bims-rimeca 2023-03-26 papers

bims-rimeca

Biomed News

on RNA methylation in cancer

Issue of 2023–03–26
34 papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics

A novel MYCN-YTHDF1 cascade contributes to retinoblastoma tumor growth by eliciting m6A -dependent activation of multiple oncogenes.
Involvement of RNA methylation modification patterns mediated by m7G, m6A, m5C and m1A regulators in immune microenvironment regulation of Sjögren's syndrome.
METTL3 activates PERK-eIF2α dependent coelomocyte apoptosis by targeting the endoplasmic reticulum degradation-related protein SEL1L in echinoderms.
The m6A reader YTHDF2 promotes bladder cancer progression by suppressing RIG-I-mediated immune response.
METTL3-METTL14 complex induces necroptosis and inflammation of vascular smooth muscle cells via promoting N6 methyladenosine mRNA methylation of receptor-interacting protein 3 in abdominal aortic aneurysms.
METTL3/YTHDF2 m6A axis promotes the malignant progression of bladder cancer by epigenetically suppressing RRAS.
Emerging Regulatory Mechanisms of N6-Methyladenosine Modification in Cancer Metastasis.
m6A-mediated nonhomologous end joining (NHEJ) pathway regulates senescence in Brachionus plicatilis (Rotifera).
N6-methyladenosine RNA modification regulates cotton drought response in a Ca2+ and ABA-dependent manner.
The m6A reader PRRC2A is essential for meiosis I completion during spermatogenesis.
Epitranscriptomic profiling of m6A RNA methylation in renal epithelial tubular cells stimulated with calcium oxalate crystals through microarray analysis.
DNER and GNL2 are differentially m6A methylated in periodontitis in comparison with periodontal health revealed by m6A microarray of human gingival tissue and transcriptomic analysis.
METTL3-mediated m6A modification of HMGA2 mRNA promotes subretinal fibrosis and epithelial-mesenchymal transition.
Roles and therapeutic implications of m6A modification in cancer immunotherapy.
Effect of N6-methyladenosine methylation-related gene signature for predicting the prognosis of hepatocellular carcinoma patients.
m6A RNA methylation modification is involved in the disease course of heart failure.
N6-methyladenosine related gene expression signatures for predicting the overall survival and immune responses of patients with colorectal cancer.
N6 - Methyladenosine defines a new checkpoint in γδ T cell development.
NAT10 drives cisplatin chemoresistance by enhancing ac4C-associated DNA repair in bladder cancer.
METTL3 (Methyltransferase Like 3)-Dependent N6-Methyladenosine Modification on Braf mRNA Promotes Macrophage Inflammatory Response and Atherosclerosis in Mice.
RNA methylation in plants: An overview.
SEPT2 crotonylation promotes metastasis and recurrence in hepatocellular carcinoma and is associated with poor survival.
m6A RNA methylation-mediated upregulation of HLF promotes intrahepatic cholangiocarcinoma progression by regulating the FZD4/β-catenin signaling pathway.
Identification and Validation of a m5C RNA Modification-Related Gene Signature for Predicting Prognosis and Immunotherapeutic Efficiency of Gastric Cancer.
Suxiao Jiuxin Pill alleviates myocardial ischemia-reperfusion injury through the ALKBH5/GSK3β/mTOR pathway.
Immune response and drug therapy based on ac4C-modified gene in pancreatic cancer typing.
Exosomal circCLIP1 regulates PM2.5-induced airway obstruction via targeting SEPT10 in vitro.
High expression of WTAP is related to poor prognosis in nasopharyngeal carcinoma.
PES1 reduces CD8+ T cell infiltration and immunotherapy sensitivity via interrupting ILF3-IL15 complex in esophageal squamous cell carcinoma.
Molecular mechanism of tRNA binding by the Escherichia coli N7 guanosine methyltransferase TrmB.
Tet methylcytosine dioxygenase 1 modulates Porphyromonas gingivalis-triggered pyroptosis by regulating glycolysis in cementoblasts.
m6A-driven SF3B1 translation control steers splicing to direct genome integrity and leukemogenesis.
Systematic analysis of the aberrances and functional implications of cuproptosis in cancer.
Expression pattern, tumor immune landscape, and prognostic value of N7‑methylguanosine regulators in bladder urothelial carcinoma.

Sci China Life Sci. 2023 Mar 17.

A novel MYCN-YTHDF1 cascade contributes to retinoblastoma tumor growth by eliciting m6A -dependent activation of multiple oncogenes.

Yingxiu Luo, Mengjia He, Jie Yang, Feifei Zhang, Jie Chen, Xuyang Wen, Jiayan Fan, Xianqun Fan, Peiwei Chai, Renbing Jia.

  Retinoblastoma, the most prevalent primary intraocular tumor in children, leads to vision impairment, disability and even death. In addition to RB1 inactivation, MYCN activation has been documented as another common oncogenic alteration in retinoblastoma and represents one of the high-risk molecular subtypes of retinoblastoma. However, how MYCN contributes to the progression of retinoblastoma is still incompletely understood. Here, we report that MYCN upregulates YTHDF1, which encodes one of the reader proteins for N6-methyladenosine (m6A) RNA modification, in retinoblastoma. We further found that this MYCN-upregulated m6A reader functions to promote retinoblastoma cell proliferation and tumor growth in an m6A binding-dependent manner. Mechanistically, YTHDF1 promotes the expression of multiple oncogenes by binding to their mRNAs and enhancing mRNA stability and translation in retinoblastoma cells. Taken together, our findings reveal a novel MYCN-YTHDF1 regulatory cascade in controlling retinoblastoma cell proliferation and tumor growth, pinpointing an unprecedented mechanism for MYCN amplification and/or activation to promote retinoblastoma progression.

Keywords:  CDK5R1; MYCN; YTHDF1; m6A; retinoblastoma

DOI:  https://doi.org/10.1007/s11427-022-2288-4
Cell Signal. 2023 Mar 17. pii: S0898-6568(23)00064-5. [Epub ahead of print] 110650

Involvement of RNA methylation modification patterns mediated by m7G, m6A, m5C and m1A regulators in immune microenvironment regulation of Sjögren's syndrome.

Yuxiu Liu, Jianing Zhu, Lin Ding.

  Keratoconjunctivitis is the most common complication of Sjögren's syndrome (SS). It has always been a hot research topic due to its complex pathogenesis. A further understanding of keratoconjunctiva xerosis can be obtained by studying the primary diseases. 7-Methylguanine (m7G), N6-methyladenosine (m6A), 5-methylcytosine (m5C), and N1-methyladenosine (m1A) are newly discovered epigenetic mechanisms involved in the development of SS. This study aimed to investigate the effects of m7G, m6A, m5C, and m1A modifications on the immune microenvironment of SS. Three microarray datasets were downloaded from the Gene Omnibus Expression (GEO) database, including 56 SS samples and 35 normal samples. Then, genes with m7G, m6A, m5C, and m1A methylation were explored, and the RNA modification patterns mediated by 59 m7G, m6A, m5C, and m1A regulators were summarized. The effects of m7G, m6A, m5C, and m1A modifications on immune infiltrating cells were discussed. Eukaryotic translation initiation factor 3 subunit D(EIF3D) was closely related to monocytes, and the expression of EIF3D was higher in SS with less monocytes. Two distinct patterns of RNA modification mediated by the 59 m7G, m6A, m5C, and m1A regulators were also identified, which infiltrated immune cells differently. Moreover, the two distinct RNA patterns were enriched in different signaling pathways, and their biological functions were explored. The findings revealed that m7G, m6A, m5C, and m1A modifications played vital roles in the diversity and complexity of the immune microenvironment in SS.

Keywords:  Epigenetics; Immune microenvironment; Methylation; Sjögren's syndrome

DOI:  https://doi.org/10.1016/j.cellsig.2023.110650
Biochim Biophys Acta Gene Regul Mech. 2023 Mar 16. pii: S1874-9399(23)00022-6. [Epub ahead of print]1866(2): 194927

METTL3 activates PERK-eIF2α dependent coelomocyte apoptosis by targeting the endoplasmic reticulum degradation-related protein SEL1L in echinoderms.

Dongdong Li, Ming Guo, Zhimeng Lv, Yina Shao, Weikang Liang, Chenghua Li.

  N6-methyladenosine (m6A) plays an important role in regulating many physiological and disease processes in vertebrates, in which methyltransferase-like 3 (METTL3) is the best-known m6A methyltransferase. However, the functional roles of invertebrate METTL3 have not yet been highlighted. In this study, we found that METTL3 from Apostichopus japonicus (AjMETTL3) was significantly induced in coelomocytes accompanied by higher levels of m6A modification in response to Vibrio splendidus challenge. Overexpression or silencing of AjMETTL3 in coelomocytes increased or decreased the m6A levels and promoted or inhibited V. splendidus-induced coelomocyte apoptosis, respectively. To further explore the molecular mechanism of AjMETTL3-mediated coelomic immunity, m6A-seq analysis revealed that the endoplasmic reticulum-related degradation (ERAD) pathway was significantly enriched, in which suppressor/enhancer of Lin-12-like (AjSEL1L) was suggested to be a target of AjMETTL3 in a negative regulatory manner. Functional analysis revealed that the increased AjMETTL3 reduced the stability of AjSEL1L mRNA by targeting the m6A modification site of 2004 bp-GGACA-2008 bp. The decreased AjSEL1L was further confirmed to be involved in AjMETTL3-mediated coelomocyte apoptosis. Mechanistically, the inhibited AjSEL1L increased the transcription of AjOS9 and Ajp97 in the EARD pathway to promote ubiquitin protein accumulation and ER stress, which further activated AjPERK-AjeIF2α pathway dependent coelomocyte apoptosis, but not the AjIRE1 or AjATF6 pathway. Taken together, our results supported invertebrate METTL3-mediated coelomocyte apoptosis by regulating the PERK-eIF2α pathway.

Keywords:  Apoptosis; Methyltransferase-like 3; N6-methyladenosine; Protein kinase RNA-like endoplasmic reticulum kinase; Suppressor/enhancer of Lin-12-like

DOI:  https://doi.org/10.1016/j.bbagrm.2023.194927
Cancer Res. 2023 Mar 20. pii: CAN-22-2485. [Epub ahead of print]

The m6A reader YTHDF2 promotes bladder cancer progression by suppressing RIG-I-mediated immune response.

Lei Zhang, Yuqing Li, Lingli Zhou, Houhong Zhou, Liefu Ye, Tong Ou, Huaishan Hong, Shiwen Zheng, Ziyu Zhou, Kang Wu, Zeqin Yan, Jean Paul Thiery, Jun Cui, Song Wu.

N6-Methyladenosine (m6A) is the most prevalent internal modification among mammalian mRNAs. Recent studies show that m6A methyltransferases, METTL3 and METTL14, play important roles in bladder carcinoma (BLCA). However, the impact of YTHDF2, a crucial m6A reader, has yet to be investigated. Here, we found that YTHDF2 is frequently up-regulated at both the RNA and protein level in bladder cancers. Functionally, YTHDF2 promotes the proliferation and tumor growth of BLCA cells in vitro and in vivo, respectively. Integrative RNA-sequencing and m6A-sequencing analyses show that RIG-I is a downstream target of YTHDF2. Mechanistically, YTHDF2 binds to the coding sequence of DDX58 mRNA and mediates its degradation in an m6A-dependent manner. Knock-down of RIG-I inhibits apoptosis and promotes the proliferation of BLCA cells. Depleting DDX58 also restores the phenotype abrogated by YTHDF2 deficiency. Moreover, bladder cancer Ythdf2-deficient cells implanted orthotopically activate an innate immune response and promote the recruitment of CD8+ T lymphocytes into the tumor bed and the urothelium. Consequently, targeting YTHDF2 may be beneficial in Bacillus Calmette-Guérin (BCG) immunotherapy. Our study reveals that YTHDF2 acts as an oncogene and RIG-I as a tumor suppressor in BLCA. These findings highlight the functional importance of the m6A modification in BLCA and implicate YTHDF2 as a potential therapeutic target of BLCA treatment.

DOI: https://doi.org/10.1158/0008-5472.CAN-22-2485
J Cell Commun Signal. 2023 Mar 22.

METTL3-METTL14 complex induces necroptosis and inflammation of vascular smooth muscle cells via promoting N6 methyladenosine mRNA methylation of receptor-interacting protein 3 in abdominal aortic aneurysms.

Kun Li, Dongbin Zhang, Shuiting Zhai, Huilin Wu, Hongzhi Liu.

  Abdominal aortic aneurysms (AAA) have the highest incidence and rupture rate of all aortic aneurysms. The N6 methyladenosine (m6A) modification is closely associated with angiotensin (Ang II)-induced aortic diseases. This study aimed to identify whether the m6A writer METTL3/METTL4 regulates rip3 mRNA expression in AAA. To induce the mouse AAA model, apolipoprotein E-deficient (ApoE-/-) mice were subcutaneously infused with Ang II, and C57BL/6 mice were infused with type I elastase. Vascular smooth muscle cells (VSMCs) were induced with Ang II. Necroptosis was detected using an Annexin V-FITC/PI apoptosis detection kit, and ELISA assays measured inflammatory cytokines. The RNA immunoprecipitation-qPCR determined the methylated rip3 mRNA level. The increased expressions of inflammatory factors, aortic adventitia injury, degradation of elastin, and CD68-positive cells suggested the successful establishment of mouse AAA models. In AAA aorta wall tissues, the m6A modification level and the expression of METTL3/METTL14 were elevated. In Ang II-induced VSMCs, necroptosis and inflammatory cytokines in the supernatants were increased. RNA immunoprecipitation and co-immunoprecipitation assays confirmed the binding between the METTL3-METTL14 complex and rip3 mRNA, the interaction between YTHDF3 and rip3 mRNA, and between the METTL3-METTL14 complex and SMAD2/3. Interference with METTL3/METTL14 attenuated VSMC necroptosis, inflammatory response, and the AAA pathological process in vivo. The METTL3-METTL14 complex, which was increased by the activation of the SMAD2/3, elevated the m6A modification of rip3 mRNA by promoting the binding between YTHDF3 and rip3 mRNA, thus contributing to the progression of AAA. The activation of SMAD2/3 in VSMCs of abdominal aortic wall tissues is stimulated by Ang II. Subsequently, it promotes METTL3 METTL14 complex mediated m6A modification of rip3 mRNA. Meanwhile, the level of rip3 mRNA becomes more stable under the m6A reader of YTHDF3, which increases the protein level of RIP3 and further induces VSMC necroptosis. In addition, cell debris induces inflammatory factors in neighboring VSMCs and recruit monocytes/macrophages to the lesion.

Keywords:  Abdominal aortic aneurysm; N6 methyladenosine; Necroptosis; RIP3; m6A modification

DOI:  https://doi.org/10.1007/s12079-023-00737-y
Oncol Rep. 2023 May;pii: 94. [Epub ahead of print]49(5):

METTL3/YTHDF2 m6A axis promotes the malignant progression of bladder cancer by epigenetically suppressing RRAS.

Jie-Xun Chen, Dong-Ming Chen, Dong Wang, Yi Xiao, Shuai Zhu, Xian-Lin Xu.

  The present study aimed to explore the potential roles of the methyltransferase‑like 3 (METTL3)‑mediated methylation of RAS related (RRAS) mRNA in the tumorigenesis and development of bladder cancer (BCa). For this purpose, the relative expression levels of METTL3 in BCa specimens and cell lines were measured using reverse transcription‑quantitative PCR (RT‑qPCR) and western blot analysis. The association between the METTL3 expression level and the clinical characteristics of patients with BCa was analyzed using The Cancer Genome Atlas (TCGA) and Gene Expression Profiling Interactive Analysis databases. Cellular experiments were performed to confirm the effects of METTL3 on the proliferative, migratory and invasive capacities of BCa cells. RT‑qPCR, western blot analysis, methylated RNA immunoprecipitation (MeRIP)‑qPCR and dual‑luciferase report assays were utilized to verify the METTL3/RRAS/YTH N6‑methyladenosine (m6A) RNA binding protein 2 (YTHDF2) regulatory axis in BCa. The results revealed that METTL3 expression was markedly increased in BCa specimens and cell lines, and was associated with poor clinical characteristics of patients with BCa. In vitro and in vivo assays demonstrated that the silencing of METTL3 markedly suppressed the proliferative, migratory and invasive capacities of BCa cells. MeRIP‑PCR and dual‑luciferase report assays indicated that METTL3 could bind to the m6A sites of RRAS mRNA and suppress the transcriptional activity of RRAS. YTHDF2 could recognize the m6A sites of RRAS and mediate RRAS degradation. On the whole, the findings of the present study reveal the pivotal role of METTL3‑catalyzed m6A modification in BCa tumorigenesis and development. The change could facilitate BCa tumor growth and metastasis by suppressing RRAS expression in an m6A YTHDF2‑dependent manner. Targeting the METTL3/RRAS/YTHDF2 regulatory axis may thus prove to be a promising strategy for the diagnosis and therapy of BCa.

Keywords:  N6‑methyladenosine; RAS related; bladder cancer; malignant progression; methyltransferase‑like 3

DOI:  https://doi.org/10.3892/or.2023.8531
Phenomics. 2023 Feb;3(1): 83-100

Emerging Regulatory Mechanisms of N6-Methyladenosine Modification in Cancer Metastasis.

Jing Zhao, Hao Xu, Yinghan Su, Junjie Pan, Sunzhe Xie, Jianfeng Xu, Lunxiu Qin.

  Cancer metastasis is the major cause of cancer-related deaths and accounts for poor therapeutic outcomes. A metastatic cascade is a series of complicated biological processes. N6-methyladenosine (m6A) is the most abundant and conserved epitranscriptomic modification in eukaryotic cells, which has great impacts on RNA production and metabolism, including RNA splicing, processing, degradation and translation. Accumulating evidence demonstrates that m6A plays a critical role in regulating cancer metastasis. However, there is a lack of studies that review the recent advances of m6A in cancer metastasis. Here, we systematically retrieved the functions and mechanisms of how the m6A axis regulates metastasis, and especially summarized the organ-specific liver, lung and brain metastasis mediated by m6A in various cancers. Moreover, we discussed the potential application of m6A modification in cancer diagnosis and therapy, as well as the present limitations and future perspectives of m6A in cancer metastasis. This review provides a comprehensive knowledge on the m6A-mediated regulation of gene expression, which is helpful to extensively understand the complexity of cancer metastasis from a new epitranscriptomic point of view and shed light on the developing novel strategies to anti-metastasis based on m6A alteration.

Keywords:  Cancer metastasis; Epitranscriptomic modification; Organ-specific metastasis; RNA metabolism; m6A

DOI:  https://doi.org/10.1007/s43657-021-00043-w
Arch Gerontol Geriatr. 2023 Mar 12. pii: S0167-4943(23)00073-0. [Epub ahead of print]111 104994

m6A-mediated nonhomologous end joining (NHEJ) pathway regulates senescence in Brachionus plicatilis (Rotifera).

Yu Zhang, Yang Zhou, Dongqi Kan, Yunhong Yang, Jing Shen, Cui Han, Xiaojie Liu, Jiaxin Yang.

  Epigenetic modifications play an important role in the regulation of senescence. N6-methyladenosine (m6A) is the most abundant modification of mRNA. However, the impact of m6A on senescence remains largely unknown at the animal individual level. Standard model organisms Caenorhabditis elegans and Drosophila melanogaster lack many gene homologs of vertebrate m6A system that are present in other invertebrates. In this study, we employed a small aquatic invertebrate Brachionus plicatilis which has been used in aging studies for nearly 100 years to study how m6A affects aging. Phylogenetic analysis confirmed that rotifers' m6A pathway has a conserved methyltransferase complex but no demethylases and the m6A reading system was more akin to that of vertebrates than that of D. melanogaster. m6A methyltransferases are highly expressed during development but reduces dramatically during aging. Knockdown of METTL3 results in decreased fecundity and premature senescence of rotifers. Furthermore, RT-qPCR analysis indicates a role for m6A in the nonhomologous end joining (NHEJ) pathway of DNA double-strand breaks (DSBs) repair. Altogether, our work reveals a senescence regulatory model for the rotifer METTL3-m6A-NHEJ pathway.

Keywords:  DNA DSBs repair; METTL3; N6-methyladenosine (m6A); Rotifer; Senescence

DOI:  https://doi.org/10.1016/j.archger.2023.104994
Plant Biotechnol J. 2023 Mar 22.

N6-methyladenosine RNA modification regulates cotton drought response in a Ca2+ and ABA-dependent manner.

Baoqi Li, Mengmeng Zhang, Weinan Sun, Dandan Yue, Yizan Ma, Boyang Zhang, Lingfeng Duan, Maojun Wang, Keith Lindsey, Xinhui Nie, Xianlong Zhang, Xiyan Yang.

  N6 -methyladenosine (m6 A) is the most prevalent internal modification present in mRNAs, and is considered to participate in a range of developmental and biological processes. Drought response is highly regulated at the genomic, transcriptional and post-transcriptional levels. However, the biological function and regulatory mechanism of m6 A modification in the drought stress response is still poorly understood. We generated a transcriptome-wide m6 A map using drought-resistant and drought-sensitive varieties of cotton under different water deficient conditions to uncover patterns of m6 A methylation in cotton response to drought stress. The results reveal that m6 A represents a common modification and exhibit dramatic changes in distribution during drought stress. More 5'UTR m6 A was deposited in the drought-resistant variety and was associated with a positive effect on drought resistance by regulating mRNA abundance. Interestingly, we observed that increased m6 A abundance was associated with increased mRNA abundance under drought, contributing to drought resistance, and vice versa. The demethylase GhALKBH10B was found to decrease m6 A levels, facilitating the mRNA decay of ABA signal-related genes (GhZEP, GhNCED4 and GhPP2CA) and Ca2+ signal-related genes (GhECA1, GhCNGC4, GhANN1 and GhCML13), and mutation of GhALKBH10B enhanced drought resistance at seedling stage in cotton. Virus-induced gene silencing (VIGS) of two Ca2+ -related genes, GhECA1 and GhCNGC4, reduced drought resistance with the decreased m6 A enrichment on silenced genes in cotton. Collectively, we reveal a novel mechanism of post-transcriptional modification involved in affecting drought response in cotton, by mediating m6 A methylation on targeted transcripts in the ABA and Ca2+ signalling transduction pathways.

Keywords:  Ca2+ signalling pathway; Cotton; Drought stress; GhALKBH10B; N6-methyladenosine; mRNA stability

DOI:  https://doi.org/10.1111/pbi.14036
Nat Commun. 2023 Mar 24. 14(1): 1636

The m6A reader PRRC2A is essential for meiosis I completion during spermatogenesis.

Xinshui Tan, Caihong Zheng, Yinghua Zhuang, Pengpeng Jin, Fengchao Wang.

N6-methyladenosine (m6A) and its reader proteins YTHDC1, YTHDC2, and YTHDF2 have been shown to exert essential functions during spermatogenesis. However, much remains unknown about m6A regulation mechanisms and the functions of specific readers during the meiotic cell cycle. Here, we show that the m6A reader Proline rich coiled-coil 2A (PRRC2A) is essential for male fertility. Germ cell-specific knockout of Prrc2a causes XY asynapsis and impaired meiotic sex chromosome inactivation in late-prophase spermatocytes. Moreover, PRRC2A-null spermatocytes exhibit delayed metaphase entry, chromosome misalignment, and spindle disorganization at metaphase I and are finally arrested at this stage. Sequencing data reveal that PRRC2A decreases the RNA abundance or improves the translation efficiency of targeting transcripts. Specifically, PRRC2A recognizes spermatogonia-specific transcripts and downregulates their RNA abundance to maintain the spermatocyte expression pattern during the meiosis prophase. For genes involved in meiotic cell division, PRRC2A improves the translation efficiency of their transcripts. Further, co-immunoprecipitation data show that PRRC2A interacts with several proteins regulating mRNA metabolism or translation (YBX1, YBX2, PABPC1, FXR1, and EIF4G3). Our study reveals post-transcriptional functions of PRRC2A and demonstrates its critical role in the completion of meiosis I in spermatogenesis.

DOI: https://doi.org/10.1038/s41467-023-37252-y
Urolithiasis. 2023 Mar 22. 51(1): 57

Epitranscriptomic profiling of m6A RNA methylation in renal epithelial tubular cells stimulated with calcium oxalate crystals through microarray analysis.

Bo-Yu Yang, Fang-Zhou Zhao, Jun Li, Xiao-Chuan Wang, Hui-Min Zhao, Ye Tian.

  The aim of this study was to investigate the relationship of m6A RNA methylation to CaOX-induced renal tubular injury. Microarray analysis was performed to detect the difference in mRNA expression and m6A methylation between the injurious groups and controls. We established injurious renal tubular epithelial cell model induced by calcium oxalate crystals (CaOX), and we validated that CaOX could increase the overall m6A methylation levels. By microarray analysis, we identified 5967 differentially expressed mRNAs (2444 were up-regulated and 3523 were down-regulated in the injurious groups) and 6853 differentially methylated mRNAs (4055 were in hypermethylation and 3688 were in hypomethylation in the injurious groups). Four clusters (hyper-up, hyper-down, hypo-up and hypo-down) were further identified via conjoint analysis. Functional analysis revealed that m6A methylation played a crucial role in the development of CaOX through participating multiple processes covering inflammation, oxidative stress, apoptosis, crystal-cell adhesion. We delineated the first transcriptome-wide m6A landscape of injurious renal tubular cells in high-CaOX environment. We identified a series of mRNAs of renal tubular epithelial cells with differential expression and m6A methylation between the CaOX-treated groups and controls.

Keywords:  Calcium oxalate; N6-Methyladenosine; Nephrolithiasis; m6A modification

DOI:  https://doi.org/10.1007/s00240-023-01425-7
J Periodontal Res. 2023 Mar 20.

DNER and GNL2 are differentially m6A methylated in periodontitis in comparison with periodontal health revealed by m6A microarray of human gingival tissue and transcriptomic analysis.

Zhenxiang Wang, Hang Chen, Limin Peng, Yujuan He, Jingjing Wei, Xiaonan Zhang.

   OBJECTIVE: This study aims to investigate the differences in the epigenomic patterns of N6-methyladenosine (m6A) methylation in gingival tissues between patients with periodontitis (PD) and healthy controls, identifying potential biomarkers.
BACKGROUND: As a multifactorial disease, PD involves multiple genetic and environmental effects. The m6A modification is the most prevalent internal mRNA modification and linked to various inflammatory diseases. However, the m6A modification pattern and m6A-related signatures in PD remain unclear.
MATERIALS AND METHODS: An m6A microarray of human gingival tissues was conducted in eight subjects: four diagnosed with PD and four healthy controls. Microarray analysis was performed to identify the differentially m6A methylated mRNAs (DMGs) and the differentially expressed mRNAs (DEGs). The differentially methylated and expressed mRNAs (DMEGs) were subjected to functional enrichment analysis by Metascape. The weighted gene co-expression network analysis (WGCNA) algorithm, the least absolute shrinkage and selection operator (LASSO) regression, and univariate logistic regression were performed to identify potential biomarkers. The cell type localization of the target genes was determined using single-cell RNA-seq (scRNA-seq) analysis. The m6A methylation level and gene expression of hub genes were subsequently verified by m6A methylated RNA immunoprecipitation (MeRIP) and quantitative real-time PCR (qRT-PCR).
RESULTS: In total, 458 DMGs, 750 DEGs, and 279 DMEGs were identified based on our microarray. Pathway analyses conducted for the DMEGs revealed that biological functions were mainly involved in the regulation of stem cell differentiation, ossification, circadian rhythm, and insulin secretion pathways. Besides, the genes involved in crucial biological processes were mainly expressed in fibroblast and epithelial cells. Furthermore, the m6A methylation and expression levels of two hub biomarkers (DNER and GNL2) were validated.
CONCLUSION: The current study exhibited a distinct m6A epitranscriptome, identified and verified two PD-related biomarkers (DNER and GNL2), which may provide novel insights into revealing the new molecular mechanisms and latent targets of PD.

Keywords:  MeRIP; bioinformatics; epigenetics; m6A; microarray; periodontitis

DOI:  https://doi.org/10.1111/jre.13117
J Mol Cell Biol. 2023 Mar 21. pii: mjad005. [Epub ahead of print]

METTL3-mediated m6A modification of HMGA2 mRNA promotes subretinal fibrosis and epithelial-mesenchymal transition.

Yuwei Wang, Yuhong Chen, Jian Liang, Mei Jiang, Ting Zhang, Xiaoling Wan, Jiahui Wu, Xiaomeng Li, Jieqiong Chen, Junran Sun, Yifan Hu, Peirong Huang, Jingyang Feng, Te Liu, Xiaodong Sun.

  Subretinal fibrosis is a major cause of the poor visual prognosis for patients with neovascular age-related macular degeneration (nAMD). Myofibroblasts originated from retinal pigment epithelial (RPE) cells through epithelial-mesenchymal transition (EMT) contribute to the fibrosis formation. N6-methyladenosine (m6A) modification has been implicated in the EMT process and multiple fibrotic diseases. The role of m6A modification in EMT-related subretinal fibrosis has not yet been elucidated. In this study, we found that during subretinal fibrosis in the mouse model of laser-induced choroidal neovascularization, METTL3 was upregulated in RPE cells. Through m6A epitranscriptomic microarray and further verification, high mobility group AT-hook 2 (HMGA2) was identified as the key downstream target of METTL3, subsequently activating potent EMT-inducing transcription factor SNAIL. Finally, by subretinal injections of adeno-associated virus vectors, we confirmed that METTL3 deficiency in RPE cells could efficiently attenuate subretinal fibrosis in vivo. In conclusion, our present research identified an epigenetic mechanism of METTL3-m6A-HMGA2 in subretinal fibrosis and EMT of RPE cells, providing a novel therapeutic target for subretinal fibrosis secondary to nAMD.

Keywords:  HMGA2; METTL3; N6-methyladenosine; epithelial–mesenchymal transition; subretinal fibrosis

DOI:  https://doi.org/10.1093/jmcb/mjad005
Front Immunol. 2023 ;14 1132601

Roles and therapeutic implications of m6A modification in cancer immunotherapy.

Juan Pan, Tuxiong Huang, Zhenjun Deng, Chang Zou.

  Recent studies have demonstrated that N6-methyladenosine (m6A), the most abundant, dynamic, and reversible epigenetic RNA modification in eukaryotes, is regulated by a series of enzymes, including methyltransferases (writers), demethylases (erasers), and m6A recognition proteins (readers). Aberrant regulation of m6A modification is pivotal for tumorigenesis, progression, invasion, metastasis, and apoptosis of malignant tumors. Immune checkpoint inhibitors (ICIs) has revolutionized cancer treatment, as recognized by the 2018 Nobel Prize in Medicine and Physiology. However, not all cancer patients response to ICI therapy, which is thought to be the result of intricate immune escape mechanisms. Recently, numerous studies have suggested a novel role for m6A epigenetic modification in the regulation of tumor immune evasion. Herein, we review the relevant mechanisms of m6A regulators in regulating various key signaling pathways in cancer biology and how m6A epigenetic modifications regulate the expression of immune checkpoints, opening a new window to understand the roles and mechanisms of m6A epigenetic modifications in regulating tumor immune evasion. In addition, we highlight the prospects and development directions of future combined immunotherapy strategies based on m6A modification targeting, providing directions for promoting the treatment outcomes of immune checkpoint inhibitors.

Keywords:  cancer; immune checkpoints; immunotherapy; m6A modification; m6A-regulator inhibitors

DOI:  https://doi.org/10.3389/fimmu.2023.1132601
Front Surg. 2023 ;10 1052100

Effect of N6-methyladenosine methylation-related gene signature for predicting the prognosis of hepatocellular carcinoma patients.

Xinyu Zheng, Yingyue Zhang, Yun Wang, Zijing He, Qiang Zhang, Dapeng Ren, Xiao Yan, Xiao Yuan.

   Background and aims: Hepatocellular carcinoma (HCC) is a common cause of cancer-related death in humans. Increasing evidence indicates that an imbalance in N6-methyladenosine (m6A) methylation is linked to the occurrence and development of cancer. We then developed a prognostic model as an independent risk factor with which predict the prognosis of HCC.
Methods: We obtained the gene expression and clinical data of HCC patients from the TCGA databases. The prognostic value of m6A methylation-related genes in patients who had HCC were subjected to comprehensive bioinformatics analysis. We use RiskScore=∑i=1nCoefi×Xi to construct the risk scoring formula. We collected pathological specimens from 68 patients who had HCC, and conducted immunohistochemical staining experiments on the specimens.
Results: There was a significant correlation between candidate m6A methylation-related genes (YTHDF2, METTL14 and ZC3H13) overall survival of HCC patients. Among the 68 HCC patient specimens that underwent immunohistochemical staining, all cancer tissues were positive for METTL14, YTHDF2, and ZC3H13 staining in contrast to the adjacent tissues. We conducted a Kaplan-Meier survival analysis. The results showed that patients who had low METTL14 expression had a longer survival time than those of patients who had high METTL14 expression. Also, patients with low YTHDF2 expression had a longer survival time than patients with high YTHDF2 expression. Finally, patients with high ZC3H13 expression lived longer than those with low ZC3H13 expression. This result is consistent with the bioinformatics analysis conclusion above.
Conclusions: Generally, the prognostic model that was based on m6A methylation-related genes in this study can effectively predict the prognosis of HCC patients.

Keywords:  TCGA database; biomarker; hepatocellular carcinoma; immunohistochemical staining experiments; m 6 a methylation-related genes; prognostic model

DOI:  https://doi.org/10.3389/fsurg.2023.1052100
Biotechnol Genet Eng Rev. 2023 Mar 21. 1-15

m6A RNA methylation modification is involved in the disease course of heart failure.

Liyan Yu, Shuxia Cai, Xiuli Guo.

  We explored N6-methyladenosine (m6A) RNA methylation as one of the gene regulatory mechanisms in heart failure (HF) biology. Understanding the different physiological mechanisms will facilitate the prevention and individualized treatment of HF. The Gene Expression Omnibus (GEO) database served as the source of the data. In GSE116250, differential analysis between ischemic cardiomyopathy (ICM), dilated cardiomyopathy (DCM) and controls yielded differentially expressed m6A regulators. Differential analysis between HF and controls in GSE131296 identifies m6A-modified genes and then performs enrichment analysis. Protein-protein interaction (PPI) network analysis was performed for the differentially expressed ICM- or DCM-associated genes in GSE116250 and GSE55296, respectively. Finally, the diagnostic genes for ICM and DCM were predicted using receiver operating characteristic (ROC) curve. YTHDC1, HNRNPC and HNRNPA2B1 were significantly downregulated in GSE116250 in DCM and ICM compared with controls. A total of 195 genes were identified in GSE131296 as subject to m6A alteration. These genes may play a role in HF through the MAPK signaling pathway and p53 signaling pathway. PPI network analysis identified CCL5, CXCR4 and CCL2 as key genes for ICM and IL-6 as a key gene for DCM. Through ROC curves, we identified m6A-modified APLP1, KLF2 as potential diagnostic genes for ICM, and m6A-modified FGF7, FREM1 and C14orf132 as potential diagnostic genes for DCM. Our findings support m6A modifying mechanisms in HF etiology that contribute to the treatment of HF. Thus, our data suggest that m6A methylation may be an interesting target for therapeutic intervention.

Keywords:  Heart failure; diagnosis; dilated cardiomyopathy; ischemic cardiomyopathy; m6A methylation modification

DOI:  https://doi.org/10.1080/02648725.2023.2191086
Front Genet. 2023 ;14 885930

N6-methyladenosine related gene expression signatures for predicting the overall survival and immune responses of patients with colorectal cancer.

Lili Yu, Lijuan Wang, Jing Sun, Xuan Zhou, Yeting Hu, Lidan Hu, Yazhou He, Chunqing Lin, Jie Chen, Xiaolin Xu, Malcolm G Dunlop, Evropi Theodoratou, Kefeng Ding, Xue Li.

  N6-methyladenosine (m6A) modification has been demonstrated to exhibit a crucial prognostic effect on colorectal cancer (CRC). Nonetheless, potential mechanism of m6A in survival rate and immunotherapeutic response remains unknown. Here we investigated the genes associated with m6A regulators and developed a risk score for predicting the overall survival (OS) of CRC patients. RNA-seq transcriptomic profiling data of COAD/READ samples were obtained from The Cancer Genome Atlas (TCGA) database. Absolute Shrinkage and Selection Operator (LASSO)- Cox regression analysis was conducted to identify the m6A-related gene expression signatures and the selected genes were inputted into stepwise regression to develop a prognostic risk score in TCGA, and its predictive performance of CRC survival was further validated in Gene Expression Omnibus (GEO) datasets. According to our results, the risk score comprising 18 m6A-related mRNAs was significantly associated with CRC survival in both TCGA and GEO datasets. And the stratified analysis also confirmed that high-risk score acted as a poor factor in different age, sex, T stage, and tumour, node, metastasis (TNM) stages. The m6A-related prognostic score in combination with clinical characteristics yielded time-dependent area under the receiver operating characteristic curve (AUCs) of 0.85 (95%CI: 0.79-0.91), 0.84 (95%CI: 0.79-0.90) and 0.80 (95%CI: 0.71-0.88) for the prediction of the 1-, 3-, 5-year OS of CRC in TCGA cohort. Furthermore, mutation of oncogenes occurred more frequently in the high-risk group and the composition of immune cells in tumour microenvironment (TME) was significantly distinct between the low- and high-risk groups. The low-risk group had a lower microsatellite instability (MSI) score, T-cell exclusion score and dysfunction score, implying that low-risk patients may have a better immunotherapy response than high-risk patients. In summary, a prognostic risk score derived from m6A-related gene expression signatures could serve as a potential prognostic predictor for CRC survival and indicator for predicting immunotherapy response in CRC patients.

Keywords:  colorectal cancer; gene expression; immune responses; overall survival; prognostic risk score

DOI:  https://doi.org/10.3389/fgene.2023.885930
Bioessays. 2023 Mar 21. e2300002

N6 - Methyladenosine defines a new checkpoint in γδ T cell development.

Jiachen Zhao, Chenbo Ding, Hua-Bing Li.

  T cells, which are derived from hematopoietic stem cells (HSCs), are the most important components of adaptive immune system. Based on the expression of αβ and γδ receptors, T cells are mainly divided into αβ and γδ T cells. In the thymus, they share common progenitor cells, while undergoing a series of well-characterized and different developmental processes. N6 -Methyladenosine (m6 A), one of the most abundant modifications in mRNAs, plays critical roles in cell development and maintenance of function. Recently, we have demonstrated that the depletion of m6 A demethylase ALKBH5 in lymphocytes specifically induces an expansion of γδ T cells through the regulation of Jag1/Notch2 signaling, but not αβ T cells, indicating a checkpoint role of ALKBH5 and m6 A modification in the early development of γδ T cells. Based on previous studies, many key pathway molecules, which exert dominant roles in γδ T cell fate determination, have been identified as the targets regulated by m6 A modification. In this review, we mainly summarize the potential regulation between m6 A modification and these key signaling molecules in the γδ T cell lineage commitment, to provide new perspectives in the checkpoint of γδ T cell development.

Keywords:  development checkpoint; m6A RNA modification; signaling molecules; γδ T cell

DOI:  https://doi.org/10.1002/bies.202300002
Cancer Res. 2023 Mar 20. pii: CAN-22-2233. [Epub ahead of print]

NAT10 drives cisplatin chemoresistance by enhancing ac4C-associated DNA repair in bladder cancer.

Ruihui Xie, Liang Cheng, Ming Huang, Lin Huang, Ziyue Chen, Qiang Zhang, Hong Li, Junlin Lu, Hongjin Wang, Qianghua Zhou, Jian Huang, Xu Chen, Tianxin Lin.

Epitranscriptomic RNA modifications constitute a critical gene regulatory component that can affect cancer progression. Among these, the RNA N4-acetylcytidine (ac4C) modification, which is mediated by the ac4C writer N-acetyltransferase 10 (NAT10), regulates the stabilization of mRNA. Here, we identified that the ac4C modification is induced upon cisplatin treatment and correlates with chemoresistance in bladder cancer (BCa). Both in vitro and in vivo, NAT10 promoted cisplatin chemoresistance in BCa cells by enhancing DNA damage repair. Mechanistically, NAT10 bound and stabilized AHNAK mRNA by protecting it from exonucleases, and AHNAK-mediated DNA damage repair was required for NAT10-induced cisplatin resistance. Clinically, NAT10 overexpression was associated with chemoresistance, recurrence, and worse clinical outcome in BCa patients. Cisplatin-induced NFκB signaling activation was required for the upregulation of NAT10 expression, and NFκB p65 directly bound to the NAT10 promoter to activate transcription. Moreover, pharmacological inhibition of NAT10 with Remodelin sensitized BCa organoids and mouse xenografts to cisplatin. Overall, the present study uncovered a mechanism of NAT10-mediated mRNA stabilization in BCa, laying the foundation for NAT10 as a therapeutic target to overcome cisplatin resistance in BCa.

DOI: https://doi.org/10.1158/0008-5472.CAN-22-2233
Arterioscler Thromb Vasc Biol. 2023 Mar 23.

METTL3 (Methyltransferase Like 3)-Dependent N6-Methyladenosine Modification on Braf mRNA Promotes Macrophage Inflammatory Response and Atherosclerosis in Mice.

Qian Li, Liwen Yu, Amy Gao, Ruiqing Ren, Jianlin Zhang, Lei Cao, Xiaohong Wang, Yapeng Liu, Wenqian Qi, Liangyu Cai, Wei Li, Weiwei Wang, Xiaobin Guo, Guohai Su, Xiao Yu, Jie Zhang, Bo Xi, Yun Zhang, Meng Zhang, Cheng Zhang.

   BACKGROUND: Atherosclerosis is a chronic inflammatory disease, in which macrophages determine the progression of atherosclerotic plaques. However, no studies have investigated how METTL3 (methyltransferase like 3) in macrophages affects atherosclerotic plaque formation in vivo. Additionally, whether Braf is modified by METTL3-dependent N6-methyladenosine (m6A) methylation remains unknown.
METHODS: We analyzed single-cell sequencing data of atherosclerotic plaques in mice fed with a high fat diet for different periods. Mettl3fl/fl Lyz2cre Apoe-/- mice and littermate control Mettl3fl/fl Apoe-/- mice were generated and fed high fat diet for 14 weeks. In vitro, we stimulated peritoneal macrophages with ox-LDL (oxidized low-density lipoprotein) and tested the mRNA and protein expression levels of inflammatory factors and molecules regulating ERK (extracellular signal-regulated kinase) phosphorylation. To find METTL3 targets in macrophages, we performed m6A-methylated RNA immunoprecipitation sequencing and m6A-methylated RNA immunoprecipitation-qPCR. Further, point mutation experiments were used to explore m6A-methylated adenine. Using RNA immunoprecipitation assay, we explored m6A methylation-writing protein bound to Braf mRNA.
RESULTS: In vivo, METTL3 expression in macrophages increased with the progression of atherosclerosis. Myeloid cell-specific METTL3 deletion negatively regulated atherosclerosis progression and the inflammatory response. In vitro, METTL3 knockdown or knockout in macrophages attenuated ox-LDL-mediated ERK phosphorylation rather than JNK (c-Jun N-terminal kinase) and p38 phosphorylation and reduced the level of inflammatory factors by affecting BRAF protein expression. The negative regulation of inflammation response caused by METTL3 knockout was rescued by overexpression of BRAF. In mechanism, METTL3 targeted adenine (39725126 in chromosome 6) on the Braf mRNA. Then, YTHDF1 could bind to m6A-methylated Braf mRNA and promote its translation.
CONCLUSIONS: Myeloid cell-specific Mettl3 deficiency suppressed hyperlipidemia-induced atherosclerotic plaque formation and attenuated atherosclerotic inflammation. We identified Braf mRNA as a novel target of METTL3 in the activation of the ox-LDL-induced ERK pathway and inflammatory response in macrophages. METTL3 may represent a potential target for the treatment of atherosclerosis.

Keywords:  ERK phosphorylation; atherosclerosis; macrophage; pathway; tumor necrosis factor

DOI:  https://doi.org/10.1161/ATVBAHA.122.318451
Front Plant Sci. 2023 ;14 1132959

RNA methylation in plants: An overview.

Harshraj Shinde, Ambika Dudhate, Ulhas S Kadam, Jong Chan Hong.

  RNA methylation is an important post-transcriptional modification that influences gene regulation. Over 200 different types of RNA modifications have been identified in plants. In animals, the mystery of RNA methylation has been revealed, and its biological role and applications have become increasingly clear. However, RNA methylation in plants is still poorly understood. Recently, plant science research on RNA methylation has advanced rapidly, and it has become clear that RNA methylation plays a critical role in plant development. This review summarizes current knowledge on RNA methylation in plant development. Plant writers, erasers, and readers are highlighted, as well as the occurrence, methods, and software development in RNA methylation is summarized. The most common and abundant RNA methylation in plants is N6-methyladenosine (m6A). In Arabidopsis, mutations in writers, erasers, and RNA methylation readers have affected the plant's phenotype. It has also been demonstrated that methylated TRANSLATIONALLY CONTROLLED TUMOR PROTEIN 1-messenger RNA moves from shoot to root while unmethylated TCTP1-mRNA does not. Methylated RNA immunoprecipitation, in conjunction with next-generation sequencing, has been a watershed moment in plant RNA methylation research. This method has been used successfully in rice, Arabidopsis, Brassica, and maize to study transcriptome-wide RNA methylation. Various software or tools have been used to detect methylated RNAs at the whole transcriptome level; the majority are model-based analysis tools (for example, MACS2). Finally, the limitations and future prospects of methylation of RNA research have been documented.

Keywords:  RNA methylations; gene regulation; plant development; software; writers

DOI:  https://doi.org/10.3389/fpls.2023.1132959
Cell Biosci. 2023 Mar 22. 13(1): 63

SEPT2 crotonylation promotes metastasis and recurrence in hepatocellular carcinoma and is associated with poor survival.

Xin-Yue Zhang, Ze-Xian Liu, Yi-Fan Zhang, Li-Xia Xu, Meng-Ke Chen, Yu-Feng Zhou, Jun Yu, Xiao-Xing Li, Ning Zhang.

   BACKGROUND: Hepatocellular carcinoma (HCC) metastasis and recurrence lead to therapy failure, which are closely associated with the proteome. However, the role of post-translational modification (PTM) in HCC, especially for the recently discovered lysine crotonylation (Kcr), is elusive.
RESULTS: We investigated the correlation between crotonylation and HCC in 100 tumor tissues and performed stable isotope labeling by amino acids and liquid chromatography tandem mass spectrometry in HCC cells, and we found that crotonylation was positively correlated with HCC metastasis, and higher crotonylation in HCC cells facilitated cell invasiveness. Through bioinformatic analysis, we found that the crotonylated protein SEPT2 was significantly hypercrotonylated in highly invasive cells, while the decrotonylated mutation of SEPT2-K74 impaired SEPT2 GTPase activity and inhibited HCC metastasis in vitro and in vivo. Mechanistically, SIRT2 decrotonylated SEPT2, and P85α was found to be the downstream effector of SEPT2. Moreover, we identified that SEPT2-K74cr was correlated with poor prognosis and recurrence in HCC patients, thus indicating its clinical potential as an independent prognostic factor.
CONCLUSIONS: We revealed the role of nonhistone protein crotonylation in regulating HCC metastasis and invasion. Crotonylation facilitated cell invasion through the crotonylated SEPT2-K74-P85α-AKT pathway. High SEPT2-K74 crotonylation predicted poor prognosis and a high recurrence rate in HCC patients. Our study revealed a novel role of crotonylation in promoting HCC metastasis.

Keywords:  Crotonylation; Hepatocellular carcinoma; Posttranslational modification; Prognosis and recurrence marker; SEPT2

DOI:  https://doi.org/10.1186/s13578-023-00996-7
Cancer Lett. 2023 Mar 21. pii: S0304-3835(23)00095-2. [Epub ahead of print] 216144

m6A RNA methylation-mediated upregulation of HLF promotes intrahepatic cholangiocarcinoma progression by regulating the FZD4/β-catenin signaling pathway.

Daimin Xiang, Mingye Gu, Junyu Liu, Wei Dong, Zhishi Yang, Kui Wang, Jing Fu, Hongyang Wang.

  Hepatic leukemia factor (HLF) is aberrantly expressed in human malignancies. However, its role in regulating intrahepatic cholangiocarcinoma (ICC) remains unclear. This study aimed to define the role of HLF in ICC progression. Here, we showed that HLF expression is upregulated in ICC and predicts the poor prognosis in patients. Mechanistically, HLF activation in ICC is mediated by METTL3-dependent m6A methylation of the HLF mRNA. As per the results from the loss- or gain-of-function experiments, HLF promoted the self-renewal, tumorigenicity, proliferation and metastasis of ICC cells. RNA-seq and CUT&Tag analyses showed that frizzled-4 (FZD4) and forkhead box Q1 (FOXQ1) are target genes of HLF. Moreover, FOXQ1 transcriptionally activates METTL3 expression, forming a positive feedback loop, which subsequently activates WNT/β-catenin signaling and downstream tumor stemness. Furthermore, HLF expression was positively correlated with METTL3, IGF2BP3, FZD4 and FOXQ1 expression in ICC tissues in a large ICC cohort. The combined IHC panels exhibited a better prognostic value for patients with ICC than any of these components alone. In conclusions, these findings demonstrated that the METTL3/HLF/FOXQ1 regulatory circuit drove FZD4-mediated WNT/β-catenin activation in ICC progression, suggesting that targeting this axis could be novel therapeutic strategy for ICC.

Keywords:  Cancer stem cells; FZD4/β-catenin; HLF; Intrahepatic cholangiocarcinoma; m6A

DOI:  https://doi.org/10.1016/j.canlet.2023.216144
J Oncol. 2023 ;2023 9931419

Identification and Validation of a m5C RNA Modification-Related Gene Signature for Predicting Prognosis and Immunotherapeutic Efficiency of Gastric Cancer.

Li Song, Shouguo Wang, Qiankun Li, Yao Lu, Rungong Yang, Xianqi Feng.

Background: 5-methylcytosine (m5C) is a major site of RNA methylation modification, and its abnormal modification is associated with the development of gastric cancer (GC). This study aimed to explore the value of m5C-related genes on the prognosis of GC patients through bioinformatics.
Methods: First, m5C-related genes were obtained by nonnegative matrix factorization (NMF) analysis and differentially expressed analysis. The m5C-related model was established and validated in distinct datasets. Moreover, a differential analysis of risk scores according to clinical characteristics was performed. The enrichment analysis was carried out to elucidate the underlying molecular mechanisms. Furthermore, we calculated the differences in immunotherapy and chemotherapy sensitivity between the high- and low-risk groups. Finally, we validated the expression levels of identified model genes by quantitative real-time polymerase chain reaction (qRT-PCR).
Results: A total of five m5C-related subtypes of GC patients in the TCGA database were identified. The m5C-related model was constructed based on APOD, ASCL2, MFAP2, and CREB3L3. Functional enrichment revealed that the m5C-related model might involve in the cell cycle and cell adhesion. Moreover, the high-risk group had a higher abundance of stromal and immune cells in malignant tumor tissues and a lower tumor purity than the low-risk group. The patients in the high-risk group were more sensitive to chemotherapy and had better sensitivity to CTLA4 inhibitors. Furthermore, qRT-PCR results from our specimens verified an over-expression of ASCL2, CREB3L3, and MFAP2 in the cancer cells compared with the normal cells.
Conclusion: A total of five GC subtypes were identified, and a risk model was constructed based on m5C modification.

DOI: https://doi.org/10.1155/2023/9931419
Chin Med. 2023 Mar 23. 18(1): 31

Suxiao Jiuxin Pill alleviates myocardial ischemia-reperfusion injury through the ALKBH5/GSK3β/mTOR pathway.

Yiping Li, Ruixia Lu, Zhenchao Niu, Dan Wang, Xiaolong Wang.

   BACKGROUND: Many studies have shown effective protection from myocardial ischemia-reperfusion injury (MIRI) in animal models, but few, if any, treatments have yielded a substantial reduction in clinical. Several studies showed significant therapeutic effects for the Chinese patent medicine Suxiao Jiuxin Pill (SJP) in MIRI, although the specific molecular mechanisms remain undefined. Recently, increasing evidence indicates an important role for m6A modification in autophagy regulation in MIRI, and SJP has not been investigated in this regard.
METHODS: In vivo experiments were performed in a Wistar rat MIRI model. In vitro assays were conducted in hypoxia/reoxygenation (H/R)-treated H9c2 cells. H9c2 cells with ALKBH5 and GSK3β silencing were constructed by lentivirus transfection. TUNEL and Annexin V/PI assays were carried out for apoptosis detection. Then, m6A modification was detected with the EpiQuik m6A RNA methylation quantification kit, and GFP-RFP-LC3B was used to observe dynamic changes in autophagy. The autophagosome structure was assessed by Transmission electron microscopy. qPCR and immunoblot were performed for mRNA and protein analyses, receptively.
RESULTS: SJP significantly mitigated MIRI in rats, reducing infarct size and myocardial apoptosis, and improving left ventricular function. In addition, SJP inhibited autophagy through the GSK3β/mTOR pathway in MIRI rats. In cultured H9c2 cells, SJP significantly inhibited H/R- related apoptosis and autophagic activity through the GSK3β/mTOR pathway. Additionally, SJP enhanced ALKBH5 expression in H/R cardiomyocytes, which is important in impaired m6A modification. Interestingly, ALKBH5 knockdown enhanced autophagy and apoptosis in H/R-induced cells, whereas SJP reversed these effects. Further experiments showed that autophagic activity and apoptosis enhanced by ALKBH5 deficiency are GSK3β/mTOR pathway dependent in H/R-treated H9c2 cells. After SJP administration the above effects were alleviated, suggesting SJP inhibited autophagy through the ALKBH5/GSK3β/mTOR pathway in H/R-induced cardiomyocytes. These effects of SJP were common to its two main constituents, including tetra-methylpyrazine (TMP) and borneol (BOR).
CONCLUSION: SJP improves MIRI in rats and alleviates autophagy and apoptosis in H9c2 cells through the ALKBH5/GSK3β/mTOR pathway, thanks to its two major constituents TMP and BOR.

Keywords:  ALKBH5/GSK3β/mTOR pathway; Autophagy; Myocardial ischemia–reperfusion injury; Suxiao Jiuxin pill

DOI:  https://doi.org/10.1186/s13020-023-00736-6
Front Immunol. 2023 ;14 1133166

Immune response and drug therapy based on ac4C-modified gene in pancreatic cancer typing.

Dong Xu, Kaige Huang, Yang Chen, Fei Yang, Cunbing Xia, Hongbao Yang.

  N-4 cytidine acetylation (ac4C) is an epitranscriptome modification catalyzed by N-acetyltransferase 10 (NAT10) and is essential for cellular mRNA stability, rRNA biosynthesis, cell proliferation, and epithelial-mesenchymal transition (EMT). Numerous studies have confirmed the inextricable link between NAT10 and the clinical characteristics of malignancies. It is unclear, however, how NAT10 might affect pancreatic ductal adenocarcinoma. We downloaded pancreatic ductal adenocarcinoma patients from the TCGA database. We obtained the corresponding clinical data for data analysis, model construction, differential gene expression analysis, and the GEO database for external validation. We screened the published papers for NAT10-mediated ac4C modifications in 2156 genes. We confirmed that the expression levels and genomic mutation rates of NAT10 differed significantly between cancer and normal tissues. Additionally, we constructed a NAT10 prognostic model and examined immune infiltration and altered biological pathways across the models. The NAT10 isoforms identified in this study can effectively predict clinical outcomes in pancreatic ductal adenocarcinoma. Furthermore, our study showed that elevated levels of NAT10 expression correlated with gemcitabine resistance, that aberrant NAT10 expression may promote the angiogenic capacity of pancreatic ductal adenocarcinoma through activation of the TGF-β pathway, which in turn promotes distal metastasis of pancreatic ductal adenocarcinoma, and that NAT10 knockdown significantly inhibited the migration and clonogenic capacity of pancreatic ductal adenocarcinoma cells. In conclusion, we proposed a predictive model based on NAT10 expression levels, a non-invasive predictive approach for genomic profiling, which showed satisfactory and effective performance in predicting patients' survival outcomes and treatment response. Medicine and electronics will be combined in more interdisciplinary areas in the future.

Keywords:  NAT10; ac4C; bioinformatics; pancreatic ductal adenocarcinoma; tumor environment

DOI:  https://doi.org/10.3389/fimmu.2023.1133166
Ecotoxicol Environ Saf. 2023 Apr 01. pii: S0147-6513(23)00254-3. [Epub ahead of print]254 114750

Exosomal circCLIP1 regulates PM2.5-induced airway obstruction via targeting SEPT10 in vitro.

Huanhuan Zhu, Xiying Tang, Huilin Zhang, Meiyu Zhou, Hanting Liu, Haiyan Chu, Zhengdong Zhang.

  Fine particulate matter (PM2.5) exposure correlates with airway obstruction, but the mechanism remains to be fully elucidated. We aim to investigate the role of exosomal circular RNAs (circRNAs)-mediated communication between airway epithelial cells and airway smooth muscle cells in PM2.5-induced airway obstruction. RNA sequencing revealed that acute PM2.5 exposure altered the expression profiles of 2904 exosomal circRNAs. Among them, exosomal hsa_circ_0029069 (spliced from CLIP1, thus termed circCLIP1 hereafter) with a loop structure was upregulated by PM2.5 exposure and mainly encapsulated in exosomes. Then, the biological functions and the underlying mechanisms were explored by Western blot, RNA immunoprecipitation and RNA pull-down, etc. Phenotypically, exosomal circCLIP1 entered recipient cells, inducing mucus secretion in recipient HBE cells and contractility of sensitive HBSMCs. Mechanistically, circCLIP1 was upregulated by METTL3-mediated N6-methyladenine (m6A) modification in PM2.5-treated producer HBE cells and exosomes, then enhancing the expression of SEPT10 in recipient HBE cells and sensitive HBSMCs. Our study revealed that exosomal circCLIP1 played a critical role in PM2.5-induced airway obstruction and provided a new potential biomarker for the assessment of PM2.5-related adverse effects.

Keywords:  Contractility; Exosome; Fine particulate matter; Mucus secretion; circCLIP1

DOI:  https://doi.org/10.1016/j.ecoenv.2023.114750
Neoplasma. 2023 Mar 24. pii: 220828N871. [Epub ahead of print]

High expression of WTAP is related to poor prognosis in nasopharyngeal carcinoma.

Chang-Juan Tao, Peng Zhang, Yuan-Yuan Chen, Ming Chen.

Wilms' tumor 1-associated protein (WTAP), a component of the m6A methyltransferase complex, recruits the m6A methyltransferases METTL3 and METTL14 to the corresponding mRNA targets to participate in the formation of N6-methyladenosine. However, the molecular mechanism of WTAP in the tumorigenesis and progression of nasopharyngeal carcinoma (NPC) remains unclear. This study aimed to explore the prognostic value and biological function of WTAP in NPC. We assessed WTAP expression and its prognostic significance using microarray datasets from the Gene Expression Omnibus (GSE12452) database and 100 NPC tissues via bioinformatics analysis and immunohistochemistry (IHC), respectively. Moreover, gene ontology (GO) and gene set enrichment analysis (GSEA) were performed. In addition, the correlation of WTAP expression with the expression of immune cell biomarkers was analyzed. The results showed that WTAP expression was significantly overexpressed in NPC tissues in GSE12452. The overexpression of WTAP was validated by the external datasets including NPC tissues (GSE150430) and NPC cell lines (GSE39826). GO analysis suggested enrichment in the nucleoplasm (cellular component) and cell cycle (biological process). The GSEA revealed that differentially expressed genes were enriched in E2F-targets, Myc_targets_v1, G2M checkpoint, Myc_targets_v2, and Interferon-alpha-response. In IHC analysis, WTAP was upregulated in NPC tissues, and high levels of WTAP expression were significantly correlated with advanced T stage (p = 0.047) and advanced N stage (p = 0.018). Cox regression demonstrated that WTAP overexpression was an independent biomarker of poor prognosis for overall survival (hazard ratio [HR], 4.747; 95% confidence interval [CI], 1.671-13.482; p = 0.003). In IHC analysis, the expression of WTAP was positively correlated with CD206 (biomarker for M2 macrophages) (p = 0.018) but negatively correlated with CD8a (biomarker for cytotoxic T cells) (p = 0.001). In conclusion, WTAP is a promising prognostic biomarker and may participate in the regulation of immune cell infiltration in NPC.

DOI: https://doi.org/10.4149/neo_2023_220828N871
J Biomed Sci. 2023 Mar 23. 30(1): 20

PES1 reduces CD8+ T cell infiltration and immunotherapy sensitivity via interrupting ILF3-IL15 complex in esophageal squamous cell carcinoma.

Ning Ma, Rong Hua, Yang Yang, Zhi-Chao Liu, Jie Pan, Bo-Yao Yu, Yi-Feng Sun, Dong Xie, Yan Wang, Zhi-Gang Li.

   BACKGROUND: Although immune checkpoint blockade (ICB) therapy has brought survival benefits to patients with specific cancer types, most of cancer patients remain refractory to the ICB therapy, which is largely attributed to the immunosuppressive tumor microenvironment. Thereby, it is urgent to profile key molecules and signal pathways responsible for modification of tumor microenvironment.
METHODS: Multiple databases of esophageal squamous cell carcinoma (ESCC) were integratively analyzed to screen candidate genes responsible for infiltration of CD8+ T cells. Expression of pescadillo ribosomal biogenesis factor 1 (PES1) in clinical ESCC samples was examined by qRT-PCR, western blotting, and immunohistochemistry. The mechanisms of PES1 were investigated via RNA sequencing and mass spectrometry followed by immunoprecipitation and proximity ligation assay. The clinical and therapeutic significance of PES1 in ESCC was comprehensively investigated using ESCC cells and mouse model.
RESULTS: PES1 was significantly upregulated and correlated with poor prognosis in ESCC patients. PES1 knockdown decreased ESCC cell growth in vitro and in vivo and enhanced the efficacy of ICB therapy in mouse model, which was established through subcutaneous inoculation with ESCC cells. Analyses on RNA sequencing and mass spectrometry suggested that PES1 expression was negatively correlated with IL15 and ILF3 was one of the PES1-associated proteins. It has been known that ILF3 interacts with and stabilizes IL15 mRNA to increase IL15 protein level. Our data further indicated that PES1 interfered with the interaction between ILF3 and IL15 mRNA and impaired ILF3-mediated stabilization of IL15 mRNA, which eventually reduced the protein level of IL15. Interestingly, the inhibitory effect of ICB therapy boosted by PES1 knockdown dramatically antagonized by knockdown of IL15, which suppressed the tumor-infiltrated CD8+ T cells in ESCC. Finally, we confirmed the relationships among PES1, IL15, and CD8+ T cell infiltration in 10 locally advanced ESCC patients receiving ICB neoadjuvant therapy and demonstrated that ICB therapy would be more effective in those with low expression of PES1.
CONCLUSIONS: Altogether, our findings herein provided novel insights on biological function and clinical significance of PES1 and suggested that high expression of PES1 could suppress ILF3-IL15 axis-mediated immunosurveillance and promote resistance to ICB through restraining tumor-infiltrated CD8+ T cells.

Keywords:  CD8+ cytotoxic T lymphocytes; IL15; ILF3; PD-1; PES1

DOI:  https://doi.org/10.1186/s12929-023-00912-8
J Biol Chem. 2023 Mar 16. pii: S0021-9258(23)00254-5. [Epub ahead of print] 104612

Molecular mechanism of tRNA binding by the Escherichia coli N7 guanosine methyltransferase TrmB.

SarahK Schultz, Kieran Meadows, Ute Kothe.

  Among the large and diverse collection of tRNA modifications, 7-methylguanosine (m7G) is frequently found in the tRNA variable loop at position 46. This modification is introduced by the TrmB enzyme, which is conserved in bacteria and eukaryotes. However, the molecular determinants and the mechanism for tRNA recognition by TrmB are not well understood. Complementing the report of various phenotypes for different organisms lacking TrmB homologs, we report here hydrogen peroxide sensitivity for the Escherichia coli ΔtrmB knockout strain. To gain insight into the molecular mechanism of tRNA binding by E. coli TrmB in real-time, we developed a new assay based on introducing a 4-thiouridine modification at position 8 of in vitro transcribed tRNAPhe enabling us to fluorescently label this unmodified tRNA. Using rapid kinetic stopped-flow measurements with this fluorescent tRNA, we examined the interaction of wildtype and single substitution variants of TrmB with tRNA. Our results reveal the role of SAM for rapid and stable tRNA binding, the rate-limiting nature of m7G46 catalysis for tRNA release, and the importance of residues R26, T127 and R155 across the entire surface of TrmB for tRNA binding.

Keywords:  7-methylguanosine; RNA binding protein; RNA methylation; RNA modification; enzyme kinetics; fluorescence; pre-steady-state kinetics; transfer RNA (tRNA)

DOI:  https://doi.org/10.1016/j.jbc.2023.104612
Ann N Y Acad Sci. 2023 Mar 18.

Tet methylcytosine dioxygenase 1 modulates Porphyromonas gingivalis-triggered pyroptosis by regulating glycolysis in cementoblasts.

Yan Peng, Huiyi Wang, Xin Huang, Heyu Liu, Junhong Xiao, Chuan Wang, Li Ma, Xiaoxuan Wang, Zhengguo Cao.

  Porphyromonas gingivalis is involved in the pathogenesis of multiple polymicrobial biofilm-induced inflammatory diseases, including apical periodontitis, and it triggers pyroptosis accompanied by robust inflammatory responses. Tet methylcytosine dioxygenase 1 (TET1), an epigenetic modifier enzyme, has been is correlated with inflammation, though an association of TET1 and P. gingivalis-related pyroptosis in cementoblasts and the molecular mechanisms has not been shown. Our study here demonstrated that P. gingivalis downregulated Tet1 expression and elicited CASP11- and GSDMD-dependent pyroptosis. Additionally, Tet1 mRNA silencing in cementoblasts appeared to result in a more severe pyroptotic phenotype, where levels of CASP11 and GSDMD cleavage, lactate dehydrogenase release, and IL-1β and IL-18 production were significantly increased. Moreover, Tet1 overexpression resulted in blockade of pyroptosis activation accompanied by inflammation moderation. Further analyses revealed that TET1 modulated glycolysis, confirmed by the application of the specific inhibitor 2-deoxy-d-glucose (2-DG). The pyroptosis phenotype enhanced by Tet1 silencing was moderated by 2-DG upon P. gingivalis invasion. Taken together, these data show the effects and underlying mechanisms of TET1 on pyroptosis and inflammatory phenotype induced by P. gingivalis in cementoblasts, and provides insight into the involvement of P. gingivalis in apical periodontitis and, possibly, other inflammatory diseases.

Keywords:  Porphyromonas gingivalis; cementoblast; glycolysis; pyroptosis; tet methylcytosine dioxygenase 1

DOI:  https://doi.org/10.1111/nyas.14979
Mol Cell. 2023 Mar 08. pii: S1097-2765(23)00151-X. [Epub ahead of print]

m6A-driven SF3B1 translation control steers splicing to direct genome integrity and leukemogenesis.

Maciej Cieśla, Phuong Cao Thi Ngoc, Sowndarya Muthukumar, Gabriele Todisco, Magdalena Madej, Helena Fritz, Marios Dimitriou, Danny Incarnato, Eva Hellström-Lindberg, Cristian Bellodi.

  SF3B1 is the most mutated splicing factor (SF) in myelodysplastic syndromes (MDSs), which are clonal hematopoietic disorders with variable risk of leukemic transformation. Although tumorigenic SF3B1 mutations have been extensively characterized, the role of "non-mutated" wild-type SF3B1 in cancer remains largely unresolved. Here, we identify a conserved epitranscriptomic program that steers SF3B1 levels to counteract leukemogenesis. Our analysis of human and murine pre-leukemic MDS cells reveals dynamic regulation of SF3B1 protein abundance, which affects MDS-to-leukemia progression in vivo. Mechanistically, ALKBH5-driven 5' UTR m6A demethylation fine-tunes SF3B1 translation directing splicing of central DNA repair and epigenetic regulators during transformation. This impacts genome stability and leukemia progression in vivo, supporting an integrative analysis in humans that SF3B1 molecular signatures may predict mutational variability and poor prognosis. These findings highlight a post-transcriptional gene expression nexus that unveils unanticipated SF3B1-dependent cancer vulnerabilities.

Keywords:  ALKBH5; MYC; SF3B1; acute myeloid leukemia; alternative splicing; genome integrity; m(6)A; myelodysplastic syndromes; p53; translation

DOI:  https://doi.org/10.1016/j.molcel.2023.02.024
iScience. 2023 Apr 21. 26(4): 106319

Systematic analysis of the aberrances and functional implications of cuproptosis in cancer.

Jiangbing Li, Boyan Li, Rongrong Zhao, Gang Li.

  Cuproptosis is a novel form of cell death driven by a copper-dependent proteotoxic stress response whose comprehensive landscape in tumors remains unclear. Here, we comprehensively characterized cuproptosis-related genes (CRGs) across 33 cancers using multi-omic data from The Cancer Genome Atlas (TCGA), showing complicated and diverse results in different cancers. We also explored the relationships between CRGs and cancer metabolic patterns, pathway activity, and tumor microenvironment (TME), suggesting that they played critical roles in tumor progression and TME cell infiltration. We further established the cuproptosis potential index (CPI) to reveal the functional roles of cuproptosis, and characterized multi-omic molecular features associated with cuproptosis. In clinical applications, we performed a combined analysis of the sensitivity of CRGs and CPI to drug response and immunotherapy. This study provides a rich resource for understanding cuproptosis, offering a broad molecular perspective for future functional and therapeutic studies of multiple cancer pathways mediated by cuproptosis.

Keywords:  Cancer systems biology; Cell biology; Omics

DOI:  https://doi.org/10.1016/j.isci.2023.106319
Oncol Lett. 2023 Apr;25(4): 169

Expression pattern, tumor immune landscape, and prognostic value of N7‑methylguanosine regulators in bladder urothelial carcinoma.

Chi Zhang, Jiangnan Xia, Simiao Zhang, Jing Li, Tian Zhou, Kaiwen Hu.

  N7-Methylguanosine (m7G) modification is important in post-transcriptional regulation. dysregulation of m7G RNA modification has been reported to be markedly associated with cancer. However, its importance in bladder urothelial carcinoma (BLCA) remains poorly characterized. The present study systematically analyzed mRNA gene expression data and clinical information from The Cancer Genome Atlas and further constructed robust risk signatures for the four regulators of m7G RNA modification (nudix hydrolase 11, gem nuclear organelle-associated protein 5, eukaryotic translation initiation factor 3 subunit D and cytoplasmic FMR1 interacting protein 1). The differential expression and cell function of m7G-related genes in bladder cancer cells were verified by reverse transcription-quantitative PCR, Cell Counting Kit-8 and colony formation assays. The four-gene-based model could accurately predict the prognosis of BLCA. Nomogram-based clinical decisions had a higher net benefit compared with that of individual predictors. Through immune infiltration analysis, it was found that immune cell infiltration affected the prognosis of patients with BLCA. Finally, the present study identified potential therapeutics that differ between high and low-risk groups based on four genes. In summary, the current findings revealed an essential role for m7G RNA modification regulators in BLCA, and developed risk signatures as promising prognostic markers in patients with BLCA.

Keywords:  N7-methyladenosine; bioinformatics; bladder cancer; immune infiltration; prognostic model

DOI:  https://doi.org/10.3892/ol.2023.13755