang, Jiangnan Qiu, Lin Zhang, Wenjun Cheng.
      Background: Lymph node (LN) metastasis is common in patients with epithelial ovarian cancer (EOC) and is associated with poor prognosis. Tumor-associated lymphangiogenesis is the first stage of LN metastasis. Research on lymphangiogenesis and lymph node metastases can help develop new anti-LN-targeted therapies. Aberrant N6-methyladenosine (m6A) modifications have been reported to be linked to LN metastasis in several cancers, however, their role in EOC lymphangiogenesis and LN metastasis remains unclear. Methods: m6A levels in EOC tissues with or without LN metastases were evaluated by dot blot analysis. Real-time polymerase chain reaction (PCR) and immunofluorescence were used to examine the expression of m6A-related enzymes. Additionally, in vitro and in vivo functional studies were performed to discover the importance of the AlkB homolog 5 (ALKBH5) gene in EOC lymphatic metastasis. To identify the downstream target genes regulated by ALKBH5, we performed RNA pulldown, RNA-binding protein immunoprecipitation-quantitative PCR, co-immunoprecipitation, m6A-modified RNA immunoprecipitation-quantitative PCR, and luciferase reporter assays. Results: m6A modification was reduced in ovarian cancers with LN metastases. ALKBH5 overexpression increased tumor-associated lymphangiogenesis and LN metastasis both in vitro and in vivo. ALKBH5 overexpression also reversed the m6A modification in ITGB1 mRNA and suppressed the YTHDF2 protein-mediated m6A-dependent ITGB1 mRNA degradation, which resulted in increased expression of ITGB1 and phosphorylation of the focal adhesion kinase (FAK) and Src proto-oncogene proteins, thereby increasing LN metastasis. Furthermore, hypoxia induced the expression of hypoxia inducible factor 1 subunit alpha, which increased ALKBH5 expression and enhanced LN metastasis in EOC. Conclusions: The ALKBH5/m6A-ITGB1/FAK signalling axis is important in ovarian cancer lymphangiogenesis and LN metastasis. Antibodies that block ITGB1 and FAK kinase-inhibitors are promising anti-metastatic agents.
    Keywords:  ALKBH5; ITGB1; N6-methyladenosine; epithelial ovarian cancer; lymphatic metastasis
    DOI:  https://doi.org/10.7150/thno.77441
  7. Int J Biol Sci. 2023 ;19(2): 705-720
    Novel Insights into The Roles of N6-methyladenosine (m6A) Modification and Autophagy in Human Diseases.
    Jiaxin Liang, Jingwen Sun, Wei Zhang, Xiwen Wang, Ying Xu, Yuan Peng, Ling Zhang, Wenqian Xiong, Yi Liu, Hengwei Liu.
      Autophagy is an evolutionarily conserved cellular degradation and recycling process. It is important for maintaining vital cellular function and metabolism. Abnormal autophagy activity can cause the development of various diseases. N6-methyladenosine (m6A) methylation is the most prevalent and abundant internal modification in eukaryotes, affecting almost all aspects of RNA metabolism. The process of m6A modification is dynamic and adjustable. Its regulation depends on the regulation of m6A methyltransferases, m6A demethylases, and m6A binding proteins. m6A methylation and autophagy are two crucial and independent cellular events. Recent studies have shown that m6A modification mediates the transcriptional and post-transcriptional regulation of autophagy-related genes, affecting autophagy regulatory networks in multiple diseases. However, the regulatory effects of m6A regulators on autophagy in human diseases are not adequately acknowledged. In the present review, we summarized the latest knowledge of m6A modification in autophagy and elucidated the molecular regulatory mechanisms underlying m6A modification in autophagy regulatory networks. Moreover, we discuss the potentiality of m6A regulators serving as promising predictive biomarkers for human disease diagnosis and targets for therapy. This review will increase our understanding of the relationship between m6A methylation and autophagy, and provide novel insights to specifically target m6A modification in autophagy-associated therapeutic strategies.
    Keywords:  Autophagy; Biomarkers; N6-methyladenosine (m6A); RNA modification; Therapeutic targets
    DOI:  https://doi.org/10.7150/ijbs.75466
  8. Exp Hematol Oncol. 2023 Jan 06. 12(1): 1
    ALKBH5 prevents hepatocellular carcinoma progression by post-transcriptional inhibition of PAQR4 in an m6A dependent manner.
    Weijian Wang, Qibo Huang, Zhibin Liao, Hongwei Zhang, Yachong Liu, Furong Liu, Xiaoping Chen, Bixiang Zhang, Yan Chen, Peng Zhu.
       BACKGROUND: N6-methyladenosine (m6A) is a prevalent modification of mRNA and is known to play important roles in tumorigenesis in many types of cancer. The function of N6-methyladenosine (m6A) RNA methylation depends on a variety of methyltransferases and demethylases. AlkB homolog 5 (ALKBH5) is a demethylase, and its biological function has not been completely explored in HCC.
    RESULTS: ALKBH5 is downregulated and has antitumor effects in HCC cells. In addition, Progestin and AdipoQ Receptor 4 (PAQR4) was identified as a downstream target of ALKBH5 based on transcriptome sequencing and validation studies. We found that ALKBH5 decreases PAQR4 mRNA and protein expression in an N6-methyladenosine (m6A)-dependent manner. The study also showed that ALKBH5 changes PAQR4 expression via the m6A reader IGF2BP1. In both in vivo and in vitro experiments, PAQR4 showed a strong association with the development of HCC. Finally, we found that PAQR4 interacts with AKT and enhances PI3K/AKT pathway activation.
    CONCLUSIONS: ALKBH5 inhibits HCC growth by downregulating PAQR4 expression in an m6A-dependent manner, therefore suppressing PI3K/AKT pathway activation.
    Keywords:  AKT; ALKBH5; HCC; Methylation; PAQR4
    DOI:  https://doi.org/10.1186/s40164-022-00370-2
  9. Cancer Res. 2023 Jan 12. pii: CAN-21-4249. [Epub ahead of print]
    The RNA m6A reader YTHDF1 is required for acute myeloid leukemia progression.
    Yun-Guang Hong, Zhigang Yang, Yan Chen, Tian Liu, Yuyuan Zheng, Chun Zhou, Guo-Cai Wu, Yinhui Chen, Juan Xia, Ruiting Wen, Wenxin Liu, Yi Zhao, Jin Chen, Xiangwei Gao, Zhanghui Chen.
      N6-methyladenosine (m6A), the most abundant modification in mRNAs, has been defined as a crucial modulator in the progression of acute myeloid leukemia (AML). Identification of the key regulators of m6A modifications in AML could provide further insights into AML biology and uncover more effective therapeutic strategies for AML patients. Here we report overexpression of YTHDF1, an m6A reader protein, in human AML samples at the protein level with enrichment in leukemia stem cells (LSCs). Whereas YTHDF1 was dispensable for normal hematopoiesis in mice, depletion of YTHDF1 attenuated self-renewal, proliferation, and leukemic capacity of primary human and mouse AML cells in vitro and in vivo. Mechanistically, YTHDF1 promoted the translation of cyclin E2 in an m6A-dependent manner. Structure-based virtual screening of FDA-approved drugs identified tegaserod as a potential YTHDF1 inhibitor. Tegaserod blocked the direct binding of YTHDF1 with m6A-modified mRNAs and inhibited YTHDF1-regulated cyclin E2 translation. Moreover, tegaserod reduced the viability of patient-derived AML cells in vitro and prolonged survival in patient-derived xenograft models. Together, our study defines YTHDF1 as an integral regulator of AML progression by regulating the expression of m6A-modified mRNAs, which might serve as a potential therapeutic target for AML.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-21-4249
  10. Int J Biol Sci. 2023 ;19(2): 449-464
    Stabilization of IGF2BP1 by USP10 promotes breast cancer metastasis via CPT1A in an m6A-dependent manner.
    Jiajun Shi, Qianyi Zhang, Xi Yin, Jiahui Ye, Shengqing Gao, Chen Chen, Yaxuan Yang, Baojuan Wu, Yuping Fu, Hongmei Zhang, Zhangding Wang, Bo Wang, Yun Zhu, Hongyan Wu, Yongzhong Yao, Guifang Xu, Qiang Wang, Shouyu Wang, Weijie Zhang.
      Metastasis leads to the vast majority of breast cancer mortality. Increasing evidence has shown that N6-methyladenosine (m6A) modification and its associated regulators play a pivotal role in breast cancer metastasis. Here, we showed that overexpression of the m6A reader IGF2BP1 was clinically correlated with metastasis in breast cancer patients. Moreover, IGF2BP1 promoted distant metastasis in vitro and in vivo. Mechanistically, we first identified USP10 as the IGF2BP1 deubiquitinase. USP10 can bind to, deubiquitinate, and stabilize IGF2BP1, resulting in its higher expression level in breast cancer. Furthermore, by MeRIP-seq and experimental verification, we found that IGF2BP1 directly recognized and bound to the m6A sites on CPT1A mRNA and enhanced its stability, which ultimately mediated IGF2BP1-induced breast cancer metastasis. In clinical samples, USP10 levels correlated with IGF2BP1 and CPT1A levels, and breast cancer patients with high levels of USP10, IGF2BP1, and CPT1A had the worst outcome. Therefore, these findings suggest that the USP10/IGF2BP1/CPT1A axis facilitates breast cancer metastasis, and this axis may be a promising prognostic biomarker and therapeutic target for breast cancer.
    Keywords:  Breast cancer; CPT1A; IGF2BP1; USP10; m6A; metastasis
    DOI:  https://doi.org/10.7150/ijbs.76798
  11. Life Sci. 2023 Jan 03. pii: S0024-3205(22)01059-1. [Epub ahead of print]315 121359
    RBM10 recruits METTL3 to induce N6-methyladenosine-MALAT1-dependent modification, inhibiting the invasion and migration of NSCLC.
    Yingshu Cao, Xin Di, Shan Cong, Chang Tian, Yan Wang, Xin Jin, Min Zhao, Xijia Zhou, Ranwei Li, Ke Wang.
       AIMS: Previous studies have shown that RNA binding motif 10 (RBM10) is a potential tumor suppressor protein that can inhibit proliferation and promote apoptosis of non-small cell lung cancer (NSCLC). Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays an important role in promoting the development of lung cancer. Inhibiting its m6A methylation can effectively inhibit the invasion and metastasis of lung cancer. There is concern that RBM10 could affect MALAT1 m6A methylation for the invasion and migration of NSCLC.
    MAIN METHODS AND FINDINGS: Transwell and wound healing assays showed that RBM10 significantly inhibited the invasion and migration of NSCLC. CLIP-Seq showed that among all RBM10 binding RNAs, MALAT1 had the highest binding peak among all non-coding RNAs. RNA immunoprecipitation verified the direct combination of RBM10 and MALAT1. The rescue experiment confirmed that RBM10 affected the phosphorylation of the PI3K/AKT/mTOR pathway protein as well as the invasion and migration ability by regulating MALAT1. MeRIP-qPCR confirmed that RBM10 could inhibit the MALAT1 m6A methylation level by recruiting Methyltransferase Like 3 (METTL3).
    SIGNIFICANCE: The study suggests that RBM10, as an RNA-binding protein, may inhibit the m6A methylation of MALAT1 by recruiting METTL3 and affecting phosphorylation of the downstream PI3K/AKT/mTOR pathway by binding and regulating MALAT1, ultimately affecting the invasion and migration of NSCLC.
    Keywords:  AKT; MALAT1; NSCLC; RBM10; m6A
    DOI:  https://doi.org/10.1016/j.lfs.2022.121359
  12. Front Oncol. 2022 ;12 1050288
    Constructing and validating of m6a-related genes prognostic signature for stomach adenocarcinoma and immune infiltration: Potential biomarkers for predicting the overall survival.
    Jing Yang, Zixuan Wu, Xiaoxi Wu, Siya Chen, Xinhua Xia, Jianguo Zeng.
       Background: Stomach adenocarcinoma (STAD) arises from the mutations of stomach cells and has poor overall survival. Chemotherapy is commonly indicated for patients with stomach cancer following surgical resection. The most prevalent alteration that affects cancer growth is N6-methyladenosine methylation (m6A), although the possible function of m6A in STAD prognosis is not recognized.
    Method: The research measured predictive FRGs in BLCA samples from the TCGA and GEO datasets. Data on the stemness indices (mRNAsi), gene mutations, copy number variations (CNV), tumor mutation burden (TMB), and corresponding clinical characteristics were obtained from TCGA and GEO. STAD from TCGA and GEO at 24 m6A was investigated. Lasso regression was used to construct the prediction model to assess the m6A prognostic signals in STAD. In addition, the correlation between m6a and immune infiltration in STAD patients was discussed using GSVA and ssGSEA analysis. Based on these genes, GO and KEGG analyses were performed to identify key biological functions and key pathways.
    Result: A significant relationship was discovered between numerous m6A clusters and the tumor immune microenvironment, as well as three m6A alteration patterns with different clinical outcomes. Furthermore, GSVA and ssGSEA showed that m6A clusters were significantly associated with immune infiltration in the STAD. The low-m6Ascore group had a lower immunotherapeutic response than the high-m6Ascore group. ICIs therapy was more effective in the group with a higher m6Ascore. Three writers (VIRMA, ZC3H13, and METTL3) showed significantly lower expression, whereas five authors (METTL14, METTL16, WTAP, RBM15, and RBM15B) showed considerably higher expression. Three readers (YTHDC2, YTHDF2, and LRPPRC) had higher levels of expression, whereas eleven readers (YTHDC1, YTHDF1, YTHDF3, HNRNPC, FMR1, HNRNPA2B1, IGFBP1, IGFBP2, IGFBP3, and RBMX) had lower levels. As can be observed, the various types of m6 encoders have varied ramifications for STAD control.
    Conclusion: STAD occurrence and progression are linked to m6A-genes. Corresponding prognostic models help forecast the prognosis of STAD patients. m6A-genes and associated immune cell infiltration in the tumor microenvironment (TME) may serve as potential therapeutic targets in STAD, which requires further trials. In addition, the m6a-related gene signature offers a viable alternative to predict bladder cancer, and these m6A-genes show a prospective research area for STAD targeted treatment in the future.
    Keywords:  CNV; M6A; SNP; immunity; predicting model; stomach adenocarcinoma (STAD)
    DOI:  https://doi.org/10.3389/fonc.2022.1050288
  13. J Clin Med. 2022 Dec 23. pii: 113. [Epub ahead of print]12(1):
    METTL3-Mediated lncSNHG7 m6A Modification in the Osteogenic/Odontogenic Differentiation of Human Dental Stem Cells.
    Yeqing Yang, Junkai Zeng, Chong Jiang, Jiawen Chen, Ci Song, Ming Chen, Buling Wu.
      Background: Human dental pulp stem cells (hDPSCs) play an important role in endodontic regeneration. N6-methyladenosine (m6A) is the most common RNA modification, and noncoding RNAs have also been demonstrated to have regulatory roles in the expression of m6A regulatory proteins. However, the study on m6A modification in hDPSCs has not yet been conducted. Methods: Single base site PCR (MazF) was used to detect the m6A modification site of lncSNHG7 before and after mineralization of hDPSCs to screen the target m6A modification protein, and bioinformatics analysis was used to analyze the related pathways rich in lncSNHG7. After knockdown and overexpression of lncSNHG7 and METTL3, the osteogenic/odontogenic ability was detected. After METTL3 knockdown, the m6A modification level and its expression of lncSNHG7 were detected by MazF, and their binding was confirmed. Finally, the effects of lncSNHG7 and METTL3 on the Wnt/β-catenin pathway were detected. Results: MazF experiments revealed that lncSNHG7 had a m6A modification before and after mineralization of hDPSCs, and the occurrence site was 2081. METTL3 was most significantly upregulated after mineralization of hDPSCs. Knockdown/ overexpression of lncSNHG7 and METTL3 inhibited/promoted the osteogenic/odontogenic differentiation of hDPSCs. The m6A modification and expression of lncSNHG7 were both regulated by METTL3. Subsequently, lncSNHG7 and METTL3 were found to regulate the Wnt/β-catenin signaling pathway. Conclusion: These results revealed that METTL3 can activate the Wnt/β-catenin signaling pathway by regulating the m6A modification and expression of lncSNHG7 in hDPSCs to enhance the osteogenic/odontogenic differentiation of hDPSCs. Our study provides new insight into stem cell-based tissue engineering.
    Keywords:  N6-methyladenosine; RNA epigenetics; human dental pulp stem cells; long noncoding RNA; osteogenic/odontogenic differentiation
    DOI:  https://doi.org/10.3390/jcm12010113
  14. J Exp Clin Cancer Res. 2023 Jan 07. 42(1): 10
    The N6-methyladenosine METTL3 regulates tumorigenesis and glycolysis by mediating m6A methylation of the tumor suppressor LATS1 in breast cancer.
    Youqin Xu, Mu Song, Ziyang Hong, Wancheng Chen, Qianbing Zhang, Jianlong Zhou, Chao Yang, Zilong He, Juanjuan Yu, Xiaolin Peng, Qiuhong Zhu, Shaotian Li, Kaiyuan Ji, Minfeng Liu, Qiang Zuo.
       BACKGROUND: Posttranscriptional modification of tumor-associated factors plays a pivotal role in breast cancer progression. However, the underlying mechanism remains unknown. M6A modifications in cancer cells are dynamic and reversible and have been found to impact tumor initiation and progression through various mechanisms. In this study, we explored the regulatory mechanism of breast cancer cell proliferation and metabolism through m6A methylation in the Hippo pathway.  METHODS: A combination of MeRIP-seq, RNA-seq and metabolomics-seq was utilized to reveal a map of m6A modifications in breast cancer tissues and cells. We conducted RNA pull-down assays, RIP-qPCR, MeRIP-qPCR, and RNA stability analysis to identify the relationship between m6A proteins and LATS1 in m6A regulation in breast cancer cells. The expression and biological functions of m6A proteins were confirmed in breast cancer cells in vitro and in vivo. Furthermore, we investigated the phosphorylation levels and localization of YAP/TAZ to reveal that the activity of the Hippo pathway was affected by m6A regulation of LATS1 in breast cancer cells.  RESULTS: We demonstrated that m6A regulation plays an important role in proliferation and glycolytic metabolism in breast cancer through the Hippo pathway factor, LATS1. METTL3 was identified as the m6A writer, with YTHDF2 as the reader protein of LATS1 mRNA, which plays a positive role in promoting both tumorigenesis and glycolysis in breast cancer. High levels of m6A modification were induced by METTL3 in LATS1 mRNA. YTHDF2 identified m6A sites in LATS1 mRNA and reduced its stability. Knockout of the protein expression of METTL3 or YTHDF2 increased the expression of LATS1 mRNA and suppressed breast cancer tumorigenesis by activating YAP/TAZ in the Hippo pathway.
    CONCLUSIONS: In summary, we discovered that the METTL3-LATS1-YTHDF2 pathway plays an important role in the progression of breast cancer by activating YAP/TAZ in the Hippo pathway.
    Keywords:  Breast cancer; Hippo-YAP/TAZ signaling pathway; LATS1; METTL3
    DOI:  https://doi.org/10.1186/s13046-022-02581-1
  15. Mol Cancer. 2023 Jan 10. 22(1): 5
    Crosstalk between 5-methylcytosine and N6-methyladenosine machinery defines disease progression, therapeutic response and pharmacogenomic landscape in hepatocellular carcinoma.
    Yu Tian, Haijuan Xiao, Yanhui Yang, Pingping Zhang, Jiahui Yuan, Wei Zhang, Lijie Chen, Yibao Fan, Jinze Zhang, Huan Cheng, Tingwei Deng, Lin Yang, Weiwei Wang, Guoyong Chen, Peiqin Wang, Peng Gong, Xing Niu, Xianbin Zhang.
       BACKGROUND: Accumulated evidence highlights the significance of the crosstalk between epigenetic and epitranscriptomic mechanisms, notably 5-methylcytosine (5mC) and N6-methyladenosine (m6A). Herein, we conducted a widespread analysis regarding the crosstalk between 5mC and m6A regulators in hepatocellular carcinoma (HCC).
    METHODS: Pan-cancer genomic analysis of the crosstalk between 5mC and m6A regulators was presented at transcriptomic, genomic, epigenetic, and other multi-omics levels. Hub 5mC and m6A regulators were summarized to define an epigenetic and epitranscriptomic module eigengene (EME), which reflected both the pre- and post-transcriptional modifications.
    RESULTS: 5mC and m6A regulators interacted with one another at the multi-omic levels across pan-cancer, including HCC. The EME scoring system enabled to greatly optimize risk stratification and accurately predict HCC patients' clinical outcomes and progression. Additionally, the EME accurately predicted the responses to mainstream therapies (TACE and sorafenib) and immunotherapy as well as hyper-progression. In vitro, 5mC and m6A regulators cooperatively weakened apoptosis and facilitated proliferation, DNA damage repair, G2/M arrest, migration, invasion and epithelial-to-mesenchymal transition (EMT) in HCC cells. The EME scoring system was remarkably linked to potential extrinsic and intrinsic immune escape mechanisms, and the high EME might contribute to a reduced copy number gain/loss frequency. Finally, we determined potential therapeutic compounds and druggable targets (TUBB1 and P2RY4) for HCC patients with high EME.
    CONCLUSIONS: Our findings suggest that HCC may result from a unique synergistic combination of 5mC-epigenetic mechanism mixed with m6A-epitranscriptomic mechanism, and their crosstalk defines therapeutic response and pharmacogenomic landscape.
    Keywords:  5-methylcytosine; Hepatocellular carcinoma; Multi-omics; N6-methyladenosine; Pharmacogenomic landscape; Therapeutic response
    DOI:  https://doi.org/10.1186/s12943-022-01706-6
  16. Cell Stem Cell. 2023 Jan 05. pii: S1934-5909(22)00490-8. [Epub ahead of print]30(1): 52-68.e13
    METTL16 drives leukemogenesis and leukemia stem cell self-renewal by reprogramming BCAA metabolism.
    Li Han, Lei Dong, Keith Leung, Zhicong Zhao, Yangchan Li, Lei Gao, Zhenhua Chen, Jianhuang Xue, Ying Qing, Wei Li, Sheela Pangeni Pokharel, Min Gao, Meiling Chen, Chao Shen, Brandon Tan, Andrew Small, Kitty Wang, Zheng Zhang, Xi Qin, Lu Yang, Mark Wunderlich, Bin Zhang, James C Mulloy, Guido Marcucci, Chun-Wei Chen, Minjie Wei, Rui Su, Jianjun Chen, Xiaolan Deng.
      N6-methyladenosine (m6A), the most prevalent internal modification in mammalian mRNAs, is involved in many pathological processes. METTL16 is a recently identified m6A methyltransferase. However, its role in leukemia has yet to be investigated. Here, we show that METTL16 is a highly essential gene for the survival of acute myeloid leukemia (AML) cells via CRISPR-Cas9 screening and experimental validation. METTL16 is aberrantly overexpressed in human AML cells, especially in leukemia stem cells (LSCs) and leukemia-initiating cells (LICs). Genetic depletion of METTL16 dramatically suppresses AML initiation/development and maintenance and significantly attenuates LSC/LIC self-renewal, while moderately influencing normal hematopoiesis in mice. Mechanistically, METTL16 exerts its oncogenic role by promoting expression of branched-chain amino acid (BCAA) transaminase 1 (BCAT1) and BCAT2 in an m6A-dependent manner and reprogramming BCAA metabolism in AML. Collectively, our results characterize the METTL16/m6A/BCAT1-2/BCAA axis in leukemogenesis and highlight the essential role of METTL16-mediated m6A epitranscriptome and BCAA metabolism reprograming in leukemogenesis and LSC/LIC maintenance.
    Keywords:  AML; BCAA metabolism; BCAT1; BCAT2; LSCs/LICs; METTL16; leukemia stem cells; leukemia-initiating cells; m6A modification; self-renewal
    DOI:  https://doi.org/10.1016/j.stem.2022.12.006
  17. J Clin Med. 2022 Dec 25. pii: 155. [Epub ahead of print]12(1):
    Prognostic Values of METTL3 and Its Roles in Tumor Immune Microenvironment in Pan-Cancer.
    Yang Guo, Yu Heng, Hui Chen, Qiang Huang, Chunping Wu, Lei Tao, Liang Zhou.
      Background: N6-methyladenosine (m6A) is among the most prevalent RNA modifications regulating RNA metabolism. The roles of methyltransferase-like 3 (METTL3), a core catalytic subunit, in various cancers remain unclear. Methods: The expression levels of METTL3 in pan-cancer were profiled and their prognostic values were examined. We assessed the relationships between METTL3 expression levels and tumor immune infiltration levels, immune checkpoint gene expression, immune neoantigens, tumor mutation burden, microsatellite instability, and DNA mismatch repair gene expression. Furthermore, a protein-protein interaction network was drawn, and gene set enrichment analysis was conducted to explore the functions of METTL3. Results: METTL3 expression levels were elevated in most cancers, with high expression associated with poorer overall and disease-free survival. METTL3 levels were significantly related to immune cell infiltration, tumor mutation burden, microsatellite instability, mismatch repair genes, and immune checkpoint gene levels. METTL3 was enriched in pathways related to RNA modification and metabolism and correlated with epithelial-mesenchymal transition. Conclusions: METTL3 serves as an oncogene in most cancer types and shows potential as a prognostic biomarker. Additionally, our comprehensive pan-cancer analysis suggested that METTL3 is involved in regulating the tumor immune microenvironments and epithelial-mesenchymal transition via modulating RNA modification and metabolism, making it a potential therapeutic target.
    Keywords:  METTL3; biomarker; pan-cancer; prognosis; tumor microenvironment
    DOI:  https://doi.org/10.3390/jcm12010155
  18. Int J Biol Sci. 2023 ;19(2): 691-704
    Upregulation of LRRC8A by m5C modification-mediated mRNA stability suppresses apoptosis and facilitates tumorigenesis in cervical cancer.
    Yanjie Chen, Xinzhao Zuo, Qinglv Wei, Jie Xu, Xiaoyi Liu, Shiling Liu, Haocheng Wang, Qingya Luo, Yuya Wang, Yu Yang, Hongyan Zhao, Jing Xu, Tao Liu, Ping Yi.
      Cervical cancer (CC) is one of the most common gynecological malignancies with poor prognosis for advanced CC patients. LRRC8A is a volume-regulated anion channel protein involved in cellular homeostasis, but its role in CC remains largely unknown. In this study, we found that LRRC8A is elevated in CC and associated with poor prognosis. LRRC8A maintains cell survivals under the hypotonic condition, and promotes tumorigenesis through apoptosis suppression in vitro and in vivo. Notably, LRRC8A is upregulated by NSUN2-mediated m5C modification. m5C modified-LRRC8A mRNA is bound by the RNA binding protein YBX1 followed by the increased RNA stability. Moreover, loss of NSUN2 suppresses the proliferation and metastasis of CC cells, and NSUN2 expression is positively correlated with LRRC8A expression in CC. Altogether, our study demonstrates that the NSUN2-m5C-LRRC8A axis is crucial and would be a potential therapeutic target for CC.
    DOI:  https://doi.org/10.7150/ijbs.79205
  19. Microbiol Spectr. 2023 Jan 10. e0394322
    Epitranscriptomic N6-Methyladenosine Profile of SARS-CoV-2-Infected Human Lung Epithelial Cells.
    Stacia Phillips, Tarun Mishra, Shaubhagya Khadka, Dana Bohan, Constanza E Espada, Wendy Maury, Li Wu.
      N6-methyladenosine (m6A) is a dynamic posttranscriptional RNA modification that plays an important role in determining transcript fate. The functional consequence of m6A deposition is dictated by a group of host proteins that specifically recognize and bind the m6A modification, leading to changes in RNA stability, transport, splicing, or translation. The cellular m6A methylome undergoes changes during certain pathogenic conditions such as viral infections. However, how m6A modification of host cell transcripts and noncoding RNAs change during severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection has not been reported. Here, we define the epitranscriptomic m6A profile of SARS-CoV-2-infected human lung epithelial cells compared to uninfected controls. We identified mRNA and long and small noncoding RNA species that are differentially m6A modified in response to SARS-CoV-2 infection. The most significantly differentially methylated transcript was the precursor of microRNA-4486 (miRNA-4486), which showed significant increases in abundance and percentage of methylated transcripts in infected cells. Pathway analyses revealed that differentially methylated transcripts were significantly associated with several cancer-related pathways, protein processing in the endoplasmic reticulum, cell death, and proliferation. Upstream regulators predicted to be associated with the proteins encoded by differentially methylated mRNAs include several proteins involved in the type-I interferon response, inflammation, and cytokine signaling. IMPORTANCE Posttranscriptional modification of viral and cellular RNA by N6-methyladenosine (m6A) plays an important role in regulating the replication of many viruses and the cellular immune response to infection. We therefore sought to define the epitranscriptomic m6A profile of human lung epithelial cells infected with SARS-CoV-2. Our analyses demonstrate the differential methylation of both coding and noncoding cellular RNAs in SARS-CoV-2-infected cells compared to uninfected controls. Pathway analyses revealed that several of these RNAs may be involved in the cellular response to infection, such as type-I interferon. Our study implicates m6A modification of infected-cell RNA as a mechanism of posttranscriptional gene regulation during SARS-CoV-2 infection.
    Keywords:  N6-methyladenosine; SARS-CoV-2; epitranscriptomics; infection; lung epithelial cells; microarray
    DOI:  https://doi.org/10.1128/spectrum.03943-22
  20. J Exp Clin Cancer Res. 2023 Jan 13. 42(1): 19
    STRIP2 motivates non-small cell lung cancer progression by modulating the TMBIM6 stability through IGF2BP3 dependent.
    Xilin Zhang, Qiuqiang Chen, Ying He, Qian Shi, Chengyi Yin, Yanping Xie, Huanming Yu, Ying Bao, Xiang Wang, Chengwu Tang, Zhaohui Dong.
       BACKGROUND: Striatin interacting protein 2 (STRIP2) is a core component of the striatin-interacting phosphatase and kinase (STRIPAK) complexes, which is involved in tumor initiation and progression via the regulation of cell contractile and metastasis. However, the underlying molecular mechanisms of STRIP2 in non-small cell lung cancer (NSCLC) progression remain largely unknown.
    METHODS: The expressions of STRIP2 and IGF2BP3 in human NSCLC specimens and NSCLC cell lines were detected using quantitative RT-PCR, western blotting, and immunohistochemistry (IHC) analyses. The roles and molecular mechanisms of STRIP2 in promoting NSCLC progression were investigated in vitro and in vivo.
    RESULTS: Here, we found that STRIP2 expression was significantly elevated in NSCLC tissues and high STRIP2 expression was associated with a poor prognosis. Knockdown of STRIP2 suppressed tumor growth and metastasis in vitro and in vivo, while STRIP2 overexpression obtained the opposite effect. Mechanistically, P300/CBP-mediated H3K27 acetylation activation in the promoter of STRIP2 induced STRIP2 transcription, which interacted with insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) and upregulated IGF2BP3 transcription. In addition, STRIP2-IGF2BP3 axis stimulated m6A modification of TMBIM6 mRNA and enhanced TMBIM6 stability. Consequently, TMBIM6 involved NSCLC cell proliferation, migration and invasion dependent on STRIP2 and IGF2BP3. In NSCLC patients, high co-expression of STRIP2, IGF2BP3 and TMBIM6 was associated with poor outcomes.
    CONCLUSIONS: Our findings indicate that STRIP2 interacts with IGF2BP3 to regulate TMBIM6 mRNA stability in an m6A-dependent manner and may represent a potential prognostic biomarker and therapeutic target for NSCLC.
    Keywords:  Insulin-like growth factor II mRNA binding protein 3; Metastasis; Non-small cell lung cancer; Striatin interacting protein 2; Transmembrane Bax inhibitor motif containing-6
    DOI:  https://doi.org/10.1186/s13046-022-02573-1
  21. Mol Cell. 2023 Jan 04. pii: S1097-2765(22)01176-5. [Epub ahead of print]
    RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia.
    Michael McMillan, Nicolas Gomez, Caroline Hsieh, Michael Bekier, Xingli Li, Roberto Miguez, Elizabeth M H Tank, Sami J Barmada.
      RNA methylation at adenosine N6 (m6A) is one of the most common RNA modifications, impacting RNA stability, transport, and translation. Previous studies uncovered RNA destabilization in amyotrophic lateral sclerosis (ALS) models in association with accumulation of the RNA-binding protein TDP43. Here, we show that TDP43 recognizes m6A RNA and that RNA methylation is critical for both TDP43 binding and autoregulation. We also observed extensive RNA hypermethylation in ALS spinal cord, corresponding to methylated TDP43 substrates. Emphasizing the importance of m6A for TDP43 binding and function, we identified several m6A factors that enhance or suppress TDP43-mediated toxicity via single-cell CRISPR-Cas9 in primary neurons. The most promising modifier-the canonical m6A reader YTHDF2-accumulated within ALS spinal neurons, and its knockdown prolonged the survival of human neurons carrying ALS-associated mutations. Collectively, these data show that m6A modifications modulate RNA binding by TDP43 and that m6A is pivotal for TDP43-related neurodegeneration in ALS.
    Keywords:  ALS; RNA modifications; TDP43; m6A; methylation; neurodegeneration
    DOI:  https://doi.org/10.1016/j.molcel.2022.12.019
  22. Immunol Cell Biol. 2023 Jan 11.
    The regulation of N6-methyladenosine modification in PD-L1-induced anti-tumor immunity.
    Wei Yu, Jinti Lin, Tao Yu, Jianan Lou, Chao Qian, Ankai Xu, Bing Liu, Huimin Tao, Libin Jin.
      There is growing evidence that programmed death ligand-1 (PD-L1) has exciting therapeutic efficacy in hematological malignancy and partial solid tumors. However, many patients still face failure with the treatment of immune checkpoint blockade because of PD-L1 expression regulation during transcription and post-transcription processes, including N6-methyladenosine (m6A). Similar to the epigenetic regulation in DNA and histones, recent research has revealed the essential regulation of m6A modification in RNA nuclear export, metabolism and translation. Recent studies have shown that m6A-induced PD-L1 expression emerges as one of the main reasons for the immunological alteration in this process and contributes to the failure of T cell-induced anti-tumor immunity. The results of preclinical studies demonstrate the potential of m6A-targeted therapy in combination with immune checkpoint blockade. Meanwhile, the comprehensive expression of m6A-related genes has provided the possibility to indicate the prognosis and optimize the treatment for patients of various cancer types. In this review, we focus on the m6A modification in PD-L1 mRNA as well as the regulation of PD-L1 expression in cancer cells and summarize its clinical value in anti-PD-L1 cancer immune therapy.
    Keywords:  N6-methyladenosine; PD-L1; RNA metabolism; immune therapy; post-transcription modification
    DOI:  https://doi.org/10.1111/imcb.12620
  23. mBio. 2023 Jan 10. e0334922
    N6-Methyladenosine and Reader Protein YTHDF2 Enhance the Innate Immune Response by Mediating DUSP1 mRNA Degradation and Activating Mitogen-Activated Protein Kinases during Bacterial and Viral Infections.
    Jian Feng, Wen Meng, Luping Chen, Xinquan Zhang, Ashley Markazi, Weiming Yuan, Yufei Huang, Shou-Jiang Gao.
      Mitogen-activated protein kinases (MAPKs) play critical roles in the induction of numerous cytokines, chemokines, and inflammatory mediators that mobilize the immune system to counter pathogenic infections. Dual-specificity phosphatase 1 (DUSP1) is a member of the dual-specificity phosphatases that inactivates MAPKs through a negative-feedback mechanism. Here, we report that in response to viral and bacterial infections, not only the DUSP1 transcript but also its N6-methyladenosine (m6A) levels rapidly increase together with that of the m6A reader protein YTHDF2, resulting in enhanced YTHDF2-mediated DUSP1 transcript degradation. The knockdown of DUSP1 promotes p38 and Jun N-terminal kinase (JNK) phosphorylation and activation, thus increasing the expression of innate immune response genes, including the interleukin-1β (IL-1β), colony-stimulating factor 3 (CSF3), transglutaminase 2 (TGM2), and proto-oncogene tyrosine-protein kinase Src (SRC) genes. Similarly, the knockdown of the m6A eraser ALKBH5 increases the DUSP1 transcript m6A level, resulting in accelerated transcript degradation, the activation of p38 and JNK, and the enhanced expression of IL-1β, CSF3, TGM2, and SRC. These results demonstrate that m6A and the reader protein YTHDF2 orchestrate optimal innate immune responses during viral and bacterial infections by downregulating the expression of a negative regulator, DUSP1, of the p38 and JNK pathways that are central to innate immune responses against pathogenic infections. IMPORTANCE Innate immunity is central to controlling pathogenic infections and maintaining the homeostasis of the host. In this study, we have revealed a novel mechanism regulating innate immune responses during viral and bacterial infections. We have found that N6-methyladenosine (m6A) and the reader protein YTHDF2 regulate dual-specificity phosphatase 1, a negative regulator of the mitogen-activated protein kinases p38 and JNK, to maximize innate immune responses during viral and bacterial infections. These results provide novel insights into the mechanism regulating innate immunity, which could help in the development of novel approaches for controlling pathogenic infections.
    Keywords:  DUSP1; JNK; MAPKs; N6-methyladenosine; YTHDF2; dual-specificity phosphatase 1; innate immunity; m6A; mitogen-activated protein kinases; p38; p38 kinases
    DOI:  https://doi.org/10.1128/mbio.03349-22
  24. Int J Mol Sci. 2023 Jan 01. pii: 773. [Epub ahead of print]24(1):
    Emerging Mutual Regulatory Roles between m6A Modification and microRNAs.
    Zongqin Mei, Yahao Mou, Nan Zhang, Xiaoyu Liu, Zuoshun He, Shiyan Gu.
      N6-metyladenosine (m6A), one of the most common RNA methylation modifications in mammals, has attracted extensive attentions owing to its regulatory roles in a variety of physiological and pathological processes. As a reversible epigenetic modification on RNAs, m6A is dynamically mediated by the functional interplay among the regulatory proteins of methyltransferases, demethylases and methyl-binding proteins. In recent years, it has become increasingly clear that m6A modification is associated with the production and function of microRNAs (miRNAs). In this review, we summarize the specific kinds of m6A modification methyltransferases, demethylases and methyl-binding proteins. In particular, we focus on describing the roles of m6A modification and its regulatory proteins in the production and function of miRNAs in a variety of pathological and physiological processes. More importantly, we further discuss the mediating mechanisms of miRNAs in m6A modification and its regulatory proteins during the occurrence and development of various diseases.
    Keywords:  N6-metyladenosine; demethylases; methyl-binding proteins; methyltransferases; miRNAs
    DOI:  https://doi.org/10.3390/ijms24010773
  25. Hepatol Commun. 2023 Jan 01. 7(1): e0004
    Integrative analysis implicates the significance of m6A in the liver fibrosis of biliary atresia by regulating THY1.
    Junfeng Wang, Min Du, Lingdu Meng, Yifan Yang, Shiwei He, Ye Zhu, Xue Ren, Meng Wei, Rui Dong, Shan Zheng, Gong Chen.
      Whether N6-methyladenosine (m6A) is involved in biliary atresia (BA) remains undefined. Herein, we comprehensively evaluated the m6A profile in BA. When compared with normal controls, BA had an elevated m6A level with upregulated m6A writers. The m6A level was correlated with liver function, stage of fibrosis and jaundice clearance in BA. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) demonstrated an altered m6A topology in BA. MeRIP-seq and RNA sequencing filtered out 130 m6A-modified genes, which were enriched in fibrogenetic pathways. MeRIP-qPCR in vivo and interventions of LX-2 and primary HSCs in vitro validated the regulatory role of m6A on COL1A1 and THY1. THY1+ myofibroblasts expanded in portal area of BA, and highly expressed profibrogenic genes (COL1A1, MMP2, PDGFRA, and DCN). THY1 was correlated with liver fibrosis and jaundice clearance in BA. Bulk array (GSE46960, GSE15235), single-cell RNA sequencing (GSE136103), primary HSC interventions, and co-immunoprecipitation revealed that THY1 was correlated with extracellular matrix organization, promoted HSC activation, showed higher interactions with integrins on myeloid cells in cholestatic fibrosis, and was correlated with native liver survival in BA. Our study highlights the significance of m6A in BA-induced liver fibrogenesis by regulating THY1, shedding new light on the novel therapies to alleviate liver fibrosis by targeting m6A/THY1 axis in BA.
    DOI:  https://doi.org/10.1097/HC9.0000000000000004
  26. Bioorg Med Chem Lett. 2023 Jan 09. pii: S0960-894X(23)00004-5. [Epub ahead of print] 129126
    Synthesis of 2'-modified N6-methyladenosine phosphoramidites and their incorporation into siRNA.
    Anna M Rydzik, Doris Riether, Dirk Gottschling.
      A synthesis of 2'-fluoro and 2'-methoxy N6-methyladenosine phosphoramidites and their successful incorporation into oligonucleotides is reported. 2'-fluoro and 2́-methoxy modifications of sugars in siRNAs are known to aid stability and N6-methylation modifies the potency of therapeutic silencing RNAs (siRNA). We demonstrate that a combination of those modifications incorporated into the antisense strand of siRNA leads to efficient knockdown of a target gene in cells. This work broadens the available pool of chemical modifications of therapeutic siRNAs and provides tools for their efficient synthesis.
    Keywords:  6-methyladenosine; RNA therapeutics; epigenetics; m6A; siRNA
    DOI:  https://doi.org/10.1016/j.bmcl.2023.129126
  27. Cell Death Dis. 2023 Jan 06. 14(1): 7
    A potassium-chloride co-transporter promotes tumor progression and castration resistance of prostate cancer through m6A reader YTHDC1.
    Shuai Yuan, Shao-Hua He, Lu-Yao Li, Shu Xi, Hong Weng, Jin-Hui Zhang, Dan-Qi Wang, Meng-Meng Guo, Haozhe Zhang, Shuang-Ying Wang, Dao-Jing Ming, Meng-Yang Liu, Hailiang Hu, Xian-Tao Zeng.
      SLC12A5, a neuron-specific potassium-chloride co-transporter, has been reported to promote tumor progression, however, the underlying mechanism remains unclear. Here we report that SLC12A5 functions as an oncogene to promote tumor progression and castration resistance of prostate cancer through the N6-methyladenosine (m6A) reader YTHDC1 and the transcription factor HOXB13. We have shown that the level of SLC12A5 was increased in prostate cancer, in comparison to its normal counterparts, and further elevated in castration-resistant prostate cancer (CRPC). The enhanced expression of SLC12A5 mRNA was associated with neuroendocrine prostate cancer (NEPC) progression and poor survival in prostate cancer. Furthermore, we demonstrated that SLC12A5 promoted the castration resistance development of prostate cancer in addition to the cell proliferation and migration. Interestingly, SLC12A5 was detected in the cell nucleus and formed a complex with nuclear m6A reader YTHDC1, which in turn upregulated HOXB13 to promote the prostate cancer progression. Therefore, our findings reveal a mechanism that how the potassium-chloride cotransporter SLC12A5 promotes the tumor progression and provide a therapeutic opportunity for prostate cancer to apply the neurological disorder drug SLC12A5 inhibitors.
    DOI:  https://doi.org/10.1038/s41419-022-05544-8
  28. Stem Cell Rev Rep. 2023 Jan 07.
    Bone Marrow Mesenchymal Stem Cells Reversed Ovarian Aging-related m6A RNA Methylation Modification Profile in Aged Granulosa Cells.
    Chuan Tian, Yuanyuan An, Jing Zhao, Xiangqing Zhu, Wei Wei, Guangping Ruan, Ye Li, Xinghua Pan.
       BACKGROUND: Ovarian ageing causes endocrine disturbances and the degeneration of systemic tissue and organ functions to seriously affect women's physical and mental health, and effective treatment methods are urgently needed. Based on our previous studies using juvenile rhesus monkey bone marrow mesenchymal stem cells (BMMSCs) to treat ovarian ageing in rhesus monkey, we found that BMMSCs improved ovarian structure and function. This study continues to explore the mechanism by which BMMSCs reversed granulosa cell (GC) ageing.
    METHODS: A GC ageing model and coculture system of BMMSCs were established, changes in the level of the N6-methyladenosine (m6A) methylation modification were detected, m6A-modified RNA immunoprecipitation sequencing (MeRIP-seq) were performed, correlations between m6A peaks and mRNA expression were determined, and the expression of hub genes was identified using Q-PCR, immunofluorescence staining, and western blot.
    RESULTS: Our results showed that H2O2 successfully induced GC ageing and that BMMSCs reversed measures of GC ageing. BMMSCs increased the expression of the FTO protein and reduced the overall level of m6A. We identified 797 m6A peaks (348 hypomethylated and 449 hypermethylated peaks) and 817 differentially expressed genes (DEGs) (412 upregulated and 405 downregulated) after aged GCs were cocultured with BMMSCs, which significantly associated with ovarian function and epigenetic modification. The epigenetic repressive mark and important cell cycle regulator lysine demethylase 8 (KDM8) was downregulated at both the mRNA and protein levels, histone H3 was upregulated in aged GCs after BMMSC coculture, and KDM8 was upregulated after FTO was inhibited through FB23.
    CONCLUSIONS: Our study revealed an essential role for m6A in BMMSCs in reversing GC ageing, and FTO regulated KDM8 mediates histone H3 changes may as a novel regulatory mechanism in BMMSCs to reverse GC ageing.
    Keywords:  BMMSCs; Lysine demethylase 8 (KDM8); Ovarian ageing; m6A
    DOI:  https://doi.org/10.1007/s12015-022-10485-y
  29. J Exp Clin Cancer Res. 2023 Jan 06. 42(1): 9
    Helicobacter pylori-induced NAT10 stabilizes MDM2 mRNA via RNA acetylation to facilitate gastric cancer progression.
    Min Deng, Long Zhang, Wenying Zheng, Jiale Chen, Nan Du, Meiqi Li, Weiqing Chen, Yonghong Huang, Ning Zeng, Yuanbin Song, Yongming Chen.
       BACKGROUND: N4-acetylcytidine (ac4C), a widespread modification in human mRNAs that is catalyzed by the N-acetyltransferase 10 (NAT10) enzyme, plays an important role in promoting mRNA stability and translation. However, the biological functions and regulatory mechanisms of NAT10-mediated ac4C were poorly defined.
    METHODS: ac4C mRNA modification status and NAT10 expression levels were analyzed in gastric cancer (GC) samples and compared with the corresponding normal tissues. The biological role of NAT10-mediated ac4C and its upstream and downstream regulatory mechanisms were determined in vitro and in vivo. The therapeutic potential of targeting NAT10 in GC was further explored.
    RESULTS: Here, we demonstrated that both ac4C mRNA modification and its acetyltransferase NAT10 were increased in GC, and increased NAT10 expression was associated with disease progression and poor patient prognosis. Functionally, we found that NAT10 promoted cellular G2/M phase progression, proliferation and tumorigenicity of GC in an ac4C-depedent manner. Mechanistic analyses demonstrated that NAT10 mediated ac4C acetylation of MDM2 transcript and subsequently stabilized MDM2 mRNA, leading to its upregulation and p53 downregulation and thereby facilitating gastric carcinogenesis. In addition, Helicobacter pylori (Hp) infection contributed to NAT10 induction, causing MDM2 overexpression and subsequent p53 degradation. Further investigations revealed that targeting NAT10 with Remodelin showed anti-cancer activity in GC and augmented the anti-tumor activity of MDM2 inhibitors in p53 wild-type GC.
    CONCLUSIONS: These results suggest the critical role of NAT10-mediated ac4C modification in GC oncogenesis and reveal a previously unrecognized signaling cascade involving the Hp-NAT10-MDM2-p53 axis during GC development.
    Keywords:  Gastric cancer; Helicobacter pylori; MDM2; N-acetyltransferase 10; N4-acetylcytidine; p53
    DOI:  https://doi.org/10.1186/s13046-022-02586-w
  30. Theranostics. 2023 ;13(2): 596-610
    Enhancer RNA promotes resistance to radiotherapy in bone-metastatic prostate cancer by m6A modification.
    Yu Zhao, Simeng Wen, Hang Li, Chun-Wu Pan, Yulei Wei, Ting Huang, Zhaochen Li, Yinhui Yang, Saijun Fan, Yingyi Zhang.
      Rationale: Prostate cancer metastasizes to the bone with the highest frequency and exhibits high resistance to 177Lu-prostate-specific membrane antigen (PSMA) radioligand therapy. Little is known about bone metastatic prostate cancer (mPCa) resistance to radiation. Methods: We filtered the metastatic eRNA using RNA-seq, MeRIP-seq, RT-qPCR and bioinformation. Western blot, RT-qPCR, CLIP, co-IP and RNA pull-down assays were used for RNA/protein interaction, RNA or protein expression examination. MTS assay was used to determine cell viability in vitro, xenograft assay was used to examine the tumor growth in mice. Results: In this study, we screened and identified bone-specific N6 adenosine methylation (m6A) on enhancer RNA (eRNA) that played a post-transcriptional functional role in bone mPCa and was correlated with radiotherapy (RT) resistance. Further data demonstrated that RNA-binding protein KHSRP recognized both m6A at eRNA and m6Am at 5'-UTR of mRNA to block RNA degradation from exoribonuclease XRN2. Depletion of the MLXIPe/KHSRP/PSMD9 regulatory complex inhibited tumor growth and RT sensitization of bone mPCa xenograft in vitro and in vivo. Conclusions: Our findings indicate that a bone-specific m6A-modified eRNA plays a vital role in regulating mPCa progression and RT resistance and might be a novel specific predictor for cancer RT.
    Keywords:  Bone metastatic prostate cancer; Enhancer RNA; Radiotherapy.; m6A; m6Am
    DOI:  https://doi.org/10.7150/thno.78687
  31. Angew Chem Int Ed Engl. 2023 Jan 11.
    A New Bacterial Adenosine-Derived Nucleoside as an Example of RNA Modification Damage.
    Larissa Bessler, Lea-Marie Vogt, Marc Lander, Christina Dal Magro, Patrick Keller, Jonas Kühlborn, Christopher J Kampf, Till Opatz, Mark Helm.
      The fields of RNA modification and RNA damage both exhibit a plethora of non-canonical nucleoside structures. While RNA modifications have evolved to improve RNA function, the term RNA damage implies detrimental effects. Based on stable isotope labelling and mass spectrometry, we report the identification and characterisation of 2-methylthio-1,N6-ethenoadenosine (ms2εA), which is related to 1,N6-ethenoadenine, a lesion resulting from exposure of nucleic acids to alkylating chemicals in vivo. In contrast, a sophisticated isoprene labelling scheme revealed that ms2eA biogenesis involves cleavage of a prenyl moiety in the known transfer RNA (tRNA) modification 2-methylthio-N6-isopentenyladenosine (ms2i6A). The relative abundance of ms2εA in tRNAs from translating ribosomes suggests reduced function in comparison to its parent RNA modification, establishing the nature of the new structure in a newly perceived overlap of the two previously separate fields, namely an RNA modification damage.
    Keywords:  epitranscriptome * isotopic labeling * mass spectrometry * nucleoside analysis * RNA modification damage
    DOI:  https://doi.org/10.1002/anie.202217128
  32. Comput Struct Biotechnol J. 2023 ;21 401-417
    Quantification of substoichiometric modification reveals global tsRNA hypomodification, preferences for angiogenin-mediated tRNA cleavage, and idiosyncratic epitranscriptomes of human neuronal cell-lines.
    Florian Pichot, Marion C Hogg, Virginie Marchand, Valérie Bourguignon, Elisabeth Jirström, Cliona Farrell, Hesham A Gibriel, Jochen H M Prehn, Yuri Motorin, Mark Helm.
      Modification of tRNA is an integral part of the epitranscriptome with a particularly pronounced potential to generate diversity in RNA expression. Eukaryotic tRNA contains modifications in up to 20% of their nucleotides, but not all sites are always fully modified. Combinations and permutations of partially modified sites in tRNAs can generate a plethora of tRNA isoforms, termed modivariants. Here, we investigate the stoichiometry of incompletely modified sites in tRNAs from human cell lines for their information content. Using a panel of RNA modification mapping methods, we assess the stoichiometry of sites that contain the modifications 5-methylcytidine (m5C), 2'-O-ribose methylation (Nm), 3-methylcytidine (m3C), 7-methylguanosine (m7G), and Dihydrouridine (D). We discovered that up to 75% of sites can be incompletely modified and that the differential modification status of a cellular tRNA population holds information that allows to discriminate e.g. different cell lines. As a further aspect, we investigated potential causal connectivity between tRNA modification and its processing into tRNA fragments (tiRNAs and tRFs). Upon exposure of cultured living cells to cell-penetrating angiogenin, the modification patterns of the corresponding RNA populations was changed. Importantly, we also found that tsRNAs were significantly less modified than their parent tRNAs at numerous sites, suggesting that tsRNAs might derive chiefly from hypomodified tRNAs.
    Keywords:  Angiogenin; Modification; Modification mapping; RNAseq; TRNA; TRNA fragments
    DOI:  https://doi.org/10.1016/j.csbj.2022.12.020
  33. Int J Biol Sci. 2023 ;19(2): 593-609
    Inhibition of IGF2BP1 attenuates renal injury and inflammation by alleviating m6A modifications and E2F1/MIF pathway.
    Yan Mao, Feng Jiang, Xue-Jiao Xu, Lan-Bo Zhou, Rui Jin, Li-Li Zhuang, Chen-Xia Juan, Guo-Ping Zhou.
      Septic acute kidney injury (AKI) is characterized by inflammation. Pyroptosis often occurs during AKI and is associated with the development of septic AKI. This study found that induction of insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) to a higher level can induce pyroptosis in renal tubular cells. Meanwhile, macrophage migration inhibitory factor (MIF), a subunit of NLRP3 inflammasomes, was essential for IGF2BP1-induced pyroptosis. A putative m6A recognition site was identified at the 3'-UTR region of E2F transcription factor 1 (E2F1) mRNA via bioinformatics analyses and validated using mutation and luciferase experiments. Further actinomycin D (Act D) chase experiments showed that IGF2BP1 stabilized E2F1 mRNA dependent on m6A. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) indicated that E2F1 acted as a transcription factor to promote MIF expression. Thus, IGF2BP1 upregulated MIF through directly upregulating E2F1 expression via m6A modification. Experiments on mice with cecum ligation puncture (CLP) surgery verified the relationships between IGF2BP1, E2F1, and MIF and demonstrated the significance of IGF2BP1 in MIF-associated pyroptosis in vivo. In conclusion, IGF2BP1 was a potent pyroptosis inducer in septic AKI through targeting the MIF component of NLRP3 inflammasomes. Inhibiting IGF2BP1 could be an alternate pyroptosis-based treatment for septic AKI.
    Keywords:  IGF2BP1; Transcriptional regulation; m6A RNA methylation; pyroptosis; septic acute kidney injury
    DOI:  https://doi.org/10.7150/ijbs.78348
  34. Am J Transl Res. 2022 ;14(12): 8800-8827
    Characterization of ligamentum flavum hypertrophy based on m6A RNA methylation modification and the immune microenvironment.
    Zhongyuan He, Zhengya Zhu, Tao Tang, Peng Guo, Manman Gao, Baoliang Li, Tran Canh Tung Nguyen, Hongkun Chen, Xizhe Liu, Zhiyu Zhou, Shaoyu Liu.
       OBJECTIVE: N6-methyladenosine (m6A) has been implicated in the progression of several diseases, and the role of epigenetic regulation in immunity is emerging, particularly for RNA m6A modification. However, it is unclear how m6A-related genes affect the immune microenvironment of ligamentum flavum hyperplasia (LFH). Therefore, we aimed to investigate the effect of m6A modification on the LFH immune microenvironment.
    METHODS: The GSE113212 dataset was downloaded from the Gene Expression Omnibus (GEO) database. We systematically analyzed m6A regulators in eight patient samples and the corresponding clinical information of the samples. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Set Enrichment Analysis (GSEA) and protein-protein interactions (PPIs) were used to explore the correlation of m6A clusters with the immune microenvironment in LFH. A least absolute shrinkage and selection operator (Lasso) regression was then used to further explore the m6A prognostic signature in LFH. The relative abundance of immune cell types was quantified using a single-sample Gene Set Enrichment Analysis (ssGSEA) algorithm. We explored the relationship between hub genes and small molecule drug sensitivity by clustering hub gene-based samples. In addition, Real-Time quantitative PCR (RT-qPCR) as well as western blotting (WB) were used to validate the gene expression of the differentially expressed genes.
    RESULTS: A total of 1259 differentially expressed genes were identified, of which 471 were upregulated and 788 were downregulated. A total of three genes showed significant differences (METTL16, PCIF1, and FTO). According to the enrichment analysis, immune factors may play a key role in LFH. ssGSEA was used to cluster the immune infiltration score, construct the hub gene diagnosis model, and screen a total of 6 LFH immune-related prediction model genes. The predictive diagnostic model of LFH was further constructed, revealing that METTL16, PCIF1, FTO and ALKBH5 had superior diagnostic efficiency. RT-qPCR results showed that 6 genes (METTL16, PCIF1, POSTN, TNNC1, MMP1 and ACTA1; P < 0.05) exhibited expression consistent with the results of the bioinformatics analysis of the mRNA microarray. Up-regulated METTL16, PCIF1, and ALKBH5 levels in LFH were validated by western blotting.
    CONCLUSION: Diversity and complexity of LFH's immune microenvironment are influenced by M6A modification, and our study provides strong evidence for predicting the diagnosis and prognosis of LFH.
    Keywords:  Ligamentum flavum hypertrophy; diagnostic model; epigenetics; immune microenvironment; m6A RNA methylation
  35. Am J Transl Res. 2022 ;14(12): 8457-8472
    Analysis of M6A associated lncRNAs in prognosis and immune response of NSCLC patients.
    Jing Lin, Xiao-Ling Gu, Chu-Ling Li, Zi-Mu Wang, Zhao-Feng Wang, Ran-Pu Wu, Yong Song, Ying Wu, Hong-Bing Liu.
      Distinguishing between N6-methyladenosine (m6A)-associated long noncoding RNAs (lncRNAs) is crucial in non-small-cell lung cancer (NSCLC) patients. In this research, the prognosis and immunotherapeutic response of lncRNAs and m6A in NSCLC were examined. lncRNAs related to m6A were identified using co-expression analyses, and their prognostic impact on patients with NSCLC was assessed using univariate Cox regression analysis. Sixty-three m6A-associated lncRNAs were determined as prognostic lncRNAs, and on this basis, 25 m6A-associated lncRNAs were screened by least absolute shrinkage and selection operator (lasso) Cox regression. Multivariable Cox analysis obtained 14 m6A-associated lncRNAs for the construction of risk model. The NSCLC patients were grouped into different risk subgroups in accordance with the median of the risk fraction in each data, and we evaluated the differences of potential immunotherapeutic characteristics and drug sensitivity prediction between the two subgroups. By using this model to recombine patients, they can be effectively distinguished in terms of the immunotherapy response. Furthermore, candidate compounds for the differentiation of NSCLC subtypes were identified. The model based on 14 m6A-associated lncRNAs is a promising prognostic biomarker, which may help to predict the efficacy of immunotherapy in NSCLC patients and provide a theoretical basis for improving the outcome of patients.
    Keywords:  M6A; NSCLC; immunotherapy response; lncRNAs; prognosis
  36. Front Cell Infect Microbiol. 2022 ;12 1074380
    Impact of m6A demethylase (ALKBH5, FTO) genetic polymorphism and expression levels on the development of pulmonary tuberculosis.
    Tian-Ping Zhang, Rui Li, Li-Jun Wang, Hong-Miao Li.
       Objective: The m6A methylation was involved in the pathogenesis of pulmonary tuberculosis (PTB), and our study aimed to reveal the potential association of m6A demethylase (ALKBH5, FTO) genes variation, expression levels and PTB.
    Methods: Eight SNPs (ALKBH5 gene rs8400, rs9913266, rs12936694, rs4925144 and FTO gene rs6499640, rs8047395, rs1121980, rs9939609) were selected for genotyping by SNPscan technique in 449 PTB patients and 463 healthy controls.
    Results: The mRNA expression levels of ALKBH5, FTO were detected by qRT-PCR. There were no significant differences in genotype, allele distributions of all SNPs between PTB patients and healthy controls. Haplotype analysis demonstrated that the frequency of FTO gene GAAA haplotype was significantly reduced in PTB patients when compared to controls. ALKBH5 rs8400 AA genotype, A allele frequencies were associated with the decreased risk of sputum smear-positive, while AA genotype frequency was related to the increased risk of hypoproteinemia in PTB patients. In addition, rs9913266 variant was linked to the occurrence of drug-induced liver injury, sputum smear-positive, and rs4925144 variant was associated with leukopenia among PTB patients. In FTO gene, rs8047395 GG genotype and G allele frequencies were significantly higher in the PTB patients with drug resistance than that in the PTB patients without drug resistance. The ALKBH5, FTO expression levels were significantly decreased in PTB patients in comparison to controls. Moreover, ALKBH5 level was increased in PTB patients with drug resistance, and FTO level was decreased in PTB patients with sputum smear-positive.
    Conclusion: FTO gene polymorphisms might be associated with PTB susceptibility, and ALKBH5, FTO levels were decreased in PTB patients, suggesting that these m6A demethylase played important roles in PTB.
    Keywords:  ALKBH5; FTO; m6A demethylase; pulmonary tuberculosis; single nucleotide polymorphisms
    DOI:  https://doi.org/10.3389/fcimb.2022.1074380
  37. Cancer Immunol Res. 2023 Jan 12. pii: CIR-22-0541. [Epub ahead of print]
    P. gingivalis infection upregulates PD-L1 expression on dendritic cells, suppresses CD8+ T cell responses, and aggravates oral cancer.
    Junling Ren, Xiao Han, Hannah Lohner, Rosalie G Hoyle, Jiong Li, Shuang Liang, Huizhi Wang.
      Accumulating evidence shows that PD-L1 expression on dendritic cells (DCs) is critical for cancer immunotherapy and that Porphyromonas gingivalis (Pg) colonization aggravates progression of upper gastrointestinal cancers. However, the effects of Pg infection on PD-L1 expression on DCs and related immune consequences in the infection milieu of oral cancer remains unexplored. Here, we found that Pg infection robustly enhanced PD-L1 expression on DCs in a gingipain-dependent manner in cultured cell and systemic infection assays. Pg infection suppressed antigen-specific CD8+ T cells through upregulation of PD-L1 expression on ovalbumin (OVA)-pulsed DCs. This suppression was manifested by decreased IFN, perforin, granzyme B, and CD107a. Further analysis showed that Pg drastically reduced CD8+ T cells' ability to lyse OVA-pulsed target cells. Additionally, Pg infection increased phosphorylation of Akt and STAT3, leading to a significant increase of PD-L1 expression. This was substantiated by using siRNA, overexpression plasmids, and pharmacological inhibitors. Consistent with the in vitro observations, in a syngeneic mouse oral cancer model, Pg infection significantly enhanced PD-L1 expression on DCs from intratumoral tissues and cervical lymph nodes and exacerbated oral cancer progression, whereas a Pg lysine-specific, gingipain-defective mutant failed to do so. These influences of Pg were largely diminished when tumor cells were pretreated with antibiotics or a STAT3 inhibitor. Therefore, we demonstrated that Pg infection upregulates PD-L1 expression on DCs through Akt-STAT3 signaling, suppresses CD8+ T-cell cytotoxicity, and aggravates oral cancer growth, suggesting targeting Pg, and/or its-mediated signaling, could be a therapeutic strategy to improve the efficacy of checkpoint blockade immunotherapy.
    DOI:  https://doi.org/10.1158/2326-6066.CIR-22-0541
  38. Plant Physiol. 2023 Jan 11. pii: kiad010. [Epub ahead of print]
    Shaping the landscape of N6-methyladenosine RNA methylation in Arabidopsis.
    Chui Eng Wong, Songyao Zhang, Tao Xu, Yu Zhang, Zhi Wei Norman Teo, An Yan, Lisha Shen, Hao Yu.
      N 6-methyladenosine (m6A) modification on mRNAs is deposited by evolutionarily conserved methyltransferases (writers). How individual m6A writers sculpt the overall landscape of the m6A methylome and the resulting biological impact in multicellular organisms remains unknown. Here, we systematically surveyed the quantitative m6A methylomes at single-nucleotide resolution and their corresponding transcriptomes in Arabidopsis (Arabidopsis thaliana) bearing respective impaired m6A writers. The m6A sites associated with the five Arabidopsis writers were located mostly within 3' UTRs with peaks at around 100 bp downstream of stop codons. m6A predominantly promoted the usage of distal poly(A) sites, but had little effect on RNA splicing. Notably, impaired m6A writers resulted in hypomethylation and downregulation of transcripts encoding ribosomal proteins, indicating a possible correlation between m6A and protein translation. Besides the common effects on mRNA metabolism and biological functions uniquely exerted by different Arabidopsis m6A writers compared with their counterparts in human cell lines, our analyses also revealed functional specificity of individual Arabidopsis m6A writers in plant development and response to stresses. Our findings thus reveal insights into the biological roles of various Arabidopsis m6A writers and their cognate counterparts in other multicellular m6A methyltransferase complexes.
    DOI:  https://doi.org/10.1093/plphys/kiad010
  39. Exp Hematol Oncol. 2023 Jan 12. 12(1): 6
    CTP synthase 2 predicts inferior survival and mediates DNA damage response via interacting with BRCA1 in chronic lymphocytic leukemia.
    Xinting Hu, Yang Han, Jiarui Liu, Hua Wang, Zheng Tian, Xin Zhang, Ya Zhang, Xin Wang.
       BACKGROUND: Cytidine triphosphate synthase 2 (CTPS2) is an essential metabolic enzyme that catalyzes the biosynthesis of CTP. CTP synthases contribute to lymphocytes proliferation and tumorigenesis, but the role of CTPS2 in chronic lymphocytic leukemia (CLL) remains undefined.
    METHODS: In silico analysis was performed to quantified the expression and clinical analysis of CTPS2 and BRCA1. The expression was then validated on the internal sets. Loss-and gain-of-function assays were conducted to investigate the physiological phenotypes in CLL. RNA-seq was employed to probe the molecular mechanism of CTPS2.
    RESULTS: Herein, significant elevated expression of CTPS2 was observed in CLL patients compared to normal CD19 + B cells, which was verified in three independent cohorts. Furthermore, overexpression of CTPS2 was closely associated with undesired prognostic indicators, including unmutated IGHV status and chromosome 11q23 deletion. Additionally, elevated CTPS2 expression predicted adverse overall survival and treatment-free survival with independent prognostic significance. Downregulation of CTPS2 in CLL cells exhibited attenuated cell proliferation, arrested G2/M cell cycle and increased apoptosis. The addition of CTP or glutamine could reverse the above effects. Since RNA-seq showed the enrichment in DNA damage and response signaling, we subsequently found that silence of CTPS2 remarkably elevated DNA damage and decreased DNA repair. It was demonstrated that CTPS2 mediated DNA damage response via interacting with Breast Cancer 1 (BRCA1) protein in CLL through CoIP assays and rescued experiments.
    CONCLUSIONS: Collectively, our study generated the novel findings that CTPS2 promoted CLL progression via DNA damage response and repair pathway. Targeting nucleotide metabolism potentially became an attractive strategy for treatment against CLL.
    Keywords:  Breast cancer associated 1; CTP synthase 2; Chronic lymphocytic leukemia; DNA damage response; Survival
    DOI:  https://doi.org/10.1186/s40164-022-00364-0
  40. Mol Ther. 2023 Jan 12. pii: S1525-0016(23)00011-4. [Epub ahead of print]
    ABRO1 arrests postnatal cardiomyocyte proliferation and myocardial repair by blocking N6-methyladenosine methylation and expression of PSPH.
    Tao Wang, Lu-Yu Zhou, Xin-Min Li, Fang Liu, Lin Liang, Xin-Zhe Chen, Jie Ju, Murugavel Ponnusamy, Kai Wang, Cui-Yun Liu, Kao-Wen Yan, Kun Wang.
      Abraxas 2 (ABRO1 or KIAA0157), a component of lysine63-linked deubiquitinating system, its role in the cardiomyocyte proliferation and myocardial regeneration is unknown. Here, we found that ABRO1 regulates cardiomyocyte proliferation and cardiac regeneration in the postnatal heart by targeting METTL3-mediated m6A methylation of Psph mRNA. The deletion of ABRO1 increased cardiomyocyte proliferation in hearts and restored the heart function after myocardial injury. On the contrary, ABRO1-overexpressing administration significantly inhibited the neonatal cardiomyocyte proliferation and cardiac regeneration in mice hearts. The mechanism that ABRO1 regulates cardiomyocyte proliferation mainly involved METTL3-mediated Psph mRNA methylation and CDK2 phosphorylation. At the early postnatal period, METTL3-dependent m6A methylation promotes cardiomyocyte proliferation by hypermethylation of Psph mRNA and upregulating PSPH expression. PSPH dephosphorylates cyclin dependent kinase 2 (CDK2), a positive regulator of cell cycle, at Thr14/Tyr15 and increases its activity. Upregulation of ABRO1 restricts METTL3 activity and halts the cardiomyocyte proliferation in the postnatal hearts. Thus, our study reveals that ABRO1 is an imperative contributor in the cell cycle withdrawal and attenuation of proliferative response in the postnatal cardiomyocytes and could act as a potential target to accelerate cardiomyocyte proliferation and cardiac repair in the adult heart.
    DOI:  https://doi.org/10.1016/j.ymthe.2023.01.011
  41. J Zhejiang Univ Sci B. 2023 Jan 15. pii: 1673-1581(2023)01-0050-14. [Epub ahead of print]24(1): 50-63
    Targeting TRMT5 suppresses hepatocellular carcinoma progression via inhibiting the HIF-1α pathways.
    Qiong Zhao, Luwen Zhang, Qiufen He, Hui Chang, Zhiqiang Wang, Hongcui Cao, Ying Zhou, Ruolang Pan, Ye Chen.
      Accumulating evidence has confirmed the links between transfer RNA (tRNA) modifications and tumor progression. The present study is the first to explore the role of tRNA methyltransferase 5 (TRMT5), which catalyzes the m1G37 modification of mitochondrial tRNAs in hepatocellular carcinoma (HCC) progression. Here, based on bioinformatics and clinical analyses, we identified that TRMT5 expression was upregulated in HCC, which correlated with poor prognosis. Silencing TRMT5 attenuated HCC proliferation and metastasis both in vivo and in vitro, which may be partially explained by declined extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Mechanistically, we discovered that knockdown of TRMT5 inactivated the hypoxia-inducible factor-1 (HIF-1) signaling pathway by preventing HIF-1α stability through the enhancement of cellular oxygen content. Moreover, our data indicated that inhibition of TRMT5 sensitized HCC to doxorubicin by adjusting HIF-‍1α. In conclusion, our study revealed that targeting TRMT5 could inhibit HCC progression and increase the susceptibility of tumor cells to chemotherapy drugs. Thus, TRMT5 might be a carcinogenesis candidate gene that could serve as a potential target for HCC therapy.
    Keywords:  Hepatocellular carcinoma (HCC); Hypoxia-inducible factor-1α (HIF-1α); Transfer RNA (tRNA); tRNA methyltransferase 5 (TRMT5)
    DOI:  https://doi.org/10.1631/jzus.B2200224
  42. Nat Commun. 2023 Jan 06. 14(1): 99
    Targeted systematic evolution of an RNA platform neutralizing DNMT1 function and controlling DNA methylation.
    Carla L Esposito, Ida Autiero, Annamaria Sandomenico, H Li, Mahmoud A Bassal, Maria L Ibba, Dongfang Wang, Lucrezia Rinaldi, Simone Ummarino, Giulia Gaggi, Marta Borchiellini, Piotr Swiderski, Menotti Ruvo, Silvia Catuogno, Alexander K Ebralidze, Marcin Kortylewski, Vittorio de Franciscis, Annalisa Di Ruscio.
      DNA methylation is a fundamental epigenetic modification regulating gene expression. Aberrant DNA methylation is the most common molecular lesion in cancer cells. However, medical intervention has been limited to the use of broadly acting, small molecule-based demethylating drugs with significant side-effects and toxicities. To allow for targeted DNA demethylation, we integrated two nucleic acid-based approaches: DNMT1 interacting RNA (DiR) and RNA aptamer strategy. By combining the RNA inherent capabilities of inhibiting DNMT1 with an aptamer platform, we generated a first-in-class DNMT1-targeted approach - aptaDiR. Molecular modelling of RNA-DNMT1 complexes coupled with biochemical and cellular assays enabled the identification and characterization of aptaDiR. This RNA bio-drug is able to block DNA methylation, impair cancer cell viability and inhibit tumour growth in vivo. Collectively, we present an innovative RNA-based approach to modulate DNMT1 activity in cancer or diseases characterized by aberrant DNA methylation and suggest the first alternative strategy to overcome the limitations of currently approved non-specific hypomethylating protocols, which will greatly improve clinical intervention on DNA methylation.
    DOI:  https://doi.org/10.1038/s41467-022-35222-4
  43. J Gastrointest Oncol. 2022 Dec;13(6): 3112-3122
    The miRNA125a-5p and miRNA125b-1-5p cluster induces cell invasion by down-regulating DDB2-reduced epithelial-to-mesenchymal transition (EMT) in colorectal cancer.
    Fang Zhu, Juan Wei, Danni He, Jing He, Ling Liu, Huyang Hou, Hengmei Shi, Shidai Jin, Jun Li, Xiaoyan Shi, Ping Liu, Mao Huang.
       Background: MicroRNA (miRNA) is a kind of non-coding RNA that regulates gene expression and is involved in tumor development. MiRNA-125 is reportedly aberrantly expressed in colorectal cancer tissue; however, its potential function and underlying mechanism remain unclear. The present study aimed to investigate the expression level and potential role of the miRNA-125 family in the invasion and migration of colorectal cancer.
    Methods: To further understand the role of the miRNA-125 family in metastatic colorectal cancer, we overexpressed miRNA-125 in the SW480 cell line by transfection with the miRNA-125 family mimics or a sponge. Methyl thiazolyl tetrazolium (MTT) assay was performed to identify the effect of the miRNA-125 family on cell proliferation, and a Transwell filter assay was used to detect the role of the miRNA-125 family in migration and invasion. A luciferase assay was carried out to confirm the binding site of miRNA-125 and the target gene, damage specific DNA binding protein 2 (DDB2). Western blot was applied to detect the expression levels of DDB2 and the markers of epithelial-to-mesenchymal transition (EMT) in colorectal cancer cells.
    Results: The real-time polymerase chain reaction (PCR) results showed that miR-125a-5p and miR-125b-1-5p were up-regulated in metastatic colorectal cancer tissues. The Transwell filter assay results appeared that miR-125a-5p and miR-125b-1-5p could promote the invasion and migration of colorectal cancer cells. The luciferase assay data confirmed the binding site of miR-125a-5p and miR-125b-1-5p on the 3' untranslated region (3'UTR) of DDB2 messenger RNA (mRNA). The real-time PCR and Western blot results indicated that miR-125a-5p and miR-125b-1-5p could regulate the expression levels of DDB2 and EMT markers, and lower DDB2 expression was observed in metastatic tissues.
    Conclusions: Our findings illustrated that miRNA125a-5p and miRNA125b-1-5p could reduce the expression of DDB2 by binding to the 3'UTR region, and then regulate the expression levels of EMT markers, leading to the enhanced invasion and metastasis of colorectal cancer cells. Thus, miRNA125a-5p and miRNA125b-1-5p might be novel markers of colorectal cancer migration and potential therapeutic targets to treat metastatic colorectal cancer patients.
    Keywords:  Colorectal cancer (CRC); damage specific DNA binding protein 2 (DDB2); endothelial-to-mesenchymal transition; metastases; miRNA-125
    DOI:  https://doi.org/10.21037/jgo-22-1222
  44. Chembiochem. 2023 Jan 14.
    Identification of 2-methylthio-methylenethio-N6-(cis-4-hydroxyisopentenyl)-adenosine (msms2io6A37) as a novel modification at adenosine 37 of tRNAs from Salmonella typhimurium.
    Marouane Libiad, Thierry Douki, Marc Fontecave, Mohamed Atta.
      Post-transcriptional modifications of tRNA nucleotide are important determinants in folding, structure and function. In this communication we successfully achieved the identification and characterization of a new modified base named 2-methylthio-methylenethio-N6-(cis-4-hydroxyisopentenyl)-adenosine present at position 37 in some tRNAs. We also showed that this new modified adenosine is derived from the known 2-methylthio-methylenethio-N6-(isopentenyl)-adenosine nucleoside by a catalytic cycle of the tRNA-diiron monooxygenase enzyme, MiaE present in Salmonella typhimurium.
    Keywords:  LC-MS, tRNA modifications, Hydroxylation, tRNA-diiron monooxygenas enzyme
    DOI:  https://doi.org/10.1002/cbic.202300019
  45. Heliyon. 2022 Dec;8(12): e12078
    The novel m6A writer METTL5 as prognostic biomarker probably associating with the regulation of immune microenvironment in kidney cancer.
    Wei Zhang, Yumei Chen, Zhipeng Zeng, Yue Peng, Lintai Li, Nan Hu, Xucan Gao, Wanxia Cai, Lianghong Yin, Yong Xu, Xinzhou Zhang, Donge Tang, Yong Dai.
      Nowadays, among all urinary system cancers, the mortality of kidney cancer (KC) has risen to the first, and the incidence has been keeping on the third. Many recent studies have demonstrated that m6A modification regulated by the methyltransferases (writers) is closely related to the tumorigenesis of multiple cancers. In our previous study, we found that the methyltransferase METTL5 had a stronger association with the hazard ratio of KC more than most tumors, indicating its special function in carcinogenesis of KC. Until now, the expression, functions and mechanism of METTL5 in KC are still unclear. In this study, we analyzed the mRNA expression of METTL5 using the data sets from public databases, and revealed that the METTL5 expression was significantly up-regulated in tumor tissues of kidney renal clear cell carcinoma (KIRC) and kidney renal papillary cell carcinoma (KIRP) compared to normal tissues. Also, the METTL5 expression was correlated with the tumor stage and grade, indicating the potential involvement of METTL5 in tumor progression. Additionally, the higher expression of METTL5 predicted poorer prognosis of KIRC and KIRP patients. Subsequently, we revealed that the functions of METTL5 in KIRC might be related to immune modulation, because its co-expressed gene were enriched in immune-relevant pathways including Th17 cell differentiation, Th1 and Th2 cell differentiation, and phosphatidylinositol 3-kinase activity. Next, we disclosed that the METTL5 expression was correlated to the microenvironment score and immune score of KIRC and KIRP, and associated with the infiltration ratios of 25 types of immune cells. Besides, we demonstrated a wide difference of the METTL5's effect on the survival of patients with high and low immune infiltration, further suggesting METTL5 might affect tumor development via modulating the immune microenvironment. The findings of our study provide a novel potential prognostic biomarker and immune drug target for KC.
    Keywords:  Immune microenvironment; Kidney cancer; METTL5; Prognostic factor; m6A
    DOI:  https://doi.org/10.1016/j.heliyon.2022.e12078
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