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BACKGROUND: RNA N6-methyladenosine (m6A) is involved in mammalian spermatogenesis. In both germ cells and Leydig cells, ALKBH5 regulates spermatogenesis and androgen synthesis in an m6A-dependent manner. However, it is unclear whether ALKBH5 plays a role in testicular Sertoli cells, which constitute the blood-testis barrier (BTB) through cell junctions between adjacent Sertoli cells. METHODS: ALKBH5 expression in the testes of humans and mice was detected by immunohistochemical staining and immunofluorescence staining. BTB integrity was evaluated by BTB assay. m6A-seq was performed to screen for BTB-related molecules regulated by ALKBH5. m6A immunoprecipitation-quantitative real-time polymerase chain reaction (qPCR), RNA immunoprecipitation-qPCR, western blot, coimmunoprecipitation, and polysome fractionation-qPCR analyses were performed to explore the mechanisms of ALKBH5 in BTB. Transmission electron microscopy was applied to observe the BTB ultrastructure. RESULTS: ALKBH5 in Sertoli cells is related to the integrity of the BTB. Subsequently, the m6A level on Cdh2 mRNA, encoding a structural protein N-cadherin in the BTB, was found to be regulated by ALKBH5. IGF2BP1/2/3 complexes and YTHDF1 promoted Cdh2 mRNA translation. In addition, we found that basal endoplasmic specialization, in which N-cadherin is a main structural protein, was severely disordered in the testes of Alkbh5-knockout mice. CONCLUSIONS: Our study revealed that ALKBH5 regulates BTB integrity via basal endoplasmic specialization by affecting Cdh2 mRNA translation.
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Barrera Hematotesticular , Células de Sertoli , Humanos , Masculino , Ratones , Animales , Barrera Hematotesticular/metabolismo , Células de Sertoli/metabolismo , Biosíntesis de Proteínas , Cadherinas/genética , Cadherinas/metabolismo , Ratones Noqueados , ARN/metabolismo , Mamíferos , Antígenos CD/metabolismo , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismoRESUMEN
N6-methyladenosine (m6A) RNA methylation is the most abundant modification on mRNAs and plays important roles in various biological processes. The formation of m6A is catalyzed by a methyltransferase complex including methyltransferase-like 3 (METTL3) as a key factor. However, the in vivo functions of METTL3 and m6A modification in mammalian development remain unclear. Here, we show that specific inactivation of Mettl3 in mouse nervous system causes severe developmental defects in the brain. Mettl3 conditional knockout (cKO) mice manifest cerebellar hypoplasia caused by drastically enhanced apoptosis of newborn cerebellar granule cells (CGCs) in the external granular layer (EGL). METTL3 depletion-induced loss of m6A modification causes extended RNA half-lives and aberrant splicing events, consequently leading to dysregulation of transcriptome-wide gene expression and premature CGC death. Our findings reveal a critical role of METTL3-mediated m6A in regulating the development of mammalian cerebellum.
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Adenosina/análogos & derivados , Cerebelo/embriología , Metiltransferasas/metabolismo , ARN Mensajero/genética , Adenosina/metabolismo , Empalme Alternativo/genética , Animales , Apoptosis/genética , Células Cultivadas , Cerebelo/anomalías , Cerebelo/patología , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/patología , Regulación de la Expresión Génica/genética , Metilación , Metiltransferasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/patología , Estabilidad del ARN/genética , ARN Mensajero/metabolismoRESUMEN
N(6)-methyladenosine (m(6)A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as another mammalian demethylase that oxidatively reverses m(6)A in mRNA in vitro and in vivo. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice have increased m(6)A in mRNA and are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1,551 differentially expressed genes that cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. The discovery of this RNA demethylase strongly suggests that the reversible m(6)A modification has fundamental and broad functions in mammalian cells.
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Dioxigenasas/metabolismo , Proteínas de la Membrana/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Desmetilasa de ARN, Homólogo 5 de AlkB , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Dioxigenasas/química , Dioxigenasas/genética , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Infertilidad Masculina/enzimología , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Tamaño de los Órganos , Oxidorreductasas N-Desmetilantes/química , Oxidorreductasas N-Desmetilantes/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Interferencia de ARN , ARN Mensajero/química , Espermatogénesis/genética , Testículo/enzimología , Testículo/patología , TranscriptomaRESUMEN
Nijmegen Breakage Syndrome (NBS) is a recessive genetic disorder characterized by immunodeficiency, elevated sensitivity to ionizing radiation, chromosomal instability, microcephaly, and high predisposition to malignancies. Since the underlying molecular mechanisms of the NBS microcephaly are still obscure, thus our group previously inactivated the Nbn gene in the central nervous system (CNS) of mice by nestin-Cre targeting gene system, and generated Nbn(CNS-del) mice. Interestingly, the newborn Nbn(CNS-del) mice exhibit obvious microcephaly, which is accompanied by severe ataxia and balance deficiency. In this study presented here, we report that Nbn-deficiency induces the enhanced apoptosis of the mature oligodendrocytes at postnatal day 7, which further affects the myelination of the nerve fibers of cerebrum and corpus callosum.The distinct regulatory roles of Ataxia telangiectasia mutated (ATM) signaling and protein kinase B(Akt)/the mammalian target of Rapamycin (AKT/mTOR) signaling are responsible for the enhanced apoptosis of the Nbn-deficient oligodendrocytes. In addition, a series of transcriptional factors including histonedeacetylase (HDAC), zinc finger protein 191 (ZFP-191) and myelin sheath regulatory factor (MRF) play distinct roles in regulating the myelination of the Nbn-deficient oligodendrocytes. Based on these results, it concludes that ATM-Chk2-P53-P21 signaling pathway and the AKT/mTOR signaling pathway are both responsible for the enhanced apoptosis of the Nbn-deficient oligodendrocytes. HDAC, ZFP-191, and MRF are also involved in the pathogenesis of the hypomyelination of the Nbn-deficient oligodendrocytes.
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Proteínas de Ciclo Celular/metabolismo , Sistema Nervioso Central/metabolismo , Corteza Cerebral/citología , Proteínas de la Mielina/metabolismo , Proteínas Nucleares/metabolismo , Oligodendroglía/fisiología , Transducción de Señal/fisiología , Animales , Animales Recién Nacidos , Apoptosis/fisiología , Proteínas de Ciclo Celular/genética , Células Cultivadas , Cuerpo Calloso/metabolismo , Proteínas de Unión al ADN , Regulación hacia Abajo/genética , Ratones , Ratones Transgénicos , Vaina de Mielina/metabolismo , Vaina de Mielina/ultraestructura , Nestina/genética , Proteínas Nucleares/genética , Oligodendroglía/metabolismo , Oligodendroglía/ultraestructura , Transducción de Señal/genéticaRESUMEN
Protein phosphatase 2A (PP2A) is an abundant heterotrimeric holoenzyme in eukaryotic cells coordinating with specific kinases to regulate spatial-temporal protein dephosphorylation in various biological processes. However, the function of PP2A in cortical neurogenesis remains largely unknown. Here, we report that neuronal-specific deletion of Pp2acα in mice displayed microcephaly, with significantly smaller brains and defective learning and memory ability. Mechanistically, neuronal Pp2acα deficiency resulted in elevated endogenous DNA damage and activation of ATR/CHK1 signaling. It was further induced by the loss of direct interaction between PP2AC and ATR as well as the function of PP2AC to dephosphorylate ATR. Importantly, ATR/CHK1 signaling dysregulation altered both the expression and activity of several critical downstream factors including P53, P21, Bcl2, and Bax, which led to decreased proliferation of cortical progenitor cells and increased apoptosis in developing cortical neurons. Taken together, our results indicate an essential function of PP2ACα in endogenous DNA damage response-mediated ATR signaling during neurogenesis, and defective PP2ACα in neurons contributes to microcephaly.
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Pancreatic ductal adenocarcinoma (PDAC) remains a highly fatal malignancy partially due to the acquired alterations related to aberrant protein glycosylation that pathologically remodel molecular biological processes and protect PDAC cells from death. Ferroptosis driven by lethal lipid peroxidation provides a targetable vulnerability for PDAC. However, the crosstalk between glycosylation and ferroptosis remains unclear. Here, we identified 4F2hc, a subunit of the glutamate-cystine antiporter system Xc-, and its asparagine (N)-glycosylation is involved in PDAC ferroptosis by N- and O-linked glycoproteomics. Knockdown of SLC3A2 (gene name of 4F2hc) or blocking the N-glycosylation of 4F2hc potentiates ferroptosis sensitization of PDAC cells by impairing the activity of system Xc- manifested by a marked decrease in intracellular glutathione. Mechanistically, we found that the glycosyltransferase B3GNT3 catalyzes the glycosylation of 4F2hc, stabilizes the 4F2hc protein, and enhances the interaction between 4F2hc and xCT. Knockout of B3GNT3 or deletion of enzymatically active B3GNT3 sensitizes PDAC cells to ferroptosis. Reconstitution of 4F2hc-deficient cells with wildtype 4F2hc restores ferroptosis resistance while glycosylation-mutated 4F2hc does not. Additionally, upon combination with a ferroptosis inducer, treatment with the classical N-glycosylation inhibitor tunicamycin (TM) markedly triggers the overactivation of lipid peroxidation and enhances the sensitivity of PDAC cells to ferroptosis. Notably, we confirmed that genetic perturbation of SLC3A2 or combination treatment with TM significantly augments ferroptosis-induced inhibition of orthotopic PDAC. Clinically, high expression of 4F2hc and B3GNT3 contributes to the progression and poor survival of PDAC patients. Collectively, our findings reveal a previously unappreciated function of N-glycosylation of 4F2hc in ferroptosis and suggest that dual targeting the vulnerabilities of N-glycosylation and ferroptosis may be an innovative therapeutic strategy for PDAC.
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Carcinoma Ductal Pancreático , Ferroptosis , Neoplasias Pancreáticas , Humanos , Glicosilación , Glicosiltransferasas/metabolismo , Línea Celular Tumoral , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/patología , N-Acetilglucosaminiltransferasas/metabolismo , Neoplasias PancreáticasRESUMEN
Pancreatic stellate cells (PSCs) are crucial for metabolism and disease progression in pancreatic ductal adenocarcinoma (PDAC). However, detailed mechanisms of PSCs in glutamine (Gln) metabolism and tumor-stromal metabolic interactions have not been well clarified. Here we showed that tumor tissues displayed Gln deficiency in orthotopic PDAC models. Single-cell RNA sequencing analysis revealed metabolic heterogeneity in PDAC, with significantly higher expression of Gln catabolism pathway in stromal cells. Significantly higher glutamine synthetase (GS) protein expression was further validated in human tissues and cells. Elevated GS levels in tumor and stroma were independently prognostic of poorer prognosis in PDAC patients. Gln secreted by PSCs increased basal oxygen consumption rate in PCCs. Depletion of GS in PSCs significantly decreased PCCs proliferation in vitro and in vivo. Mechanistically, activation of Wnt signaling induced directly binding of ß-catenin/TCF7 complex to GS promoter region and upregulated GS expression. Rescue experiments testified that GS overexpression recovered ß-catenin knockdown-mediated function on Gln synthesis and tumor-promoting ability of PSCs. Overall, these findings identify the Wnt/ß-catenin/TCF7/GS-mediated growth-promoting effect of PSCs and provide new insights into stromal Gln metabolism, which may offer novel therapeutic strategies for PDAC.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Glutamina/metabolismo , Células Estrelladas Pancreáticas/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/patología , Transformación Celular Neoplásica/metabolismo , Línea Celular Tumoral , Proliferación Celular , Factor 1 de Transcripción de Linfocitos T/metabolismo , Neoplasias PancreáticasRESUMEN
Mitochondrial encephalomyopathies (ME) are frequently associated with mutations of mitochondrial DNA, but the pathogenesis of a subset of ME (sME) remains elusive. Here we report that haploinsufficiency of a mitochondrial inner membrane protein, Mic60, causes progressive neurological abnormalities with insulted mitochondrial structure and neuronal loss in mice. In addition, haploinsufficiency of Mic60 reduces mitochondrial membrane potential and cellular ATP production, increases reactive oxygen species, and alters mitochondrial oxidative phosphorylation complexes in neurons in an age-dependent manner. Moreover, haploinsufficiency of Mic60 compromises brain glucose intake and oxygen consumption in mice, resembling human ME syndrome. We further discover that MIC60 protein expression declined significantly in human sME, implying that insufficient MIC60 may contribute for pathogenesis of human ME. Notably, systemic administration of antioxidant N-acetylcysteine largely reverses mitochondrial dysfunctions and metabolic disorders in haplo-insufficient Mic60 mice, also restores neurological abnormal symptom. These results reveal Mic60 is required in the maintenance of mitochondrial integrity and function, and likely a potential therapeutics target for mitochondrial encephalomyopathies.
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Encefalomiopatías Mitocondriales , Animales , Ratones , Humanos , Encefalomiopatías Mitocondriales/genética , Encefalomiopatías Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Mitocondrias/metabolismo , ADN Mitocondrial , AntioxidantesRESUMEN
Nijmegen breakage syndrome (NBS), ataxia telangiectasia and ataxia telangiectasia-like disorder (ATLD) show overlapping phenotypes such as growth retardation, microcephaly, cerebellar developmental defects and ataxia. However, the molecular pathogenesis of these neurological defects remains elusive. Here we show that inactivation of the Nbn gene (also known as Nbs1) in mouse neural tissues results in a combination of the neurological anomalies characteristic of NBS, ataxia telangiectasia and ATLD, including microcephaly, growth retardation, cerebellar defects and ataxia. Loss of Nbn causes proliferation arrest of granule cell progenitors and apoptosis of postmitotic neurons in the cerebellum. Furthermore, Nbn-deficient neuroprogenitors show proliferation defects (but not increased apoptosis) and contain more chromosomal breaks, which are accompanied by ataxia telangiectasia mutated protein (ATM)-mediated p53 activation. Notably, depletion of p53 substantially rescues the neurological defects of Nbn mutant mice. This study gives insight into the physiological function of NBS1 (the Nbn gene product) and the function of the DNA damage response in the neurological anomalies of NBS, ataxia telangiectasia and ATLD.
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Apoptosis/genética , Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/genética , Proliferación Celular , Reparación del ADN , Proteínas Nucleares/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Ácido Anhídrido Hidrolasas , Animales , Ataxia Telangiectasia/prevención & control , Proteínas de la Ataxia Telangiectasia Mutada , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiología , Cerebelo/patología , Cartilla de ADN , Enzimas Reparadoras del ADN , Proteínas de Unión al ADN/metabolismo , Inmunohistoquímica , Proteína Homóloga de MRE11 , Ratones , Ratones Noqueados , Actividad Motora/fisiología , Mutación/genética , Neuronas/patología , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
INTRODUCTION: Pituitary growth hormone-secreting (GH) pituitary adenomas (PAs) cause mass effects and dysregulated hypersecretion of GH. However, somatic mutation burden is low in PAs. While progress has been made in identifying the epigenetic changes involved in GH-PA initiation, the precise details of its tumorigenesis in GH-PA patients remains to be elucidated. As N6-methyladenosine (m6A) has been shown to often play a critical role in various tumors, it represents a possible initiation point for the tumorigenesis of pituitary adenomas. However, the role of RNA methylation in GH adenomas remains unclear. METHODS: Protein expression of m6A regulators was measured by immunohistochemistry. Global levels and distribution of m6A methylation were separately analyzed by m6A enzyme-linked immunosorbent assay and m6A sequencing (m6A-seq). RNA interference and lentivirus knockdown system were used to investigate the role of methyltransferase-like 3 (METTL3) and its m6A- dependent regulatory mechanism in tumor progression and GH secretion. RESULTS: We show that both METTL3 messenger RNA and protein expression are elevated in GH-PA samples when compared with both normal pituitary tissue specimens and nonsecreting pituitary adenomas. Levels of m6A modification increased in GH-PAs, and hypermethylated RNAs are involved in hormone secretion and cell development. Knockdown of METTL3 in GH3 cell line resulted in decreased cell growth and GH secretion. Importantly, we found that GNAS and GADD45γ act as the downstream targets in this process. CONCLUSION: Our findings strongly suggest that m6A methyltransferase METTL3 promotes tumor growth and hormone secretion by increasing expression of GNAS and GADD45γ in a m6A-dependent manner. Thus, METTL3 and the methylated RNAs constitute suitable targets for clinical treatment of GH-PAs.
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Adenoma/patología , Adenosina/metabolismo , Carcinogénesis , Adenoma Hipofisario Secretor de Hormona del Crecimiento/genética , Hormona de Crecimiento Humana/metabolismo , Metiltransferasas/metabolismo , Neoplasias Hipofisarias/patología , ARN/metabolismo , Adenoma/genética , Adenoma/metabolismo , Adenosina/análogos & derivados , Adulto , Anciano , Anciano de 80 o más Años , Carcinogénesis/genética , Línea Celular Tumoral , Proliferación Celular , Cromograninas/genética , Epigénesis Genética , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Adenoma Hipofisario Secretor de Hormona del Crecimiento/metabolismo , Adenoma Hipofisario Secretor de Hormona del Crecimiento/patología , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Metilación , Metiltransferasas/genética , Persona de Mediana Edad , Hipófisis/metabolismo , Hipófisis/patología , Neoplasias Hipofisarias/genética , Neoplasias Hipofisarias/metabolismo , Adulto Joven , Proteinas GADD45RESUMEN
SHH subgroup medulloblastoma (SHH-MB) is one of the most common malignant pediatric tumors that arises in the cerebellum. Previously, we showed that RNA m6A methylation participates in regulation of cerebellar development. Here we investigate whether dysregulated m6A methylation contributes to tumorigenesis of SHH-MB. We show that high expression of m6A methyltransferase METTL3 associates with worse survival in the patients with SHH-MB. A large number of hypermethylated transcripts are identified in SHH-MB tumor cells by m6A-seq. We find that METTL3 promotes tumor progression via activating Sonic hedgehog signaling. Mechanistically, METTL3 methylates PTCH1 and GLI2 RNAs and further regulates their RNA stability and translation. Importantly, targeting METTL3 by depleting METTL3 expression or treatment with its catalytic inhibitor STM2457 restrains tumor progression. Collectively, this study shows a critical function for METTL3 and m6A methylation in SHH-MB, indicative of a potential role of METTL3 as therapeutic target in SHH-MB.
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Neoplasias Cerebelosas , Meduloblastoma , Niño , Humanos , Neoplasias Cerebelosas/patología , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Meduloblastoma/metabolismo , Metilación , Metiltransferasas/genética , Metiltransferasas/metabolismo , Proteínas Nucleares/metabolismo , ARN/metabolismo , Proteína Gli2 con Dedos de Zinc/metabolismoRESUMEN
Exploring the cross-talk between the immune system and advanced biomaterials to treat SARS-CoV-2 infection is a promising strategy. Here, we show that ACE2-overexpressing A549 cell-derived microparticles (AO-MPs) are a potential therapeutic agent against SARS-CoV-2 infection. Intranasally administered AO-MPs dexterously navigate the anatomical and biological features of the lungs to enter the alveoli and are taken up by alveolar macrophages (AMs). Then, AO-MPs increase the endosomal pH but decrease the lysosomal pH in AMs, thus escorting bound SARS-CoV-2 from phago-endosomes to lysosomes for degradation. This pH regulation is attributable to oxidized cholesterol, which is enriched in AO-MPs and translocated to endosomal membranes, thus interfering with proton pumps and impairing endosomal acidification. In addition to promoting viral degradation, AO-MPs also inhibit the proinflammatory phenotype of AMs, leading to increased treatment efficacy in a SARS-CoV-2-infected mouse model without side effects. These findings highlight the potential use of AO-MPs to treat SARS-CoV-2-infected patients and showcase the feasibility of MP therapies for combatting emerging respiratory viruses in the future.
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Enzima Convertidora de Angiotensina 2/administración & dosificación , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/metabolismo , COVID-19/terapia , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Micropartículas Derivadas de Células/metabolismo , Colesterol/metabolismo , Endosomas/química , Macrófagos Alveolares/metabolismo , SARS-CoV-2/metabolismo , Células A549 , Enzima Convertidora de Angiotensina 2/genética , Animales , COVID-19/virología , Chlorocebus aethiops , Modelos Animales de Enfermedad , Femenino , Humanos , Concentración de Iones de Hidrógeno , Lisosomas/química , Ratones , Ratones Endogámicos ICR , Ratones Transgénicos , Oxidación-Reducción , Células RAW 264.7 , Resultado del Tratamiento , Células VeroRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) originates from normal pancreatic ducts where digestive juice is regularly produced. It remains unclear how PDAC can escape autodigestion by digestive enzymes. Here we show that human PDAC tumour cells use gasdermin E (GSDME), a pore-forming protein, to mediate digestive resistance. GSDME facilitates the tumour cells to express mucin 1 and mucin 13, which form a barrier to prevent chymotrypsin-mediated destruction. Inoculation of GSDME-/- PDAC cells results in subcutaneous but not orthotopic tumour formation in mice. Inhibition or knockout of mucin 1 or mucin 13 abrogates orthotopic PDAC growth in NOD-SCID mice. Mechanistically, GSDME interacts with and transports YBX1 into the nucleus where YBX1 directly promotes mucin expression. This GSDME-YBX1-mucin axis is also confirmed in patients with PDAC. These findings uncover a unique survival mechanism of PDAC cells in pancreatic microenvironments.
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Adenocarcinoma , Neoplasias Pancreáticas , Proteínas Citotóxicas Formadoras de Poros , Adenocarcinoma/genética , Animales , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mucina-1 , Mucinas , Neoplasias Pancreáticas/patología , Proteínas Citotóxicas Formadoras de Poros/fisiología , Microambiente Tumoral , Proteína 1 de Unión a la Caja YRESUMEN
The entity of DNA N6-methyladenine (6mA) in mammals remains elusive and subsequently its roles in diseases are poorly understood. Here we exploited a bacterial DNA contamination-free and ultrasensitive UHPLC-MS/MS assay to reassess DNA 6mA in human glioblastomas and unveiled that DNA 6mA (~0.08 ppm) is extremely rare. By the use of two independent heavy stable isotope-labeling strategies, we further prove that the observed 6mA is solely generated by DNA polymerase-mediated misinocorporation. In vitro experiments point toward that the generation of misincorporated DNA 6mA is associated with the cellular stresses-caused release of RNA N6-methyladenine (m6A) nucleoside, which is profoundly inhibited by hypoxia milieu. Consistently, compared with normal brain tissues, DNA 6mA decreases in hypoxic human gliomas. Our data also strongly support that rare DNA 6mA rather than relatively abundant DNA 5-methylcytosine and 5-hydroxymethylcytosine is a hallmark of poor prognosis of IDH1/2 mutation-absent glioblastoma patients, reflecting the incidence of cytotoxic stresses and subsequent release of m6A nucleoside. The released m6A nucleoside may selectively preserve a subset of the glioblastoma cells and stimulate their stemness and proliferation. Noteworthily, demethylation-inhibiting IDH1 mutation increases the DNA 6mA content in human gliomas, but the depletion of the demethylase candidate ALKBH1 fails to do so, together suggesting the presence of other unknown 6mA demethylase for erasing misincorporated DNA 6mA. This is the first report on the identification of the misincorporated 6mA together with its origin and roles in diseases.
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SARS-CoV-2 infection causes complicated clinical manifestations with variable multi-organ injuries, however, the underlying mechanism, in particular immune responses in different organs, remains elusive. In this study, comprehensive transcriptomic alterations of 14 tissues from rhesus macaque infected with SARS-CoV-2 were analyzed. Compared to normal controls, SARS-CoV-2 infection resulted in dysregulation of genes involving diverse functions in various examined tissues/organs, with drastic transcriptomic changes in cerebral cortex and right ventricle. Intriguingly, cerebral cortex exhibited a hyperinflammatory state evidenced by significant upregulation of inflammation response-related genes. Meanwhile, expressions of coagulation, angiogenesis and fibrosis factors were also up-regulated in cerebral cortex. Based on our findings, neuropilin 1 (NRP1), a receptor of SARS-CoV-2, was significantly elevated in cerebral cortex post infection, accompanied by active immune response releasing inflammatory factors and signal transmission among tissues, which enhanced infection of the central nervous system (CNS) in a positive feedback way, leading to viral encephalitis. Overall, our study depicts a multi-tissue/organ transcriptomic landscapes of rhesus macaque with early infection of SARS-CoV-2, and provides important insights into the mechanistic basis for COVID-19-associated clinical complications.
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COVID-19 , SARS-CoV-2 , Animales , COVID-19/genética , Macaca mulatta , SARS-CoV-2/genética , TranscriptomaRESUMEN
Although RNA m6A regulators have been implicated in the tumorigenesis of several different types of tumors, including pancreatic cancer, their clinical relevance and intrinsic regulatory mechanism remain elusive. This study analyzed eight m6A regulators (METTL3, METTL14, WTAP, FTO, ALKBH5, and YTHDF1-3) in pancreatic ductal adenocarcinoma (PDAC) and found that only RNA m6A demethylase ALKBH5 serves as an independent favorable prognostic marker for this tumor. To better understand the molecular mechanism underlying the protective effect conferred by ALKBH5 against pancreatic tumorigenesis, we performed a transcriptome-wide analysis of m6A methylation, gene expression, and alternative splicing (AS) using the MIA PaCa-2 stable cell line with ALKBH5 overexpression. We demonstrated that ALKBH5 overexpression induced a reduction in RNA m6A levels globally. Furthermore, mRNAs encoding ubiquitin ligase FBXL5, and mitochondrial iron importers SLC25A28 and SLC25A37, were identified as substrates of ALKBH5. Mechanistically, the RNA stabilities of FBXL5 and SLC25A28, and the AS of SLC25A37 were affected, which led to their upregulation in pancreatic cancer cell line. Particularly, we observed that downregulation of FBXL5 in tumor samples correlated with shorter survival time of patients. Owing to FBXL5-mediated degradation, ALKBH5 overexpression incurred a significant reduction in iron-regulatory protein IRP2 and the modulator of epithelial-mesenchymal transition (EMT) SNAI1. Notably, ALKBH5 overexpression led to a significant reduction in intracellular iron levels as well as cell migratory and invasive abilities, which could be rescued by knocking down FBXL5. Overall, our results reveal a previously uncharacterized mechanism of ALKBH5 in protecting against PDAC through modulating regulators of iron metabolism and underscore the multifaceted role of m6A in pancreatic cancer.
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Medulloblastoma, as the most common malignant brain tumor in children, exhibits highly dysregulated DNA methylation. The novel epigenetic marker-5-hydroxymethylcytosine (5hmC) plays essential role in gene regulation during brain development and in brain tumors. However, the biological and clinical implications of 5hmC in medulloblastoma are still unclear. Here, we detected global 5hmC levels in two independent medulloblastoma patient cohorts (discovery cohort: n = 81; validation cohort: n = 171) using ultra-high performance liquid chromatography-tandem mass spectrometry analysis. Immunohistochemistry was used to identify the cell proliferation and expression of Ten-eleven translocation 1 and 2 (TET1/2). The prognostic impacts of covariates on progression-free survival (PFS) and overall survival (OS) were evaluated using multivariate Cox hazards regression models. We observed that global 5hmC levels were decreased in medulloblastomas compared to normal cerebellums (P < 0.001). Multivariate analysis showed that low global 5hmC levels correlated with poor PFS and OS rates (discovery cohort: PFS: P = 0.003, OS: P = 0.002; validation cohort: PFS: P = 0.0002, OS: P = 0.001). Immunohistochemistry showed an inverse correlation between 5hmC score and Ki-67 index (r = -0.747, P < 0.0001). Moreover, 5hmC score in MB samples was associated with nuclear expression of TET1 (r = -0.419, P = 0.003) and TET2 (r = -0.399, P = 0.005) proteins. Our study demonstrates that loss of 5hmC is an epigenetic biomarker in medulloblastomas. Our results indicate that 5hmC could be a candidate prognostic indicator for improving survival prediction of risk stratification in patients with medulloblastoma.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) invades the alveoli, where abundant alveolar macrophages (AMs) reside. How AMs respond to SARS-CoV-2 invasion remains elusive. Here, we show that classically activated M1 AMs facilitate viral spread; however, alternatively activated M2 AMs limit the spread. M1 AMs utilize cellular softness to efficiently take up SARS-CoV-2. Subsequently, the invaded viruses take over the endo-lysosomal system to escape. M1 AMs have a lower endosomal pH, favoring membrane fusion and allowing the entry of viral RNA from the endosomes into the cytoplasm, where the virus achieves replication and is packaged to be released. In contrast, M2 AMs have a higher endosomal pH but a lower lysosomal pH, thus delivering the virus to lysosomes for degradation. In hACE2 transgenic mouse model, M1 AMs are found to facilitate SARS-CoV-2 infection of the lungs. These findings provide insights into the complex roles of AMs during SARS-CoV-2 infection, along with potential therapeutic targets.
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Although DNA 5-hydroxymethylcytosine (5hmC) is recognized as an important epigenetic mark in cancer, its precise role in lymph node metastasis remains elusive. In this study, we investigated how 5hmC associates with lymph node metastasis in breast cancer. Accompanying with high expression of TET1 and TET2 proteins, large numbers of genes in the metastasis-positive primary tumors exhibit higher 5hmC levels than those in the metastasis-negative primary tumors. In contrast, the TET protein expression and DNA 5hmC decrease significantly within the metastatic lesions in the lymph nodes compared to those in their matched primary tumors. Through genome-wide analysis of 8 sets of primary tumors, we identified 100 high-confidence metastasis-associated 5hmC signatures, and it is found that increased levels of DNA 5hmC and gene expression of MAP7D1 associate with high risk of lymph node metastasis. Furthermore, we demonstrate that MAP7D1, regulated by TET1, promotes tumor growth and metastasis. In conclusion, the dynamic 5hmC profiles during lymph node metastasis suggest a link between DNA 5hmC and lymph node metastasis. Meanwhile, the role of MAP7D1 in breast cancer progression suggests that the metastasis-associated 5hmC signatures are potential biomarkers to predict the risk for lymph node metastasis, which may serve as diagnostic and therapeutic targets for metastatic breast cancer.