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1.
Gene ; 722: 144076, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31454538

RESUMO

N6-methyladenosine (m6A) is the most prevalent internal modification in mammalian mRNAs and methyltransferase-like 3 (METTL3) is a vital methyltransferase in m6A modification. Here, this study tries to discover the regulatory role of METTL3 and its mechanism in the breast cancer tumorigenesis. Results found that METTL3 was up-regulated in the breast cancer tissue and cells. In vivo and vitro, METTL3 knockdown could decrease the methylation level, reduce the proliferation, accelerate the apoptosis and inhibited the tumor growth. Moreover, we found that Bcl-2 acted as the target of METTL3, thereby regulating the proliferation and apoptosis of breast cancer. This study could reveal the potential mechanism of m6A modification in the breast cancer tumorigenesis, providing potential drug targets in the treatment.


Assuntos
Neoplasias da Mama/enzimologia , Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Apoptose , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Feminino , Humanos , Metiltransferases/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
2.
Cell Physiol Biochem ; 53(4): 731-745, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31613064

RESUMO

BACKGROUND/AIMS: 3-Deazaneplanocin, DZNep, has been reported to inhibit the EZH2 histone methylase and to induce cell apoptosis in chondrosarcomas (CS). The present study aims to confirm the therapeutic potential of EZH2 inhibitors and investigate the molecular mechanisms of DZNep in chondrosarcomas. METHODS: CS cell lines and primary cultures were used. Apoptosis was investigated using PARP cleavage, caspase 3/7 activity, or Apo2.7 expression. S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM) were quantified by UHPLC-MS/MS. Differentially expressed genes in treated-chondrosarcomas and chondrocytes were researched by microarray analysis. RESULTS: DZNep induced apoptosis in chondrosarcomas both in vivo and in vitro. However, this effect was not correlated to EZH2 expression nor activity, and EZH2 knock-down by siRNA did not reduce CS viability. Additionally, the reduction of H3K27me3 induced by GSK126 or tazemetostat (EPZ-6438) did not provoke chondrosarcoma death. However, as expected, DZNep induced SAH accumulation and reduced SAM:SAH ratio. Further, microarray analysis suggests a key role of EGFR in antitumoral effect of DZNep, and pharmacological inhibition of EGFR reduced chondrosarcoma survival. CONCLUSION: EZH2 is not an adequate target for chondrosarcoma treatment. However, DZNep induces apoptosis in chondrosarcomas in vitro and in vivo, by a mechanism likely mediated though EGFR expression. Consequently, it would be worth initiating clinical trials to evaluating efficiency to S-adenosylhomocysteine hydrolase or EGFR inhibitors in patients with chondrosarcomas.


Assuntos
Adenosina/análogos & derivados , Regulação para Baixo/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Adenosina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Condrossarcoma/metabolismo , Condrossarcoma/patologia , Dano ao DNA/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Mapas de Interação de Proteínas/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , S-Adenosil-Homocisteína/metabolismo
3.
Genome Biol ; 20(1): 156, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387610

RESUMO

BACKGROUND: Methylation of nucleotides, notably in the forms of 5-methylcytosine (5mC) in DNA and N6-methyladenosine (m6A) in mRNA, carries important information for gene regulation. 5mC has been elucidated to participate in the regulation of fruit ripening, whereas the function of m6A in this process and the interplay between 5mC and m6A remain uncharacterized. RESULTS: Here, we show that mRNA m6A methylation exhibits dynamic changes similar to DNA methylation during tomato fruit ripening. RNA methylome analysis reveals that m6A methylation is a prevalent modification in the mRNA of tomato fruit, and the m6A sites are enriched around the stop codons and within the 3' untranslated regions. In the fruit of the ripening-deficient epimutant Colorless non-ripening (Cnr) which harbors DNA hypermethylation, over 1100 transcripts display increased m6A levels, while only 134 transcripts show decreased m6A enrichment, suggesting a global increase in m6A. The m6A deposition is generally negatively correlated with transcript abundance. Further analysis demonstrates that the overall increase in m6A methylation in Cnr mutant fruit is associated with the decreased expression of RNA demethylase gene SlALKBH2, which is regulated by DNA methylation. Interestingly, SlALKBH2 has the ability to bind the transcript of SlDML2, a DNA demethylase gene required for tomato fruit ripening, and modulates its stability via m6A demethylation. Mutation of SlALKBH2 decreases the abundance of SlDML2 mRNA and delays fruit ripening. CONCLUSIONS: Our study identifies a novel layer of gene regulation for key ripening genes and establishes an essential molecular link between DNA methylation and mRNA m6A methylation during fruit ripening.


Assuntos
Adenosina/análogos & derivados , Regulação da Expressão Gênica de Plantas , Lycopersicon esculentum/genética , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Metilação de DNA , Retículo Endoplasmático/enzimologia , Frutas/genética , Frutas/metabolismo , Regulação Enzimológica da Expressão Gênica , Lycopersicon esculentum/enzimologia , Lycopersicon esculentum/metabolismo , Metilação , Mutação , Motivos de Nucleotídeos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estabilidade de RNA , RNA Mensageiro/química
5.
Nat Chem Biol ; 15(9): 865-871, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31383972

RESUMO

RNA modification in the form of N6-methyladenosine (m6A) regulates nearly all the post-transcriptional processes. The asymmetric m6A deposition suggests that regional methylation may have distinct functional consequences. However, current RNA biology tools do not distinguish the contribution of individual m6A modifications. Here we report the development of 'm6A editing', a powerful approach that enables m6A installation and erasure from cellular RNAs without changing the primary sequence. We engineered fusions of CRISPR-Cas9 and a single-chain m6A methyltransferase that can be programmed with a guide RNA. The resultant m6A 'writers' allow functional comparison of single site methylation in different messenger RNA regions. We further engineered m6A 'erasers' by fusing CRISPR-Cas9 with ALKBH5 or FTO to achieve site-specific demethylation of RNAs. The development of programmable m6A editing not only expands the scope of RNA engineering, but also facilitates mechanistic understanding of epitranscriptome.


Assuntos
Adenosina/análogos & derivados , Sistemas CRISPR-Cas , Edição de Genes/métodos , Metiltransferases/metabolismo , RNA Mensageiro/metabolismo , Adenosina/química , Adenosina/metabolismo , Sequência de Bases , Linhagem Celular , Humanos , Metiltransferases/classificação , RNA Mensageiro/química , RNA Mensageiro/genética
6.
Science ; 365(6454)2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31320558

RESUMO

DNA viruses typically eject genomic DNA into the nuclei of host cells after entry. It is unclear, however, how nuclear pathogen-derived DNA triggers innate immune responses. We report that heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) recognizes pathogenic DNA and amplifies interferon-α/ß (IFN-α/ß) production. Upon DNA virus infection, nuclear-localized hnRNPA2B1 senses viral DNA, homodimerizes, and is then demethylated at arginine-226 by the arginine demethylase JMJD6. This results in hnRNPA2B1 translocation to the cytoplasm where it activates the TANK-binding kinase 1-interferon regulatory factor 3 (TBK1-IRF3) pathway, leading to IFN-α/ß production. Additionally, hnRNPA2B1 facilitates N 6-methyladenosine (m6A) modification and nucleocytoplasmic trafficking of CGAS, IFI16, and STING messenger RNAs. This, in turn, amplifies the activation of cytoplasmic TBK1-IRF3 mediated by these factors. Thus, hnRNPA2B1 plays important roles in initiating IFN-α/ß production and enhancing stimulator of interferon genes (STING)-dependent cytoplasmic antiviral signaling.


Assuntos
Núcleo Celular/imunologia , Núcleo Celular/virologia , Infecções por Vírus de DNA/imunologia , DNA Viral/imunologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Imunidade Inata , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Citoplasma/metabolismo , Células HEK293 , Herpesvirus Humano 1/imunologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Fator Regulador 3 de Interferon , Interferon-alfa/metabolismo , Interferon beta/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas Nucleares/metabolismo , Nucleotidiltransferases/metabolismo , Fosfoproteínas/metabolismo , Transporte Proteico , Proteínas Serina-Treonina Quinases/metabolismo , Células RAW 264.7
7.
Nature ; 571(7765): 424-428, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31292544

RESUMO

N6-methyladenosine (m6A) is the most prevalent modified nucleotide in mRNA1,2, with around 25% of mRNAs containing at least one m6A. Methylation of mRNA to form m6A is required for diverse cellular and physiological processes3. Although the presence of m6A in an mRNA can affect its fate in different ways, it is unclear how m6A directs this process and why the effects of m6A can vary in different cellular contexts. Here we show that the cytosolic m6A-binding proteins-YTHDF1, YTHDF2 and YTHDF3-undergo liquid-liquid phase separation in vitro and in cells. This phase separation is markedly enhanced by mRNAs that contain multiple, but not single, m6A residues. Polymethylated mRNAs act as a multivalent scaffold for the binding of YTHDF proteins, juxtaposing their low-complexity domains and thereby leading to phase separation. The resulting mRNA-YTHDF complexes then partition into different endogenous phase-separated compartments, such as P-bodies, stress granules or neuronal RNA granules. m6A-mRNA is subject to compartment-specific regulation, including a reduction in the stability and translation of mRNA. These studies reveal that the number and distribution of m6A sites in cellular mRNAs can regulate and influence the composition of the phase-separated transcriptome, and suggest that the cellular properties of m6A-modified mRNAs are governed by liquid-liquid phase separation principles.


Assuntos
Adenosina/análogos & derivados , Compartimento Celular , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , Humanos , Metilação , Metiltransferases/deficiência , Camundongos , Transição de Fase , RNA Mensageiro/análise , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Estresse Fisiológico
8.
Int Arch Allergy Immunol ; 180(2): 120-127, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31256157

RESUMO

BACKGROUND: Allergen immunotherapy (AIT) is the only etiological and potentially curative therapy for allergic rhinitis (AR). OBJECTIVES: We sought to investigate the role of epigenetic regulator enhancer of zeste homolog 2 (EZH2) in the activation of dendritic cells (DCs) in AIT. METHOD: In this study, EZH2 expression in circulating myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) were evaluated using flow cytometry. Clinical information from 56 AR patients receiving AIT was collected, including 30 subjects with subcutaneous immunotherapy (SCIT) and 26 subjects with sublingual immunotherapy (SLIT). In vitro, the effect of EZH2 inhibitor, 3 Deazaneplanocin A (DZNep), on the phenotypic and functional activation of monocyte-derived DCs (moDCs) was evaluated. RESULTS: EZH2 expression in circulating mDCs and pDCs were both negatively correlated to treatment time of AIT (r = -0.39, p = 0.003 and r = -0.47, p = 0.0002, respectively). Furthermore, there was a higher correlation between EZH2 expression and AIT treatment time in the SCIT group compared to that of the SLIT group in mDCs (r = -0.42, p = 0.02 vs. r = -0.23, p = 0.26)and pDCs (r = -0.52, p = 0.003 vs. r = -0.33, p = 0.10). In vitro, the co-stimulatory molecules on moDCs, such as CD80, CD86, and CD83, were significantly inhibited by DZNep in a dose-dependent manner. The -DC-driven T-cell proliferation was suppressed by DZNep (MD = 22.88, 95% CI 7.809-37.96, p < 0.05). CONCLUSIONS: Our study shows that EZH2, which is required in the activation of DCs, mediates the epigenetic modification in AIT and stresses the importance of patient adherence during AIT.


Assuntos
Adenosina/análogos & derivados , Células Dendríticas/imunologia , Dessensibilização Imunológica/métodos , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/imunologia , Rinite Alérgica/imunologia , Adenosina/uso terapêutico , Adulto , Proliferação de Células/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Epigênese Genética/genética , Epigênese Genética/imunologia , Feminino , Humanos , Interleucina-10/análise , Interleucina-6/análise , Masculino , Cooperação do Paciente , Rinite Alérgica/patologia , Adulto Jovem
9.
Chemistry ; 25(58): 13309-13317, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31328310

RESUMO

The synthesis of the protected form of 2-methylthio-N6 -threonylcarbamoyl adenosine (ms2 t6 A) was developed starting from adenosine or guanosine by using the optimized carbamate method and, for the first time, an isocyanate route. The hypermodified nucleoside was subsequently transformed into the protected ms2 t6 A-phosphoramidite monomer and used in a large-scale synthesis of the precursor 17nt ms2 t6 A-oligonucleotide (the anticodon stem and loop fragment of tRNALys from T. brucei). Finally, stereochemically secure ms2 t6 A→ms2 ct6 A cyclization at the oligonucleotide level efficiently afforded a tRNA fragment bearing the ms2 ct6 A unit. The applied post-synthetic approach provides two sequentially homologous ms2 t6 A- and ms2 ct6 A-oligonucleotides that are suitable for further comparative structure-activity relationship studies.


Assuntos
Adenosina/análogos & derivados , Oligorribonucleotídeos/síntese química , RNA de Transferência/química , Treonina/análogos & derivados , Adenosina/química , Sequência de Bases , Carbamatos/química , Ciclização , Guanosina/química , Isocianatos/química , Conformação de Ácido Nucleico , Compostos Organofosforados/química , Relação Estrutura-Atividade , Treonina/síntese química , Treonina/química
10.
Biochim Biophys Acta Gene Regul Mech ; 1862(8): 796-806, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31295563

RESUMO

N6-methyladenosine (m6A), the most abundant internal mRNA modification in eukaryotes, plays a vital role in regulating adipogenesis. However, its underlying mechanism remains largely unknown. Here, we reveal that deletion of m6A demethylase FTO in porcine and mouse preadipocytes inhibits adipogenesis through JAK2-STAT3-C/EBPß signaling. Mechanistically, FTO deficiency suppresses JAK2 expression and STAT3 phosphorylation, leading to attenuated transcription of C/EBPß, which is essential for the early stage of adipocyte differentiation. Using dual-luciferase assay, we validate that knockdown of FTO reduces expression of JAK2 in an m6A-dependent manner. Furthermore, we find that m6A "reader" protein YTHDF2 directly targets m6A-modified transcripts of JAK2 and accelerates mRNA decay, which results in decreased JAK2 expression and inactivated JAK2-STAT3-C/EBPß signaling, thereby inhibiting adipogenesis. Collectively, our results provide a novel insight into the molecular mechanism of m6A methylation in post-transcriptional regulation of JAK2-STAT3-C/EBPß signaling axis and highlight the crucial role of m6A modification and its modulators in adipogenesis.


Assuntos
Adenosina/análogos & derivados , Adipogenia , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Janus Quinase 2/genética , Fator de Transcrição STAT3/metabolismo , Células 3T3-L1 , Adenosina/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Animais , Diferenciação Celular , Deleção de Genes , Regulação da Expressão Gênica , Camundongos , Fosforilação , Estabilidade de RNA , Proteínas de Ligação a RNA , Transdução de Sinais , Suínos
11.
Eur J Med Chem ; 179: 310-324, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31255928

RESUMO

To obtain potential A2A adenosine receptor agonists, a series of 2-hydrazinyladenosine derivatives were synthesized and assayed for adenosine receptors activity using radioligand binding activity assays. The binding activity of the subtypes was examined, and the structure-activity relationship of this class of compounds at the A2A receptor was investigated. A fragment-based computer-aided design method was used to modify the 2-position side chain structures with different structural fragments, and the newly generated molecules were docked to the A2A receptor to assess scoring and screening activity. To synthesize compounds with better scoring activity, the newly synthesized compounds were tested for in vitro receptor binding activity. 2-Hydrazinyladenosine derivatives of 32 new structural types were designed and synthesized, with the most potent adenosine derivative 23 exhibiting a Ki value of 1.8 nM for A2AAR and significant selectivity for the A2A receptor compared to the A1 receptor. In addition to, compound 23, 24, 30, 31, and 42 also exhibited potent A2A receptor selectivity, with Ki values for the A2A receptor of 6.4, 20, 67 and 6.3 nM, respectively. We also found that compound 35 has a high A1 receptor selectivity, with a Ki value for the A1 receptor of 4.5 nM. Further functional assays also demonstrated that these compounds have potent A2A receptor agonist activity. The study shows the applicability of an in silico fragment-based molecular design for rational lead optimization in A2AAR.


Assuntos
Adenosina/farmacologia , Desenho de Drogas , Hidrazinas/farmacologia , Receptor A2A de Adenosina/metabolismo , Adenosina/análogos & derivados , Adenosina/química , AMP Cíclico/análise , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Hidrazinas/síntese química , Hidrazinas/química , Ligantes , Estrutura Molecular , Relação Estrutura-Atividade
12.
Talanta ; 202: 198-206, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171170

RESUMO

Investigation of the in vivo drug action and metabolic differences of epimer drugs is challenging. Whole-body MSI analysis can visually present the stereoscopic distribution of molecules related to the interaction of drugs and organisms, and can provide more comprehensive organ-specific profiling information. Herein, we developed a whole-body spatially-resolved imaging metabolomics method based on an air flow-assisted ionisation desorption electrospray ionisation (AFADESI)-MSI system coupled with a high-resolution mass spectrometer and highly discriminating imaging software. The epimeric sedative-hypnotic drug candidates YZG-331 and YZG-330 were selected as examples, and rats administered normal or high oral doses were used. By performing multivariate statistical data-mining on the combined MSI data, organ-specific differential ions were screened. By comparing the variations in the relative contents of the drugs, their metabolites, and endogenous neurotransmitters throughout whole-body tissue sections of the rats, rich information that could potentially explain the more significant sedative-hypnotic effects of YZG-330 compared to YZG-331 was obtained. Such as the increased ratio of gamma-aminobutyric acid in the brain and stomach of the rats (0.25, 0.47, 0.68, 0.30, and 0.89 for the control and YZG-331-H, YZG-330-H, YZG-331-L, and YZG-330-L, respectively) were interesting. This study provided a convenient and visual method to investigate in vivo molecular metabolic differences and provide insight towards a better understanding of the pharmacodynamic mechanisms of these sedative-hypnotic drug-candidates.


Assuntos
Adenosina/análogos & derivados , Metabolômica , Adenosina/análise , Adenosina/metabolismo , Animais , Masculino , Espectrometria de Massas , Ratos , Software
13.
Genomics Proteomics Bioinformatics ; 17(2): 154-168, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31154015

RESUMO

N6-methyladenosine (m6A), catalyzed by the methyltransferase complex consisting of Mettl3 and Mettl14, is the most abundant RNA modification in mRNAs and participates in diverse biological processes. However, the roles and precise mechanisms of m6A modification in regulating neuronal development and adult neurogenesis remain unclear. Here, we examined the function of Mettl3, the key component of the complex, in neuronal development and adult neurogenesis of mice. We found that the depletion of Mettl3 significantly reduced m6A levels in adult neural stem cells (aNSCs) and inhibited the proliferation of aNSCs. Mettl3 depletion not only inhibited neuronal development and skewed the differentiation of aNSCs more toward glial lineage, but also affected the morphological maturation of newborn neurons in the adult brain. m6A immunoprecipitation combined with deep sequencing (MeRIP-seq) revealed that m6A was predominantly enriched in transcripts related to neurogenesis and neuronal development. Mechanistically, m6A was present on the transcripts of histone methyltransferase Ezh2, and its reduction upon Mettl3 knockdown decreased both Ezh2 protein expression and consequent H3K27me3 levels. The defects of neurogenesis and neuronal development induced by Mettl3 depletion could be rescued by Ezh2 overexpression. Collectively, our results uncover a crosstalk between RNA and histone modifications and indicate that Mettl3-mediated m6A modification plays an important role in regulating neurogenesis and neuronal development through modulating Ezh2.


Assuntos
Adenosina/análogos & derivados , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Neurogênese , Neurônios/metabolismo , Adenosina/metabolismo , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Encéfalo/metabolismo , Diferenciação Celular/genética , Proliferação de Células , Regulação da Expressão Gênica , Metiltransferases/metabolismo , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Neurônios/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
14.
Nat Commun ; 10(1): 2782, 2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31239444

RESUMO

Melanoma is one of the most deadly and therapy-resistant cancers. Here we show that N6-methyladenosine (m6A) mRNA demethylation by fat mass and obesity-associated protein (FTO) increases melanoma growth and decreases response to anti-PD-1 blockade immunotherapy. FTO level is increased in human melanoma and enhances melanoma tumorigenesis in mice. FTO is induced by metabolic starvation stress through the autophagy and NF-κB pathway. Knockdown of FTO increases m6A methylation in the critical protumorigenic melanoma cell-intrinsic genes including PD-1 (PDCD1), CXCR4, and SOX10, leading to increased RNA decay through the m6A reader YTHDF2. Knockdown of FTO sensitizes melanoma cells to interferon gamma (IFNγ) and sensitizes melanoma to anti-PD-1 treatment in mice, depending on adaptive immunity. Our findings demonstrate a crucial role of FTO as an m6A demethylase in promoting melanoma tumorigenesis and anti-PD-1 resistance, and suggest that the combination of FTO inhibition with anti-PD-1 blockade may reduce the resistance to immunotherapy in melanoma.


Assuntos
Adenosina/análogos & derivados , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Anticorpos Monoclonais/administração & dosagem , Melanoma/enzimologia , Melanoma/terapia , Adenosina/genética , Adenosina/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Animais , Carcinogênese , Linhagem Celular Tumoral , Proliferação de Células , Metilação de DNA , Desmetilação , Feminino , Humanos , Imunoterapia , Melanoma/genética , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
15.
Cancer Sci ; 110(8): 2318-2327, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31187550

RESUMO

Precision medicine places significant emphasis on techniques for the identification of DNA mutations and gene expression by deep sequencing of gene panels to obtain medical data. However, other diverse information that is not easily readable using bioinformatics, including RNA modifications, has emerged as a novel diagnostic and innovative therapy owing to its multifunctional aspects. It is suggested that this breakthrough innovation might open new avenues for the elucidation of uncharacterized cancer cellular functions to develop more precise medical applications. The functional characteristics and regulatory mechanisms of RNA modifications, ie, the epitranscriptome (ETR), which reflects RNA metabolism, remains unclear, mainly due to detection methods being limited. Recent studies have revealed that N6-methyl adenosine, the most common modification in mRNA in eukaryotes, is affected in various types of cancer and, in some cases, cancer stem cells, but also affects cellular responses to viral infections. The ETR can control cancer cell fate through mRNA splicing, stability, nuclear export, and translation. Here we report on the recent progress of ETR detection methods, and biological findings regarding the significance of ETR in cancer precision medicine.


Assuntos
Neoplasias/genética , Transcriptoma/genética , Adenosina/análogos & derivados , Adenosina/genética , Animais , Eucariotos/genética , Humanos , Medicina de Precisão/métodos , RNA Mensageiro/genética
16.
Sheng Wu Gong Cheng Xue Bao ; 35(5): 775-783, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31222996

RESUMO

Messenger RNA (mRNA) can be modified by more than 100 chemical modifications. Among these modifications, N6-methyladenosine (m6A) is one of the most prevalent modifications. During the processes of cells differentiation, embryo development or stress, m6A can be modified on key mRNAs and regulate the progress of cells through modulating mRNA metabolism and translation. Other mRNA modifications, including N1-methyladenosine (m¹A), 5-methylcytosine (m5C) and pseudouridine, together with m6A form the epitranscriptome of mRNA that accurately modulate the mRNA translation. Here we review the types and characteristic of mRNA epigenetic modifications, especially the recent progresses of the function of m6A, we also expect the main research direction of m6A epigenetic modification in the future.


Assuntos
Adenosina/análogos & derivados , Epigênese Genética , Regulação da Expressão Gênica , RNA Mensageiro , Adenosina/genética , Adenosina/metabolismo , Diferenciação Celular/genética , Desenvolvimento Embrionário/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo
17.
PLoS Pathog ; 15(6): e1007796, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31226160

RESUMO

Epstein-Barr virus (EBV) is a ubiquitous oncogenic virus that induces many cancers. N6-Methyladenosine (m6A) modification regulates many cellular processes. We explored the role of m6A in EBV gene regulation and associated cancers. We have comprehensively defined m6A modification of EBV latent and lytic transcripts. Furthermore, m6A modification demonstrated a functional role in regulation of the stability of viral transcripts. The methyltransferase METTL14 was induced at the transcript and protein levels, and knock-down of METTL14 led to decreased expression of latent EBV transcripts. METTL14 was also significantly induced in EBV-positive tumors, promoted growth of EBV-transformed cells and tumors in Xenograft animal models. Mechanistically, the viral-encoded latent oncoprotein EBNA3C activated transcription of METTL14, and directly interacted with METTL14 to promote its stability. This demonstrated that EBV hijacks METTL14 to drive EBV-mediated tumorigenesis. METTL14 is now a new target for development of therapeutics for treatment of EBV-associated cancers.


Assuntos
Transformação Celular Viral , Infecções por Vírus Epstein-Barr/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/metabolismo , Metiltransferases/biossíntese , Proteínas de Neoplasias/biossíntese , Neoplasias/metabolismo , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Animais , Linhagem Celular Tumoral , Infecções por Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Células HEK293 , Humanos , Masculino , Metiltransferases/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Neoplasias/virologia
18.
Biol Pharm Bull ; 42(6): 968-976, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31155593

RESUMO

Previously, we reported that adenosine N1-oxide (ANO), which is found in royal jelly, inhibited the secretion of inflammatory mediators by activated macrophages and reduced lethality in lipopolysaccharide (LPS)-induced endotoxin shock. Here, we examined the regulatory mechanisms of ANO on the release of pro-inflammatory cytokines, with a focus on the signaling pathways activated by toll-like receptor (TLR)4 in response to LPS. ANO inhibited both tumor necrosis factor (TNF)-α and interleukin (IL)-6 secretion from LPS-stimulated RAW264.7 cells without affecting cell proliferation. In this response, phosphorylation of mitogen-activated protein kinase (MAPK) family members (extracellular signal-regulated kinase (ERK)1/2, p38 and SAPK/c-Jun N-terminal kinase (JNK)) and nuclear factor-κB (NF-κB) p65 was not affected by treatment with ANO. In contrast, phosphorylation of Akt (Ser473) and its downstream molecule glycogen synthase kinase-3ß (GSK-3ß) (Ser9) was up-regulated by ANO, suggesting that ANO stimulated GSK-3ß phosphorylation via phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. The phosphorylation of GSK-3ß on Ser9 has been shown to negatively regulate the LPS-induced inflammatory response. Activation of PI3K/Akt signaling pathway has also been implicated in differentiation of mesenchymal stem cells into osteoblasts and adipocytes. As expected, ANO induced alkaline phosphatase activity and promoted calcium deposition in a mouse pre-osteoblastic MC3T3-E1 cell line. The ANO-induced differentiation into osteoblasts was abrogated by coincubation with Wortmannin. Furthermore, ANO promoted insulin/dexamethasone-induced differentiation of mouse 3T3-L1 preadipocytes into adipocytes at much lower concentrations than adenosine. The protective roles of PI3K/Akt/GSK-3ß signaling pathway in inflammatory disorders have been well documented. Our data suggest that ANO may serve as a potential candidate for the treatment of inflammatory disorders. Promotion of osteogenic and adipocyte differentiation further suggests its application for regenerative medicine.


Assuntos
Adenosina/análogos & derivados , Adipócitos/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Óxidos N-Cíclicos/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Adenosina/farmacologia , Adipócitos/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
19.
Nat Cell Biol ; 21(5): 651-661, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31036937

RESUMO

A single genome gives rise to diverse tissues through complex epigenomic mechanisms, including N6-methyladenosine (m6A), a widespread RNA modification that is implicated in many biological processes. Here, to explore the global landscape of m6A in human tissues, we generated 21 whole-transcriptome m6A methylomes across major fetal tissues using m6A sequencing. These data reveal dynamic m6A methylation, identify large numbers of tissue differential m6A modifications and indicate that m6A is positively correlated with gene expression homeostasis. We also report m6A methylomes of long intergenic non-coding RNA (lincRNA), finding that enhancer lincRNAs are enriched for m6A. Tissue m6A regions are often enriched for single nucleotide polymorphisms that are associated with the expression of quantitative traits and complex traits including common diseases, which may potentially affect m6A modifications. Finally, we find that m6A modifications preferentially occupy genes with CpG-rich promoters, features of which regulate RNA transcript m6A. Our data indicate that m6A is widely regulated by human genetic variation and promoters, suggesting a broad involvement of m6A in human development and disease.


Assuntos
Adenosina/análogos & derivados , Elementos Facilitadores Genéticos , Desenvolvimento Fetal/genética , Feto , Adenosina/genética , Epigenômica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Humanos , Metilação , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , Transcriptoma/genética
20.
Clin Drug Investig ; 39(8): 765-773, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31140114

RESUMO

BACKGROUND AND OBJECTIVE: Ticagrelor is a P2Y12 receptor inhibitor approved as an antiplatelet drug for patients with acute coronary syndrome or a history of myocardial infarction. Ticagrelor is also being investigated for the reduction of vaso-occlusive crises in pediatric patients with sickle cell disease. A pediatric formulation suitable for this age range was developed; the development strategy is described. Primary objectives were determining the relative bioavailability of ticagrelor pediatric tablets and granules for oral suspension to the adult immediate-release tablet, and the pediatric tablets taken whole and dispersed/suspended in water to the granules for oral suspension. Bioequivalence between the pediatric tablet taken whole or suspended in water was also assessed. Secondary objectives were comparing the formulations' safety and tolerability. METHODS: We conducted a randomized, four-period, cross-over, single-dose study. Pharmacokinetic parameters were assessed for ticagrelor and its active metabolite AR-C124910XX. Bioequivalence was concluded if the 90% confidence intervals of the maximum plasma concentration and area under the plasma concentration-time curve ratios were contained completely within the 80.00-125.00% limits for ticagrelor/AR-C124910XX. RESULTS: Forty-four healthy adults (95% white; 57% male) were included. Similar bioavailability of ticagrelor (and AR-C124910XX) was demonstrated for all comparisons tested. Ticagrelor pediatric tablets taken whole were bioequivalent to pediatric tablets suspended in water. The plasma concentration-time profiles for ticagrelor and AR-C124910XX were similar, showing rapid ticagrelor absorption and AR-C124910XX formation. All formulations were well tolerated. CONCLUSION: Similar bioavailability of a new pediatric dispersible tablet formulation of ticagrelor for use across a wide age range of pediatric patients was demonstrated compared with other oral ticagrelor formulations. CLINICALTRIALS. GOV IDENTIFIER: NCT03126695. EUDRACT: 2017-000371-93.


Assuntos
Inibidores da Agregação de Plaquetas/farmacocinética , Antagonistas do Receptor Purinérgico P2Y/farmacocinética , Comprimidos , Ticagrelor/farmacocinética , Adenosina/análogos & derivados , Adolescente , Disponibilidade Biológica , Criança , Estudos Cross-Over , Feminino , Humanos , Masculino , Inibidores da Agregação de Plaquetas/administração & dosagem , Antagonistas do Receptor Purinérgico P2Y/administração & dosagem , Equivalência Terapêutica , Ticagrelor/administração & dosagem
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