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1.
J Exp Clin Cancer Res ; 43(1): 154, 2024 May 31.
Article in English | MEDLINE | ID: mdl-38822363

ABSTRACT

BACKGROUND: RNA modifications of transfer RNAs (tRNAs) are critical for tRNA function. Growing evidence has revealed that tRNA modifications are related to various disease processes, including malignant tumors. However, the biological functions of methyltransferase-like 1 (METTL1)-regulated m7G tRNA modifications in breast cancer (BC) remain largely obscure. METHODS: The biological role of METTL1 in BC progression were examined by cellular loss- and gain-of-function tests and xenograft models both in vitro and in vivo. To investigate the change of m7G tRNA modification and mRNA translation efficiency in BC, m7G-methylated tRNA immunoprecipitation sequencing (m7G tRNA MeRIP-seq), Ribosome profiling sequencing (Ribo-seq), and polysome-associated mRNA sequencing were performed. Rescue assays were conducted to decipher the underlying molecular mechanisms. RESULTS: The tRNA m7G methyltransferase complex components METTL1 and WD repeat domain 4 (WDR4) were down-regulated in BC tissues at both the mRNA and protein levels. Functionally, METTL1 inhibited BC cell proliferation, and cell cycle progression, relying on its enzymatic activity. Mechanistically, METTL1 increased m7G levels of 19 tRNAs to modulate the translation of growth arrest and DNA damage 45 alpha (GADD45A) and retinoblastoma protein 1 (RB1) in a codon-dependent manner associated with m7G. Furthermore, in vivo experiments showed that overexpression of METTL1 enhanced the anti-tumor effectiveness of abemaciclib, a cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor. CONCLUSION: Our study uncovered the crucial tumor-suppressive role of METTL1-mediated tRNA m7G modification in BC by promoting the translation of GADD45A and RB1 mRNAs, selectively blocking the G2/M phase of the cell cycle. These findings also provided a promising strategy for improving the therapeutic benefits of CDK4/6 inhibitors in the treatment of BC patients.


Subject(s)
Breast Neoplasms , Methyltransferases , RNA, Transfer , Humans , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Female , Mice , Animals , Methyltransferases/metabolism , Methyltransferases/genetics , RNA, Transfer/genetics , RNA, Transfer/metabolism , Methylation , Cell Line, Tumor , Cell Proliferation , Carcinogenesis/genetics , Cell Cycle Checkpoints , Protein Biosynthesis , Xenograft Model Antitumor Assays , Mice, Nude
3.
Theor Appl Genet ; 137(7): 146, 2024 Jun 04.
Article in English | MEDLINE | ID: mdl-38834825

ABSTRACT

KEY MESSAGE: The major QTL Sdp1.1+ controlling seed dormancy in cowpea was finely mapped, and two CCoAOMT1 genes were identified as candidate genes for the dormancy. Seed dormancy in wild cowpea may be useful in breeding cultivated cowpea with pre-harvest sprouting resistance. A previous study identified a major quantitative trait locus (QTL) for seed dormancy, Sdp1.1+ , using the population of the cross between cultivated cowpea 'JP81610' and wild cowpea 'JP89083.' However, the molecular basis of seed dormancy in cowpea is not yet known. In this study, we aimed to finely map the locus Sdp1.1+ and identify candidate gene(s) for it. Germination tests demonstrated that the seed coat is the major factor controlling seed dormancy in the wild cowpea JP89083. Microscopic observations revealed that wild cowpea seeds, unlike cultivated cowpea seeds, possessed a palisade cuticle layer. Fine mapping using a large F2 population of the cross JP81610 × JP89083 grown in Thailand revealed a single QTL, Sdp1.1+ , controlling seed dormancy. The Sdp1.1+ was confirmed using a small F2 population of the same cross grown in Japan. The Sdp1.1+ was mapped to a 37.34-Kb region containing three genes. Two closely linked genes, Vigun03g278900 (VuCCoAOMT1a) and Vigun03g290000 (VuCCoAOMT1b), located 4.844 Kb apart were considered as candidate genes for seed dormancy. The two genes encoded caffeoyl coenzyme A O-methyltransferase 1 (CCoAOMT1). DNA sequencing and alignment of VuCCoAOMT1a and VuCCoAOMT1b between JP89083 and JP81610 revealed a single nucleotide polymorphism (SNP) causing an amino acid change in VuCCoAOMT1a and several SNPs leading to six amino acid changes in VuCCoAOMT1b. Altogether, these results indicate that VuCCoAOMT1a and VuCCoAOMT1b are candidate genes controlling physical seed dormancy in the wild cowpea JP89083.


Subject(s)
Chromosome Mapping , Germination , Methyltransferases , Plant Dormancy , Quantitative Trait Loci , Seeds , Vigna , Plant Dormancy/genetics , Vigna/genetics , Vigna/growth & development , Vigna/physiology , Seeds/genetics , Seeds/growth & development , Methyltransferases/genetics , Methyltransferases/metabolism , Germination/genetics , Genes, Plant , Phenotype , Plant Proteins/genetics , Plant Proteins/metabolism
4.
Breast Cancer Res ; 26(1): 94, 2024 Jun 06.
Article in English | MEDLINE | ID: mdl-38844963

ABSTRACT

BACKGROUND: RNA m5C methylation has been extensively implicated in the occurrence and development of tumors. As the main methyltransferase, NSUN2 plays a crucial regulatory role across diverse tumor types. However, the precise impact of NSUN2-mediated m5C modification on breast cancer (BC) remains unclear. Our study aims to elucidate the molecular mechanism underlying how NSUN2 regulates the target gene HGH1 (also known as FAM203) through m5C modification, thereby promoting BC progression. Additionally, this study targets at preliminarily clarifying the biological roles of NSUN2 and HGH1 in BC. METHODS: Tumor and adjacent tissues from 5 BC patients were collected, and the m5C modification target HGH1 in BC was screened through RNA sequencing (RNA-seq) and single-base resolution m5C methylation sequencing (RNA-BisSeq). Methylation RNA immunoprecipitation-qPCR (MeRIP-qPCR) and RNA-binding protein immunoprecipitation-qPCR (RIP-qPCR) confirmed that the methylation molecules NSUN2 and YBX1 specifically recognized and bound to HGH1 through m5C modification. In addition, proteomics, co-immunoprecipitation (co-IP), and Ribosome sequencing (Ribo-Seq) were used to explore the biological role of HGH1 in BC. RESULTS: As the main m5C methylation molecule, NSUN2 is abnormally overexpressed in BC and increases the overall level of RNA m5C. Knocking down NSUN2 can inhibit BC progression in vitro or in vivo. Combined RNA-seq and RNA-BisSeq analysis identified HGH1 as a potential target of abnormal m5C modifications. We clarified the mechanism by which NSUN2 regulates HGH1 expression through m5C modification, a process that involves interactions with the YBX1 protein, which collectively impacts mRNA stability and protein synthesis. Furthermore, this study is the first to reveal the binding interaction between HGH1 and the translation elongation factor EEF2, providing a comprehensive understanding of its ability to regulate transcript translation efficiency and protein synthesis in BC cells. CONCLUSIONS: This study preliminarily clarifies the regulatory role of the NSUN2-YBX1-m5C-HGH1 axis from post-transcriptional modification to protein translation, revealing the key role of abnormal RNA m5C modification in BC and suggesting that HGH1 may be a new epigenetic biomarker and potential therapeutic target for BC.


Subject(s)
Breast Neoplasms , Disease Progression , Gene Expression Regulation, Neoplastic , Methyltransferases , RNA Stability , Y-Box-Binding Protein 1 , Humans , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Female , Methylation , Methyltransferases/metabolism , Methyltransferases/genetics , Y-Box-Binding Protein 1/metabolism , Y-Box-Binding Protein 1/genetics , Mice , Animals , Cell Line, Tumor , RNA, Messenger/genetics , RNA, Messenger/metabolism , Cell Proliferation
5.
Microb Cell Fact ; 23(1): 162, 2024 Jun 01.
Article in English | MEDLINE | ID: mdl-38824548

ABSTRACT

BACKGROUND: Syringic acid (SA) is a high-value natural compound with diverse biological activities and wide applications, commonly found in fruits, vegetables, and herbs. SA is primarily produced through chemical synthesis, nonetheless, these chemical methods have many drawbacks, such as considerable equipment requirements, harsh reaction conditions, expensive catalysts, and numerous by-products. Therefore, in this study, a novel biotransformation route for SA production was designed and developed by using engineered whole cells. RESULTS: An O-methyltransferase from Desulfuromonas acetoxidans (DesAOMT), which preferentially catalyzes a methyl transfer reaction on the meta-hydroxyl group of catechol analogues, was identified. The whole cells expressing DesAOMT can transform gallic acid (GA) into SA when S-adenosyl methionine (SAM) is used as a methyl donor. We constructed a multi-enzyme cascade reaction in Escherichia coli, containing an endogenous shikimate kinase (AroL) and a chorismate lyase (UbiC), along with a p-hydroxybenzoate hydroxylase mutant (PobA**) from Pseudomonas fluorescens, and DesAOMT; SA was biosynthesized from shikimic acid (SHA) by using whole cells catalysis. The metabolic system of chassis cells also affected the efficiency of SA biosynthesis, blocking the chorismate metabolism pathway improved SA production. When the supply of the cofactor NADPH was optimized, the titer of SA reached 133 µM (26.2 mg/L). CONCLUSION: Overall, we designed a multi-enzyme cascade in E. coli for SA biosynthesis by using resting or growing whole cells. This work identified an O-methyltransferase (DesAOMT), which can catalyze the methylation of GA to produce SA. The multi-enzyme cascade containing four enzymes expressed in an engineered E. coli for synthesizing of SA from SHA. The metabolic system of the strain and biotransformation conditions influenced catalytic efficiency. This study provides a new green route for SA biosynthesis.


Subject(s)
Biocatalysis , Escherichia coli , Gallic Acid , Metabolic Engineering , Gallic Acid/metabolism , Gallic Acid/analogs & derivatives , Escherichia coli/metabolism , Escherichia coli/genetics , Metabolic Engineering/methods , Methyltransferases/metabolism , Methyltransferases/genetics , Shikimic Acid/metabolism , Pseudomonas fluorescens/metabolism , Pseudomonas fluorescens/enzymology , Pseudomonas fluorescens/genetics , Biotransformation
6.
Oncol Res ; 32(6): 1047-1061, 2024.
Article in English | MEDLINE | ID: mdl-38827317

ABSTRACT

Background: Colorectal cancer (CRC) is one of the most frequently diagnosed cancers. In many cases, the poor prognosis of advanced CRC is associated with resistance to treatment with chemotherapeutic drugs such as 5-Fluorouracil (5-FU). The epithelial-to-mesenchymal transition (EMT) and dysregulation in protein methylation are two mechanisms associated with chemoresistance in many cancers. This study looked into the effect of 5-FU dose escalation on EMT and protein methylation in CRC. Materials and Methods: HCT-116, Caco-2, and DLD-1 CRC cell lines were exposed to dose escalation treatment of 5-FU. The motility and invasive potentials of the cells before and after treatment with 5-FU were investigated through wound healing and invasion assays. This was followed by a Western blot which analyzed the protein expressions of the epithelial marker E-cadherin, mesenchymal marker vimentin, and the EMT transcription factor (EMT-TF), the snail family transcriptional repressor 1 (Snail) in the parental and desensitized cells. Western blotting was also conducted to study the protein expressions of the protein methyltransferases (PMTs), Euchromatic histone lysine methyltransferase 2 (EHMT2/G9A), protein arginine methyltransferase (PRMT5), and SET domain containing 7/9 (SETD7/9) along with the global lysine and arginine methylation profiles. Results: The dose escalation method generated 5-FU desensitized CRC cells with distinct morphological features and increased tolerance to high doses of 5-FU. The 5-FU desensitized cells experienced a decrease in migration and invasion when compared to the parental cells. This was reflected in the observed reduction in E-cadherin, vimentin, and Snail in the desensitized cell lines. Additionally, the protein expressions of EHMT2/G9A, PRMT5, and SETD7/9 also decreased in the desensitized cells and global protein lysine and arginine methylation became dysregulated with 5-FU treatment. Conclusion: This study showed that continuous, dose-escalation treatment of 5-FU in CRC cells generated 5-FU desensitized cancer cells that seemed to be less aggressive than parental cells.


Subject(s)
Cell Movement , Colorectal Neoplasms , Epithelial-Mesenchymal Transition , Fluorouracil , Humans , Fluorouracil/pharmacology , Fluorouracil/administration & dosage , Epithelial-Mesenchymal Transition/drug effects , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Colorectal Neoplasms/metabolism , Cell Movement/drug effects , Cell Line, Tumor , Antimetabolites, Antineoplastic/pharmacology , Drug Resistance, Neoplasm , Dose-Response Relationship, Drug , Methyltransferases/metabolism , Methyltransferases/genetics , Gene Expression Regulation, Neoplastic/drug effects , Methylation , Protein-Arginine N-Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/genetics
7.
FASEB J ; 38(11): e23729, 2024 Jun 15.
Article in English | MEDLINE | ID: mdl-38847786

ABSTRACT

Diabetic nephropathy (DN) is one of the common microvascular complications in diabetic patients. Marrow mesenchymal stem cells (MSCs) have attracted attention in DN therapy but the underlying mechanism remains unclear. Here, we show that MSC administration alleviates high glucose (HG)-induced human kidney tubular epithelial cell (HK-2 cell) injury and ameliorates renal injury in DN mice. We identify that Smad2/3 is responsible for MSCs-regulated DN progression. The activity of Smad2/3 was predominantly upregulated in HG-induced HK-2 cell and DN mice and suppressed with MSC administration. Activation of Smad2/3 via transforming growth factor-ß1 (TGF-ß1) administration abrogates the protective effect of MSCs on HG-induced HK-2 cell injury and renal injury of DN mice. Smad2/3 has been reported to interact with methyltransferase of N6-methyladenosine (m6A) complex and we found a methyltransferase, Wilms' tumor 1-associating protein (WTAP), is involved in MSCs-Smad2/3-regulated DN development. Moreover, WTAP overexpression abrogates the improvement of MSCs on HG-induced HK-2 cell injury and renal injury of DN mice. Subsequently, α-enolase (ENO1) is the downstream target of WTAP-mediated m6A modification and contributes to the MSCs-mediated regulation. Collectively, these findings reveal a molecular mechanism in DN progression and indicate that Smad2/3/WTAP/ENO1 may present a target for MSCs-mediated DN therapy.


Subject(s)
Diabetic Nephropathies , Mesenchymal Stem Cells , Smad2 Protein , Smad3 Protein , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/pathology , Animals , Mesenchymal Stem Cells/metabolism , Smad2 Protein/metabolism , Mice , Humans , Smad3 Protein/metabolism , Male , Mice, Inbred C57BL , Adenosine/metabolism , Adenosine/analogs & derivatives , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Signal Transduction , Methyltransferases/metabolism , Methyltransferases/genetics , Mesenchymal Stem Cell Transplantation/methods , Transforming Growth Factor beta1/metabolism , Cell Line
8.
Int J Biol Sci ; 20(7): 2491-2506, 2024.
Article in English | MEDLINE | ID: mdl-38725850

ABSTRACT

Colon inflammation is characterized by disturbances in the intestinal microbiota and inflammation. Melatonin (Mel) can improve colon inflammation. However, the underlying mechanism remains unclear. Recent studies suggest that m6A methylation modification may play an important role in inflammatory responses. This study aimed to explore the effects of melatonin and LPS-mediated m6A methylation on colon inflammation. Our study found that melatonin inhibits M1 macrophages, activates M2 macrophages, inhibit the secretion of pro-inflammatory factors, maintain colon homeostasis and improves colon inflammation through MTNR1B. In addition, the increased methylation level of m6A is associated with the occurrence of colon inflammation, and melatonin can also reduce the level of colon methylation to improve colon inflammation. Among them, the main methylated protein METTL3 can be inhibited by melatonin through MTNR1B. In a word, melatonin regulates m6A methylation by improving abnormal METTL3 protein level to reshape the microflora and activate macrophages to improve colon inflammation, mainly through MTNR1B.


Subject(s)
Adenosine , Lipopolysaccharides , Macrophages , Melatonin , Melatonin/pharmacology , Melatonin/metabolism , Animals , Mice , Adenosine/metabolism , Adenosine/analogs & derivatives , Adenosine/pharmacology , Methylation/drug effects , Macrophages/metabolism , Macrophages/drug effects , Methyltransferases/metabolism , Methyltransferases/genetics , Inflammation/metabolism , Colon/metabolism , Colon/drug effects , Male , Mice, Inbred C57BL , Colitis/chemically induced , Colitis/metabolism , Receptor, Melatonin, MT2/metabolism , Receptor, Melatonin, MT2/genetics , RAW 264.7 Cells
9.
Clin Exp Pharmacol Physiol ; 51(7): e13868, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38745265

ABSTRACT

Cervical cancer (CC) is a gynaecological malignancy tumour that seriously threatens women's health. Recent evidence has identified that interferon regulatory factor 5 (IRF5), a nucleoplasm shuttling protein, is a pivotal transcription factor regulating the growth and metastasis of various human tumours. This study aimed to investigate the function and molecular basis of IRF5 in CC development. IRF5, protein phosphatase 6 catalytic subunit (PPP6C) and methyltransferase-like 3 (METTL3) mRNA levels were evaluated by quantitative real-time (qRT)-polymerase chain reaction (PCR). IRF5, PPP6C, METTL3, B-cell lymphoma 2 and Bax protein levels were detected using western blot. Cell proliferation, migration, invasion, angiogenesis and apoptosis were determined by using colony formation, 5-ethynyl-2'-deoxyuridine (EdU), transwell, tube formation assay and flow cytometry assay, respectively. Glucose uptake and lactate production were measured using commercial kits. Xenograft tumour assay in vivo was used to explore the role of IRF5. After JASPAR predication, binding between IRF5 and PPP6C promoter was verified using chromatin immunoprecipitation and dual-luciferase reporter assays. Moreover, the interaction between METTL3 and IRF5 was verified using methylated RNA immunoprecipitation (MeRIP). IRF5, PPP6C and METTL3 were highly expressed in CC tissues and cells. IRF5 silencing significantly inhibited cell proliferation, migration, invasion, angiogenesis and glycolytic metabolism in CC cells, while induced cell apoptosis. Furthermore, the absence of IRF5 hindered tumour growth in vivo. At the molecular level, IRF5 might bind with PPP6C to positively regulate the expression of PPP6C mRNA. Meanwhile, IRF5 was identified as a downstream target of METTL3-mediated m6A modification. METTL3-mediated m6A modification of mRNA might promote CC malignant progression by regulating PPP6C, which might provide a promising therapeutic target for CC treatment.


Subject(s)
Cell Proliferation , Disease Progression , Interferon Regulatory Factors , Methyltransferases , Up-Regulation , Uterine Cervical Neoplasms , Humans , Female , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/metabolism , Methyltransferases/genetics , Methyltransferases/metabolism , Interferon Regulatory Factors/genetics , Interferon Regulatory Factors/metabolism , Cell Line, Tumor , Animals , Cell Proliferation/genetics , Mice , Gene Expression Regulation, Neoplastic , Apoptosis/genetics , Cell Movement/genetics , Mice, Nude , Neoplasm Invasiveness , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/pathology , Neovascularization, Pathologic/metabolism
10.
Nat Commun ; 15(1): 3991, 2024 May 11.
Article in English | MEDLINE | ID: mdl-38734724

ABSTRACT

Citrus reticulata cv. Chachiensis (CRC) is an important medicinal plant, its dried mature peels named "Guangchenpi", has been used as a traditional Chinese medicine to treat cough, indigestion, and lung diseases for several hundred years. However, the biosynthesis of the crucial natural products polymethoxylated flavonoids (PMFs) in CRC remains unclear. Here, we report a chromosome-scale genome assembly of CRC with the size of 314.96 Mb and a contig N50 of 16.22 Mb. Using multi-omics resources, we discover a putative caffeic acid O-methyltransferase (CcOMT1) that can transfer a methyl group to the 3-hydroxyl of natsudaidain to form 3,5,6,7,8,3',4'-heptamethoxyflavone (HPMF). Based on transient overexpression and virus-induced gene silencing experiments, we propose that CcOMT1 is a candidate enzyme in HPMF biosynthesis. In addition, a potential gene regulatory network associated with PMF biosynthesis is identified. This study provides insights into PMF biosynthesis and may assist future research on mining genes for the biosynthesis of plant-based medicines.


Subject(s)
Citrus , Flavonoids , Methyltransferases , Citrus/genetics , Citrus/metabolism , Flavonoids/biosynthesis , Flavonoids/metabolism , Methyltransferases/metabolism , Methyltransferases/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Gene Expression Regulation, Plant , Genome, Plant , Gene Regulatory Networks , Multiomics
11.
Cell Death Dis ; 15(5): 338, 2024 May 14.
Article in English | MEDLINE | ID: mdl-38744809

ABSTRACT

Epitranscriptomic RNA modifications are crucial for the maintenance of glioma stem cells (GSCs), the most malignant cells in glioblastoma (GBM). 3-methylcytosine (m3C) is a new epitranscriptomic mark on RNAs and METTL8 represents an m3C writer that is dysregulated in cancer. Although METTL8 has an established function in mitochondrial tRNA (mt-tRNA) m3C modification, alternative splicing of METTL8 can also generate isoforms that localize to the nucleolus where they may regulate R-loop formation. The molecular basis for METTL8 dysregulation in GBM, and which METTL8 isoform(s) may influence GBM cell fate and malignancy remain elusive. Here, we investigated the role of METTL8 in regulating GBM stemness and tumorigenicity. In GSC, METTL8 is exclusively localized to the mitochondrial matrix where it installs m3C on mt-tRNAThr/Ser(UCN) for mitochondrial translation and respiration. High expression of METTL8 in GBM is attributed to histone variant H2AZ-mediated chromatin accessibility of HIF1α and portends inferior glioma patient outcome. METTL8 depletion impairs the ability of GSC to self-renew and differentiate, thus retarding tumor growth in an intracranial GBM xenograft model. Interestingly, METTL8 depletion decreases protein levels of HIF1α, which serves as a transcription factor for several receptor tyrosine kinase (RTK) genes, in GSC. Accordingly, METTL8 loss inactivates the RTK/Akt axis leading to heightened sensitivity to Akt inhibitor treatment. These mechanistic findings, along with the intimate link between METTL8 levels and the HIF1α/RTK/Akt axis in glioma patients, guided us to propose a HIF1α/Akt inhibitor combination which potently compromises GSC proliferation/self-renewal in vitro. Thus, METTL8 represents a new GBM dependency that is therapeutically targetable.


Subject(s)
Glioblastoma , Hypoxia-Inducible Factor 1, alpha Subunit , Methyltransferases , Neoplastic Stem Cells , Proto-Oncogene Proteins c-akt , Humans , Glioblastoma/metabolism , Glioblastoma/pathology , Glioblastoma/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Proto-Oncogene Proteins c-akt/metabolism , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Animals , Methyltransferases/metabolism , Methyltransferases/genetics , Mice , Brain Neoplasms/pathology , Brain Neoplasms/metabolism , Brain Neoplasms/genetics , Cell Line, Tumor , Carcinogenesis/genetics , Carcinogenesis/pathology , Carcinogenesis/metabolism , Signal Transduction , RNA, Transfer/metabolism , RNA, Transfer/genetics , Mitochondria/metabolism , Gene Expression Regulation, Neoplastic , Mice, Nude , Cell Proliferation
12.
Ecotoxicol Environ Saf ; 278: 116435, 2024 Jun 15.
Article in English | MEDLINE | ID: mdl-38714084

ABSTRACT

The compound known as Sodium arsenite (NaAsO2), which is a prevalent type of inorganic arsenic found in the environment, has been strongly associated with liver fibrosis (LF), a key characteristic of nonalcoholic fatty liver disease (NAFLD), which has been demonstrated in our previous study. Our previous research has shown that exposure to NaAsO2 triggers the activation of hepatic stellate cells (HSCs), a crucial event in the development of LF. However, the molecular mechanism is still unknown. N6-methyladenosine (m6A) modification is the most crucial post-transcriptional modification in liver disease. Nevertheless, the precise function of m6A alteration in triggering HSCs and initiating LF caused by NaAsO2 remains unknown. Here, we found that NaAsO2 induced LF and HSCs activation through TGF-ß/Smad signaling, which could be reversed by TGF-ß1 knockdown. Furthermore, NaAsO2 treatment enhanced the m6A modification level both in vivo and in vitro. Significantly, NaAsO2 promoted the specific interaction of METTL14 and IGF2BP2 with TGF-ß1 and enhanced the TGF-ß1 mRNA stability. Notably, NaAsO2-induced TGF-ß/Smad pathway and HSC-t6 cells activation might be avoided by limiting METTL14/IGF2BP2-mediated m6A modification. Our findings showed that the NaAsO2-induced activation of HSCs and LF is made possible by the METTL14/IGF2BP2-mediated m6A methylation of TGF-ß1, which may open up new therapeutic options for LF brought on by environmental hazards.


Subject(s)
Adenosine , Arsenites , Hepatic Stellate Cells , Liver Cirrhosis , Sodium Compounds , Transforming Growth Factor beta1 , Arsenites/toxicity , Hepatic Stellate Cells/drug effects , Sodium Compounds/toxicity , Liver Cirrhosis/pathology , Liver Cirrhosis/chemically induced , Animals , Transforming Growth Factor beta1/metabolism , Adenosine/analogs & derivatives , Methyltransferases/genetics , Methyltransferases/metabolism , Male , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Signal Transduction/drug effects , Mice , Humans , Mice, Inbred C57BL
13.
Nat Commun ; 15(1): 4358, 2024 May 22.
Article in English | MEDLINE | ID: mdl-38778058

ABSTRACT

3C-based methods have significantly advanced our understanding of 3D genome organization. However, it remains a formidable task to precisely capture long-range chromosomal interactions between individual loci, such as those between promoters and distal enhancers. Here, we present Methyltransferase Targeting-based chromosome Architecture Capture (MTAC), a method that maps the contacts between a target site (viewpoint) and the rest of the genome in budding yeast with high resolution and sensitivity. MTAC detects hundreds of intra- and inter-chromosomal interactions within nucleosome-depleted regions (NDRs) that cannot be captured by 4C, Hi-C, or Micro-C. By applying MTAC to various viewpoints, we find that (1) most long-distance chromosomal interactions detected by MTAC reflect tethering by the nuclear pore complexes (NPCs), (2) genes co-regulated by methionine assemble into inter-chromosomal clusters near NPCs upon activation, (3) mediated by condensin, the mating locus forms a highly specific interaction with the recombination enhancer (RE) in a mating-type specific manner, and (4) correlation of MTAC signals among NDRs reveal spatial mixing and segregation of the genome. Overall, these results demonstrate MTAC as a powerful tool to resolve fine-scale long-distance chromosomal interactions and provide insights into the 3D genome organization.


Subject(s)
Chromosomes, Fungal , DNA Methylation , Nucleosomes , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Nucleosomes/metabolism , Nucleosomes/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Chromosomes, Fungal/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Chromosome Mapping/methods , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/genetics , Genome, Fungal , Promoter Regions, Genetic/genetics , Multiprotein Complexes/metabolism , Multiprotein Complexes/genetics , Nuclear Pore/metabolism , Nuclear Pore/genetics , Methyltransferases/metabolism , Methyltransferases/genetics
14.
Oncol Rep ; 51(6)2024 06.
Article in English | MEDLINE | ID: mdl-38757383

ABSTRACT

Prostate cancer (PCa) affects males of all racial and ethnic groups, and leads to higher rates of mortality in those belonging to a lower socioeconomic status due to the late detection of the disease. PCa affects middle­aged males between the ages of 45 and 60 years, and is the highest cause of cancer­associated mortality in Western countries. As the most abundant and common mRNA modification in higher eukaryotes, N6­methyladenosine (m6A) is widely distributed in mammalian cells and influences various aspects of mRNA metabolism. Recent studies have found that abnormal expression levels of various m6A regulators significantly affect the development and progression of various types of cancer, including PCa. The present review discusses the influence of m6A regulatory factors on the pathogenesis and progression of PCa through mRNA modification based on the current state of research on m6A methylation modification in PCa. It is considered that the treatment of PCa with micro­molecular drugs that target the epigenetics of the m6A regulator to correct abnormal m6A modifications is a direction for future research into current diagnostic and therapeutic approaches for PCa.


Subject(s)
Adenosine , Prostatic Neoplasms , Humans , Male , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/diagnosis , Prostatic Neoplasms/therapy , Adenosine/analogs & derivatives , Adenosine/metabolism , Adenosine/therapeutic use , Gene Expression Regulation, Neoplastic/drug effects , Epigenesis, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , Methyltransferases/metabolism , Methyltransferases/genetics
15.
FASEB J ; 38(11): e23693, 2024 Jun 15.
Article in English | MEDLINE | ID: mdl-38809685

ABSTRACT

N6-methylated adenosine (m6A) is a crucial RNA modification in eukaryotes, particularly in cancer. However, its role in cervical cancer (CC) is unclear. We aimed to elucidate the part of m6A in CC by analyzing methyltransferase-like 3 (METTL3) expression, identifying downstream targets, and exploring the underlying mechanism. We assessed METTL3 expression in CC using western blotting, quantitative polymerase chain reaction (qPCR), and immunohistochemistry. In vitro and in vivo experiments examined METTL3's role in CC. We employed RNA sequencing, methylated RNA immunoprecipitation sequencing, qPCR, and RNA immunoprecipitation qPCR to explore METTL3's mechanism in CC. METTL3 expression was upregulated in CC, promoting cell proliferation and metastasis. METTL3 knockdown inhibited human cervical cancer by inactivating AKT/mTOR signaling pathway. METTL3-mediated m6A modification was observed in CC cells, targeting phosphodiesterase 3A (PDE3A). METTL3 catalyzed m6A modification on PDE3A mRNA through YTH domain family protein 3 (YTHDF3). Our study indicated the mechanism of m6A modification in CC and suggested the METTL3/YTHDF3/PDE3A axis as a potential clinical target for CC treatment.


Subject(s)
Adenosine , Cell Proliferation , Methyltransferases , Uterine Cervical Neoplasms , Methyltransferases/metabolism , Methyltransferases/genetics , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathology , Humans , Female , Adenosine/analogs & derivatives , Adenosine/metabolism , Animals , Mice , Gene Expression Regulation, Neoplastic , Cell Line, Tumor , Mice, Nude , Signal Transduction , Mice, Inbred BALB C
16.
Zhongguo Zhong Yao Za Zhi ; 49(10): 2680-2688, 2024 May.
Article in Chinese | MEDLINE | ID: mdl-38812168

ABSTRACT

Methyleugenol is one of the main active constituents in the volatile oil of the traditional Chinese medicine Asari Radix et Rhizoma. It possesses various pharmacological effects such as analgesic, anesthetic, and anti-inflammatory properties. In biosynthesis, the initial precursor phenylalanine is finally converted into methyleugenol through a series of intermediate compounds including coniferyl acid, courmaryl acid, caffeic acid, ferulic acid/ferulic-CoA, coniferyl aldehyde, conferyl alcohol, cnfiferyl acetate, and eugenol/isoeugenol, which are produced through catalysis of a large number of enzymes. Eugenol O-methyltransferase(EOMT) is one of the key enzymes in the biosynthesis pathway, capable of methylating eugenol on the para-site hydroxyl group of the benzene ring, thereby generating methyleugenol. Here, an(iso)eugenol O-methyltransferase(IEMT) gene was cloned for the first time from Asarum siebo-ldii, holding an open reading frame that consisted of 1 113 bp and encoded a protein containing 370 amino acid residues. Bioinformatics analysis results showed that this protein was equipped with the characteristic structural domains of methyltransferases such as S-adenosylmethionine(SAM) binding sites and dimerization domains. The prokaryotic expression recombinant plasmid pET28a(+)-AsIEMT was constructed, and the candidate protein was induced and purified. In vitro enzyme assays confirmed that AsIEMT had dual functions. The enzyme could catalyze the production either of methyleugenol from eugenol or of methylisoeugenol from isoeugenol, although the latter was more prevalent. When isoeugenol was used as the substrate, the kinetics parameters K_m and V_(max) of catalytic reaction were(0.90±0.06) mmol·L~(-1) and(1.32±0.04)nmol·s~(-1)·mg~(-1), respectively. This study expanded our understandings of critical enzyme genes involved in phenylpropanoid metabolic pathways, and would facilitate the elucidation of quality formation mechanisms of the TCM Asari Radix et Rhizoma.


Subject(s)
Asarum , Eugenol , Methyltransferases , Methyltransferases/genetics , Methyltransferases/chemistry , Methyltransferases/metabolism , Eugenol/analogs & derivatives , Eugenol/metabolism , Eugenol/chemistry , Asarum/genetics , Asarum/chemistry , Asarum/enzymology , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Proteins/chemistry , Phylogeny , Amino Acid Sequence , Cloning, Molecular
17.
Int J Oral Sci ; 16(1): 42, 2024 May 23.
Article in English | MEDLINE | ID: mdl-38782892

ABSTRACT

Bisphosphonate-related osteonecrosis of jaw (BRONJ) is characterized by impaired osteogenic differentiation of orofacial bone marrow stromal cells (BMSCs). Corin has recently been demonstrated to act as a key regulator in bone development and orthopedic disorders. However, the role of corin in BRONJ-related BMSCs dysfunction remains unclarified. A m6A epitranscriptomic microarray study from our group shows that the CORIN gene is significantly upregulated and m6A hypermethylated during orofacial BMSCs osteogenic differentiation. Corin knockdown inhibits BMSCs osteogenic differentiation, whereas corin overexpression or soluble corin (sCorin) exerts a promotion effect. Furthermore, corin expression is negatively regulated by bisphosphonates (BPs). Corin overexpression or sCorin reverses BPs-impaired BMSCs differentiation ability. Mechanistically, we find altered expression of phos-ERK in corin knockdown/overexpression BMSCs and BMSCs under sCorin stimulation. PD98059 (a selective ERK inhibitor) blocks the corin-mediated promotion effect. With regard to the high methylation level of corin during osteogenic differentiation, we apply a non-selective m6A methylase inhibitor, Cycloleucine, which also blocks the corin-mediated promotion effect. Furthermore, we demonstrate that METTL7A modulates corin m6A modification and reverses BPs-impaired BMSCs function, indicating that METTL7A regulates corin expression and thus contributes to orofacial BMSCs differentiation ability. To conclude, our study reveals that corin reverses BPs-induced BMSCs dysfunction, and METTL7A-mediated corin m6A modification underlies corin promotion of osteogenic differentiation via the ERK pathway. We hope this brings new insights into future clinical treatments for BRONJ.


Subject(s)
Cell Differentiation , Mesenchymal Stem Cells , Osteogenesis , Cell Differentiation/drug effects , Osteogenesis/drug effects , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Diphosphonates/pharmacology , Humans , Methyltransferases/metabolism , Bisphosphonate-Associated Osteonecrosis of the Jaw , Animals , Up-Regulation , Blotting, Western , Cells, Cultured
18.
Biochem Biophys Res Commun ; 716: 150039, 2024 Jul 05.
Article in English | MEDLINE | ID: mdl-38701556

ABSTRACT

The objective of this study was to better characterize the role of the glutamine transporter SLC38A1 in cervical cancer and explore the underlying mechanisms. Data from public databases and clinical cervical cancer tissue samples were used to assess the expression of SLC38A1 and its prognostic significance. Immunohistochemical staining, qRT-PCR, and Western blotting were used to evaluate the expression of relevant genes and proteins. Cell viability, cell cycle, apoptosis, and intracellular glutamine content were measured using CCK-8, flow cytometry, and biochemical assays. Additionally, the RNA immunoprecipitation (RIP) assay was used to examine the impact of METTL3/IGF2BP3 on the m6A modification of the SLC38A1 3'UTR. Both cervical cancer specimens and cells showed significantly increased expression of SLC38A1 and its expression correlated with an unfavorable prognosis. Knockdown of SLC38A1 inhibited cell viability and cell cycle progression, induced apoptosis, and suppressed tumor growth in vivo. Glutaminase-1 inhibitor CB-839 reversed the effects of SLC38A1 overexpression. METTL3 promoted m6A modification of SLC38A1 and enhanced its mRNA stability through IGF2BP3 recruitment. Moreover, METTL3 silencing inhibited cell viability, cell cycle progression, intracellular glutamine content, and induced apoptosis, but these effects were reversed by SLC38A1 overexpression. In conclusion, METTL3-mediated m6A methylation of SLC38A1 stimulates cervical cancer progression. SLC38A1 inhibition is a potential therapeutic strategy for cervical cancer.


Subject(s)
Adenosine , Methyltransferases , Uterine Cervical Neoplasms , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathology , Humans , Female , Methyltransferases/metabolism , Methyltransferases/genetics , Adenosine/analogs & derivatives , Adenosine/metabolism , Adenosine/genetics , Methylation , Cell Line, Tumor , Cell Proliferation/genetics , Animals , Amino Acid Transport System A/metabolism , Amino Acid Transport System A/genetics , Apoptosis/genetics , Gene Expression Regulation, Neoplastic , Mice, Nude , Mice , Prognosis , Cell Survival/genetics
19.
Biochem Biophys Res Commun ; 716: 150011, 2024 Jul 05.
Article in English | MEDLINE | ID: mdl-38704890

ABSTRACT

Methionine adenosyltransferase 2 A (MAT2A) mediates the synthesis of methyl donor S-Adenosylmethionine (SAM), providing raw materials for methylation reactions in cells. MAT2A inhibitors are currently used for the treatment of tumors with methylthioadenosine phosphorylase (MTAP) deficiency in clinical research. Methyltransferase like 3 (METTL3) catalyzes N6-methyladenosine (m6A) modification of mRNA in mammalian cells using SAM as the substrate which has been shown to affect the tumorigenesis of non-small cell lung cancer (NSCLC) from multiple perspectives. MAT2A-induced SAM depletion may have the potential to inhibit the methyl transfer function of METTL3. Therefore, in order to expand the applicability of inhibitors, improve anti-tumor effects and reduce toxicity, the combinational effect of MAT2A inhibitor AG-270 and METTL3 inhibitor STM2457 was evaluated in NSCLC. The results showed that this combination induced cell apoptosis rather than cell cycle arrest, which was non-tissue-specific and was independent of MTAP expression status, resulting in a significant synergistic anti-tumor effect. We further elucidated that the combination-induced enhanced apoptosis was associated with the decreased m6A level, leading to downregulation of PI3K/AKT protein, ultimately activating the apoptosis-related proteins. Unexpectedly, although combination therapy resulted in metabolic recombination, no significant change in methionine metabolic metabolites was found. More importantly, the combination also exerted synergistic effects in vivo. In summary, the combination of MAT2A inhibitor and METTL3 inhibitor showed synergistic effects both in vivo and in vitro, which laid a theoretical foundation for expanding the clinical application research of the two types of drugs.


Subject(s)
Apoptosis , Carcinoma, Non-Small-Cell Lung , Drug Synergism , Lung Neoplasms , Methionine Adenosyltransferase , Methyltransferases , Methionine Adenosyltransferase/metabolism , Methionine Adenosyltransferase/antagonists & inhibitors , Methionine Adenosyltransferase/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/metabolism , Humans , Apoptosis/drug effects , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Animals , Methyltransferases/metabolism , Methyltransferases/antagonists & inhibitors , Cell Line, Tumor , Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Mice , Mice, Nude , Mice, Inbred BALB C , Xenograft Model Antitumor Assays
20.
Cancer Biol Ther ; 25(1): 2349429, 2024 Dec 31.
Article in English | MEDLINE | ID: mdl-38738555

ABSTRACT

Cervical cancer (CC) is a prevalent malignancy among women worldwide. This study was designed to investigate the role of METTL14 in sorafenib-induced ferroptosis in CC. METTL14 expression and m6A methylation were determined in CC tissues, followed by analyzes correlating these factors with clinical features. Subsequently, METTL14 was knocked down in CC cell lines, and the effects on cell proliferation, mitochondrial morphology and ferroptosis were assessed using CCK-8, microscopy, and markers associated with ferroptosis, respectively. The regulatory relationship between METTL14 and FTH1 was verified using qRT-PCR and luciferase reporter assays. The functional significance of this interaction was further investigated both in vitro and in vivo by co-transfecting cells with overexpression vectors or shRNAs targeting METTL14 and FTH1 after sorafenib treatment. METTL14 expression and m6A methylation were significantly reduced in CC tissues, and lower METTL14 expression levels were associated with a poorer CC patients' prognosis. Notably, METTL14 expression increased during sorafenib-induced ferroptosis, and METTL14 knockdown attenuated the ferroptotic response induced by sorafenib in CC cells. FTH1 was identified as a direct target of METTL14, with METTL14 overexpression leading to increased m6A methylation of FTH1 mRNA, resulting in reduced stability and expression of FTH1 in CC. Furthermore, FTH1 overexpression or treatment with LY294002 partially counteracted the promotion of sorafenib-induced ferroptosis by METTL14. In vivo xenograft experiments demonstrated that inhibiting METTL14 reduced the anticancer effects of sorafenib, whereas suppression of FTH1 significantly enhanced sorafenib-induced ferroptosis and increased its anticancer efficacy. METTL14 reduces FTH1 mRNA stability through m6A methylation, thereby enhancing sorafenib-induced ferroptosis, which contributes to suppressing CC progression via the PI3K/Akt signaling pathway.


Subject(s)
Ferroptosis , Methyltransferases , RNA Stability , Sorafenib , Uterine Cervical Neoplasms , Humans , Sorafenib/pharmacology , Sorafenib/therapeutic use , Female , Ferroptosis/drug effects , Ferroptosis/genetics , Uterine Cervical Neoplasms/drug therapy , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/genetics , Mice , Animals , Methyltransferases/metabolism , Methyltransferases/genetics , RNA Stability/drug effects , Mice, Nude , Gene Expression Regulation, Neoplastic/drug effects , Methylation/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Xenograft Model Antitumor Assays , RNA, Messenger/genetics , RNA, Messenger/metabolism , Prognosis , Ferritins , Oxidoreductases
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