RESUMEN
Atherosclerosis is characterized by the plaque formation that restricts intraarterial blood flow. The disturbed blood flow with the associated oscillatory stress (OS) at the arterial curvatures and branch points can trigger endothelial activation and is one of the risk factors of atherosclerosis. Many studies reported the mechanotransduction related to OS and atherogenesis; however, the transcriptional and posttranscriptional regulatory mechanisms of atherosclerosis remain unclear. Herein, we investigated the role of N6-methyladenosine (m6A) RNA methylation in mechanotransduction in endothelial cells (ECs) because of its important role in epitranscriptome regulation. We have identified m6A methyltransferase METTL3 as a responsive hub to hemodynamic forces and atherogenic stimuli in ECs. OS led to an up-regulation of METTL3 expression, accompanied by m6A RNA hypermethylation, increased NF-κB p65 Ser536 phosphorylation, and enhanced monocyte adhesion. Knockdown of METTL3 abrogated this OS-induced m6A RNA hypermethylation and other manifestations, while METTL3 overexpression led to changes resembling the OS effects. RNA-sequencing and m6A-enhanced cross-linking and immunoprecipitation (eCLIP) experiments revealed NLRP1 and KLF4 as two hemodynamics-related downstream targets of METTL3-mediated hypermethylation. The METTL3-mediated RNA hypermethylation up-regulated NLRP1 transcript and down-regulated KLF4 transcript through YTHDF1 and YTHDF2 m6A reader proteins, respectively. In the in vivo atherosclerosis model, partial ligation of the carotid artery led to plaque formation and up-regulation of METTL3 and NLRP1, with down-regulation of KLF4; knockdown of METTL3 via repetitive shRNA administration prevented the atherogenic process, NLRP3 up-regulation, and KLF4 down-regulation. Collectively, we have demonstrated that METTL3 serves a central role in the atherogenesis induced by OS and disturbed blood flow.
Asunto(s)
Adenosina/análogos & derivados , Aterosclerosis/metabolismo , Endotelio Vascular/metabolismo , Metiltransferasas/metabolismo , Procesamiento Postranscripcional del ARN , Adenosina/metabolismo , Animales , Aterosclerosis/genética , Endotelio Vascular/patología , Epigénesis Genética , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Metiltransferasas/genética , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Proteínas NLR/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Células THP-1 , TranscriptomaRESUMEN
Epigenetic regulation plays a critical role in glioblastoma (GBM) tumorigenesis. However, how microRNAs (miRNAs) and cytokines cooperate to regulate GBM tumor progression is still unclear. Here, we show that interleukin-6 (IL-6) inhibits miR142-3p expression and promotes GBM propagation by inducing DNA methyltransferase 1-mediated hypermethylation of the miR142-3p promoter. Interestingly, miR142-3p also suppresses IL-6 secretion by targeting the 3' UTR of IL-6. In addition, miR142-3p also targets the 3' UTR and suppresses the expression of high-mobility group AT-hook 2 (HMGA2), leading to inhibition of Sox2-related stemness. We further show that HMGA2 enhances Sox2 expression by directly binding to the Sox2 promoter. Clinically, GBM patients whose tumors present upregulated IL-6, HMGA2, and Sox2 protein expressions and hypermethylated miR142-3p promoter also demonstrate poor survival outcome. Orthotopic delivery of miR142-3p blocks IL-6/HMGA2/Sox2 expression and suppresses stem-like properties in GBM-xenotransplanted mice. Collectively, we discovered an IL-6/miR142-3p feedback-loop-dependent regulation of GBM malignancy that could be a potential therapeutic target.
Asunto(s)
Neoplasias Encefálicas/genética , Glioblastoma/genética , Interleucina-6/genética , MicroARNs/genética , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Línea Celular Tumoral , Metilación de ADN , Epigénesis Genética , Femenino , Proteína HMGA2/genética , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Factores de Transcripción SOXB1/genética , Regulación hacia ArribaRESUMEN
Pluripotency and cell fates can be modulated through the regulation of super-enhancers; however, the underlying mechanisms are unclear. Here, we showed a novel mechanism in which Ash2l directly binds to super-enhancers of several stemness genes to regulate pluripotency and self-renewal in pluripotent stem cells. Ash2l recruits Oct4/Sox2/Nanog (OSN) to form Ash2l/OSN complex at the super-enhancers of Jarid2, Nanog, Sox2 and Oct4, and further drives enhancer activation, upregulation of stemness genes, and maintains the pluripotent circuitry. Ash2l knockdown abrogates the OSN recruitment to all super-enhancers and further hinders the enhancer activation. In addition, CRISPRi/dCas9-mediated blocking of Ash2l-binding motifs at these super-enhancers also prevents OSN recruitment and enhancer activation, validating that Ash2l directly binds to super-enhancers and initiates the pluripotency network. Transfection of Ash2l with W118A mutation to disrupt Ash2l-Oct4 interaction fails to rescue Ash2l-driven enhancer activation and pluripotent gene upregulation in Ash2l-depleted pluripotent stem cells. Together, our data demonstrated Ash2l formed an enhancer-bound Ash2l/OSN complex that can drive enhancer activation, govern pluripotency network and stemness circuitry.
Asunto(s)
Proteínas de Unión al ADN/genética , Elementos de Facilitación Genéticos , Células Madre Embrionarias de Ratones/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factores de Transcripción/genética , Animales , Sistemas CRISPR-Cas/genética , Diferenciación Celular/genética , Linaje de la Célula/genética , Autorrenovación de las Células/genética , Reprogramación Celular/genética , Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Ratones , Mutación/genética , Proteína Homeótica Nanog/genética , Células Madre Pluripotentes/metabolismo , Factores de Transcripción SOXB1/genética , TransfecciónRESUMEN
Heart failure (HF) is a syndrome encompassing several important etiologies that lead to the imbalance between oxygen demand and supply. Despite the usage of guideline-directed medical therapy for HF has shown better outcomes, novel therapeutic strategies are desirable, especially for patients with preserved or mildly reduced left ventricular ejection fraction. In this regard, understanding the molecular basis for cardiomyopathies is expected to fill in the knowledge gap and generate new therapies to improve prognosis for HF. This review discusses an evolutionary mechanism designed to regulate cardiac contraction and relaxation through the most often genetically determined cardiomyopathies associated with HF. In addition, both the myosin inhibitor and myosin activator are promising new treatments for cardiomyopathies. A comprehensive review from genetic mutations to the molecular basis of direct sarcomere modulators will help shed light on future studies for a better characterization of HF etiologies and potential therapeutic targets.
Asunto(s)
Bencilaminas/uso terapéutico , Miosinas Cardíacas/genética , Insuficiencia Cardíaca/tratamiento farmacológico , Terapia Molecular Dirigida , Uracilo/análogos & derivados , Urea/análogos & derivados , Bencilaminas/farmacología , Miosinas Cardíacas/antagonistas & inhibidores , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Humanos , Miocitos Cardíacos/patología , Uracilo/farmacología , Uracilo/uso terapéutico , Urea/farmacología , Urea/uso terapéuticoRESUMEN
Lung cancer is the leading cause of death from cancer in Taiwan and throughout the world. Immunotherapy has revealed promising and significant efficacy in NSCLC, through immune checkpoint inhibition by blocking programmed cell death protein (PD)-1/PD-1 ligand (PD-L1) signaling pathway to restore patients' T-cell immunity. One novel type of long, non-coding RNAs, circular RNAs (circRNAs), are endogenous, stable, and widely expressed in tissues, saliva, blood, urine, and exosomes. Our previous results revealed that the plasma level of hsa_circ_0000190 can be monitored by liquid-biopsy-based droplet digital PCR and may serve as a valuable blood-based biomarker to monitor the disease progression and the efficacy of immunotherapy. In this study, hsa_circ_0000190 was shown to increase the PD-L1 mRNA-mediated soluble PD-L1 (sPD-L1) expression, consequently interfering with the efficacy of anti-PD-L1 antibody and T-cell activation, which may result in immunotherapy resistance and poor outcome. Our results unraveled that hsa_circ_0000190 facilitated the tumorigenesis and immune evasion of NSCLC by upregulating sPD-L1 expression, potentially developing a different aspect in elucidating the molecular immunopathogenesis of NSCLC. Hsa_circ_0000190 upregulation can be an effective indicator for the progression of NSCLC, and hsa_circ_0000190 downregulation may possess a potential therapeutic value for the treatment of NSCLC in combination with immunotherapy.
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Antígeno B7-H1/genética , Carcinogénesis/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Evasión Inmune/genética , Neoplasias Pulmonares/genética , ARN Circular/genética , Regulación hacia Arriba/genética , Células A549 , Animales , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Regulación hacia Abajo/genética , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Linfocitos T/fisiología , TaiwánRESUMEN
The late-onset type of Fabry disease (FD) with GLA IVS4 + 919G > A mutation has been shown to lead to cardiovascular dysfunctions. In order to eliminate variations in other aspects of the genetic background, we established the isogenic control of induced pluripotent stem cells (iPSCs) for the identification of the pathogenetic factors for FD phenotypes through CRISPR/Cas9 genomic editing. We adopted droplet digital PCR (ddPCR) to efficiently capture mutational events, thus enabling isolation of the corrected FD from FD-iPSCs. Both of these exhibited the characteristics of pluripotency and phenotypic plasticity, and they can be differentiated into endothelial cells (ECs). We demonstrated the phenotypic abnormalities in FD iPSC-derived ECs (FD-ECs), including intracellular Gb3 accumulation, autophagic flux impairment, and reactive oxygen species (ROS) production, and these abnormalities were rescued in isogenic control iPSC-derived ECs (corrected FD-ECs). Microarray profiling revealed that corrected FD-derived endothelial cells reversed the enrichment of genes in the pro-inflammatory pathway and validated the downregulation of NF-κB and the MAPK signaling pathway. Our findings highlighted the critical role of ECs in FD-associated vascular dysfunctions by establishing a reliable isogenic control and providing information on potential cellular targets to reduce the morbidity and mortality of FD patients with vascular complications.
Asunto(s)
Células Endoteliales , Enfermedad de Fabry/terapia , Edición Génica , Células Madre Pluripotentes Inducidas , Mutación , alfa-Galactosidasa/genética , Proteína 9 Asociada a CRISPR , Enfermedad de Fabry/enzimología , Enfermedad de Fabry/genética , Enfermedad de Fabry/patología , Humanos , Inflamación , FenotipoRESUMEN
Lung cancer is the leading cause of cancer-related mortality worldwide, and its tumorigenesis involves the accumulation of genetic and epigenetic events in the respiratory epithelium. Epigenetic modifications, such as DNA methylation, RNA modification, and histone modifications, have been widely reported to play an important role in lung cancer development and in other pulmonary diseases. Whereas the functionality of DNA and chromatin modifications referred to as epigenetics is widely characterized, various modifications of RNA nucleotides have recently come into prominence as functionally important. N6-methyladosine (m6A) is the most prevalent internal modification in mRNAs, and its machinery of writers, erasers, and readers is well-characterized. However, several other nucleotide modifications of mRNAs and various noncoding RNAs have also been shown to play an important role in the regulation of biological processes and pathology. Such epitranscriptomic modifications play an important role in regulating various aspects of RNA metabolism, including transcription, translation, splicing, and stability. The dysregulation of epitranscriptomic machinery has been implicated in the pathological processes associated with carcinogenesis including uncontrolled cell proliferation, migration, invasion, and epithelial-mesenchymal transition. In recent years, with the advancement of RNA sequencing technology, high-resolution maps of different modifications in various tissues, organs, or disease models are being constantly reported at a dramatic speed. This facilitates further understanding of the relationship between disease development and epitranscriptomics, shedding light on new therapeutic possibilities. In this review, we summarize the basic information on RNA modifications, including m6A, m1A, m5C, m7G, pseudouridine, and A-to-I editing. We then demonstrate their relation to different kinds of lung diseases, especially lung cancer. By comparing the different roles RNA modifications play in the development processes of different diseases, this review may provide some new insights and offer a better understanding of RNA epigenetics and its involvement in pulmonary diseases.
Asunto(s)
Epigénesis Genética , Enfermedades Pulmonares/genética , Neoplasias Pulmonares/genética , Procesamiento Postranscripcional del ARN , ARN/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Animales , Humanos , Enfermedades Pulmonares/metabolismo , Neoplasias Pulmonares/metabolismo , ARN/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic with high infectivity and mortality has caused severe social and economic impacts worldwide. Growing reports of COVID-19 patients with multi-organ damage indicated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) may also disturb the cardiovascular system. Herein, we used human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs) as the in vitro platform to examine the consequence of SARS-CoV2 infection on iCMs. Differentiated iCMs expressed the primary SARS-CoV2 receptor angiotensin-converting enzyme-II (ACE2) and the transmembrane protease serine type 2 (TMPRSS2) receptor suggesting the susceptibility of iCMs to SARS-CoV2. Following the infection of iCMs with SARS-CoV2, the viral nucleocapsid (N) protein was detected in the host cells, demonstrating the successful infection. Bioinformatics analysis revealed that the SARS-CoV2 infection upregulates several inflammation-related genes, including the proinflammatory cytokine tumor necrosis factor-α (TNF-α). The pretreatment of iCMs with TNF-α for 24 h, significantly increased the expression of ACE2 and TMPRSS2, SASR-CoV2 entry receptors. The TNF-α pretreatment enhanced the entry of GFP-expressing SARS-CoV2 pseudovirus into iCMs, and the neutralization of TNF-α ameliorated the TNF-α-enhanced viral entry. Collectively, SARS-CoV2 elevated TNF-α expression, which in turn enhanced the SARS-CoV2 viral entry. Our findings suggest that, TNF-α may participate in the cytokine storm and aggravate the myocardial damage in COVID-19 patients.
Asunto(s)
COVID-19/complicaciones , Enfermedades Cardiovasculares/inmunología , Síndrome de Liberación de Citoquinas/inmunología , SARS-CoV-2/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/inmunología , COVID-19/patología , COVID-19/virología , Enfermedades Cardiovasculares/virología , Diferenciación Celular , Línea Celular , Biología Computacional , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Síndrome de Liberación de Citoquinas/patología , Síndrome de Liberación de Citoquinas/virología , Humanos , Células Madre Pluripotentes Inducidas , Miocardio/citología , Miocardio/inmunología , Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/virología , Fosfoproteínas/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Serina Endopeptidasas/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Regulación hacia Arriba/inmunología , Internalización del Virus/efectos de los fármacosRESUMEN
Angiotensin-converting enzyme 2 (ACE2) was identified as the main host cell receptor for the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its subsequent infection. In some coronavirus disease 2019 (COVID-19) patients, it has been reported that the nervous tissues and the eyes were also affected. However, evidence supporting that the retina is a target tissue for SARS-CoV-2 infection is still lacking. This present study aimed to investigate whether ACE2 expression plays a role in human retinal neurons during SARS-CoV-2 infection. Human induced pluripotent stem cell (hiPSC)-derived retinal organoids and monolayer cultures derived from dissociated retinal organoids were generated. To validate the potential entry of SARS-CoV-2 infection in the retina, we showed that hiPSC-derived retinal organoids and monolayer cultures endogenously express ACE2 and transmembrane serine protease 2 (TMPRSS2) on the mRNA level. Immunofluorescence staining confirmed the protein expression of ACE2 and TMPRSS2 in retinal organoids and monolayer cultures. Furthermore, using the SARS-CoV-2 pseudovirus spike protein with GFP expression system, we found that retinal organoids and monolayer cultures can potentially be infected by the SARS-CoV-2 pseudovirus. Collectively, our findings highlighted the potential of iPSC-derived retinal organoids as the models for ACE2 receptor-based SARS-CoV-2 infection.
Asunto(s)
Enzima Convertidora de Angiotensina 2/genética , COVID-19/genética , Expresión Génica , Células Madre Pluripotentes Inducidas/citología , Retina/citología , SARS-CoV-2/fisiología , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/metabolismo , Técnicas de Cultivo de Célula , Línea Celular , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Organoides/citología , Organoides/metabolismo , Retina/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Internalización del VirusRESUMEN
BACKGROUND: Glioblastoma (GBM) is the most lethal brain tumor characterized by high morbidity and limited treatment options. Tumor malignancy is usually associated with the epigenetic marks, which coordinate gene expression to ascertain relevant phenotypes. One of such marks is m6A modification of RNA, whose functional effects are dependent on the YTH family m6A reader proteins. METHODS AND RESULTS: In this study, we investigated the expression of five YTH family proteins in different GBM microarray datasets from the Oncomine database, and identified YTHDF1 as the most highly overexpressed member of this family in GBM. By performing the knockdown of YTHDF1 in a GBM cell line, we found that it positively regulates proliferation, chemoresistance and cancer stem cell-like properties. Musashi-1 (MSI1) is a postranscriptional gene expression regulator associated with high oncogenicity in GBM. By knocking down and overexpressing MSI1, we found that it positively regulates YTHDF1 expression. The inhibitory effects imposed on the processes of proliferation and migration by YTHDF1 knockdown were shown to be partially rescued by concomitant overexpression of MSI1. MSI1 and YTHDF1 were shown to be positively correlated in clinical glioma samples, and their concomitant upregulation was associated with decreased survival of glioma patients. We identified the direct regulation of YTHDF1 by MSI1. CONCLUSIONS: Given the fact that both proteins are master regulators of gene expression, and both of them are unfavorable factors in GBM, we suggest that in any future studies aimed to uncover the prognostic value and therapy potential, these two proteins should be considered together.
RESUMEN
Neuromuscular diseases (NMDs) belong to a class of functional impairments that cause dysfunctions of the motor neuron-muscle functional axis components. Inherited monogenic neuromuscular disorders encompass both muscular dystrophies and motor neuron diseases. Understanding of their causative genetic defects and pathological genetic mechanisms has led to the unprecedented clinical translation of genetic therapies. Challenged by a broad range of gene defect types, researchers have developed different approaches to tackle mutations by hijacking the cellular gene expression machinery to minimize the mutational damage and produce the functional target proteins. Such manipulations may be directed to any point of the gene expression axis, such as classical gene augmentation, modulating premature termination codon ribosomal bypass, splicing modification of pre-mRNA, etc. With the soar of the CRISPR-based gene editing systems, researchers now gravitate toward genome surgery in tackling NMDs by directly correcting the mutational defects at the genome level and expanding the scope of targetable NMDs. In this article, we will review the current development of gene therapy and focus on NMDs that are available in published reports, including Duchenne Muscular Dystrophy (DMD), Becker muscular dystrophy (BMD), X-linked myotubular myopathy (XLMTM), Spinal Muscular Atrophy (SMA), and Limb-girdle muscular dystrophy Type 2C (LGMD2C).
Asunto(s)
Edición Génica/métodos , Terapia Genética/métodos , Enfermedades Neuromusculares/genética , Animales , Sistemas CRISPR-Cas , Ensayos Clínicos como Asunto , Humanos , Enfermedades Neuromusculares/terapiaRESUMEN
The sudden outbreak of 2019 novel coronavirus (2019-nCoV, later named SARS-CoV-2) in Wuhan, China, which rapidly grew into a global pandemic, marked the third introduction of a virulent coronavirus into the human society, affecting not only the healthcare system, but also the global economy. Although our understanding of coronaviruses has undergone a huge leap after two precedents, the effective approaches to treatment and epidemiological control are still lacking. In this article, we present a succinct overview of the epidemiology, clinical features, and molecular characteristics of SARS-CoV-2. We summarize the current epidemiological and clinical data from the initial Wuhan studies, and emphasize several features of SARS-CoV-2, which differentiate it from SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), such as high variability of disease presentation. We systematize the current clinical trials that have been rapidly initiated after the outbreak of COVID-19 pandemic. Whereas the trials on SARS-CoV-2 genome-based specific vaccines and therapeutic antibodies are currently being tested, this solution is more long-term, as they require thorough testing of their safety. On the other hand, the repurposing of the existing therapeutic agents previously designed for other virus infections and pathologies happens to be the only practical approach as a rapid response measure to the emergent pandemic, as most of these agents have already been tested for their safety. These agents can be divided into two broad categories, those that can directly target the virus replication cycle, and those based on immunotherapy approaches either aimed to boost innate antiviral immune responses or alleviate damage induced by dysregulated inflammatory responses. The initial clinical studies revealed the promising therapeutic potential of several of such drugs, including favipiravir, a broad-spectrum antiviral drug that interferes with the viral replication, and hydroxychloroquine, the repurposed antimalarial drug that interferes with the virus endosomal entry pathway. We speculate that the current pandemic emergency will be a trigger for more systematic drug repurposing design approaches based on big data analysis.
Asunto(s)
Antivirales/uso terapéutico , Betacoronavirus , Infecciones por Coronavirus , Factores Inmunológicos/uso terapéutico , Pandemias , Neumonía Viral , Vacunas Virales , Betacoronavirus/química , Betacoronavirus/genética , Betacoronavirus/inmunología , Betacoronavirus/fisiología , COVID-19 , Vacunas contra la COVID-19 , Ensayos Clínicos como Asunto , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/terapia , Infecciones por Coronavirus/virología , Genoma Viral , Humanos , Inmunización Pasiva , Neumonía Viral/diagnóstico , Neumonía Viral/epidemiología , Neumonía Viral/terapia , Neumonía Viral/virología , SARS-CoV-2 , Tratamiento Farmacológico de COVID-19 , Sueroterapia para COVID-19RESUMEN
PARP1 and poly(ADP-ribosyl)ation (PARylation) have been shown to be essential for the initial steps of cellular reprogramming. However, the mechanism underlying PARP1/PARylation-regulated activation of pluripotency loci remains undetermined. Here, we demonstrate that CHD1L, a DNA helicase, possesses chromatin remodeling activity and interacts with PARP1/PARylation in regulating pluripotency during reprogramming. We found that this interaction is mediated through the interplay of the CHD1L macro-domain and the PAR moiety of PARylated-PARP1. Chromatin immunoprecipitation assays demonstrated the co-occupancy of CHD1L and PARP1 at Pou5f1, Nanog, and Esrrb pluripotency loci. Knockdown of CHD1L significantly blocked the binding activity of PARP1 at pluripotency loci and inhibited the efficiency of PARP1-driven reprogramming. Notably, we found that CHD1L-promoted reprogramming requires both a PARP1-interacting domain and DNA helicase activity, partly contributing to the chromatin-remodeling states of pluripotency loci. Taken together, these results identify CHD1L as a key chromatin remodeler involved in PARP1/PARylation-regulated early-stage reprogramming and pluripotency in stem cells.
Asunto(s)
Reprogramación Celular/genética , Ensamble y Desensamble de Cromatina/genética , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Células Madre Pluripotentes , Poli(ADP-Ribosa) Polimerasas/genética , Animales , Diferenciación Celular/genética , ADN Helicasas/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/biosíntesis , Ratones , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/biosíntesis , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/biosíntesis , Receptores de Estrógenos/biosíntesisRESUMEN
The CRISPR/Cas9 Genome-editing system has revealed promising potential for generating gene mutation, deletion, and correction in human cells. Application of this powerful tool in Fabry disease (FD), however, still needs to be explored. Enzyme replacement therapy (ERT), a regular administration of recombinant human α Gal A (rhα-GLA), is a currently available and effective treatment to clear the accumulated Gb3 in FD patients. However, the short half-life of rhα-GLA in human body limits its application. Moreover, lack of an appropriate in vitro disease model restricted the high-throughput screening of drugs for improving ERT efficacy. Therefore, it is worth establishing a large-expanded in vitro FD model for screening potential candidates, which can enhance and prolong ERT potency. Using CRISPR/Cas9-mediated gene knockout of GLA in HEK-293T cells, we generated GLA-null cells to investigate rhα-GLA cellular pharmacokinetics. The half-life of administrated rhα-GLA was around 24 h in GLA-null cells; co-administration of proteasome inhibitor MG132 and rhα-GLA significantly restored the GLA enzyme activity by two-fold compared with rhα-GLA alone. Furthermore, co-treatment of rhα-GLA/MG132 in patient-derived fibroblasts increased Gb3 clearance by 30%, compared with rhα-GLA treatment alone. Collectively, the CRISPR/Cas9-mediated GLA-knockout HEK-293T cells provide an in vitro FD model for evaluating the intracellular pharmacokinetics of the rhα-GLA as well as for screening candidates to prolong rhα-GLA potency. Using this model, we demonstrated that MG132 prolongs rhα-GLA half-life and enhanced Gb3 clearance, shedding light on the direction of enhancing ERT efficacy in FD treatment.
Asunto(s)
Sistemas CRISPR-Cas/genética , Evaluación Preclínica de Medicamentos , Enfermedad de Fabry/tratamiento farmacológico , Técnicas de Inactivación de Genes , alfa-Galactosidasa/metabolismo , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Secuencia de Bases , Muerte Celular/efectos de los fármacos , Estabilidad de Enzimas/efectos de los fármacos , Fibroblastos/metabolismo , Edición Génica , Marcación de Gen , Células HEK293 , Humanos , Espacio Intracelular/metabolismo , Leupeptinas/administración & dosificación , Leupeptinas/farmacología , Modelos Biológicos , Proteínas Recombinantes/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismoRESUMEN
Coronaviruses (CoVs) are enveloped single-stranded RNA viruses that predominantly attack the human respiratory system. In recent decades, several deadly human CoVs, including SARS-CoV, SARS-CoV-2, and MERS-CoV, have brought great impact on public health and economics. However, their high infectivity and the demand for high biosafety level facilities restrict the pathogenesis research of CoV infection. Exacerbated inflammatory cell infiltration is associated with poor prognosis in CoV-associated diseases. In this study, we used human CoV 229E (HCoV-229E), a CoV associated with relatively fewer biohazards, to investigate the pathogenesis of CoV infection and the regulation of neutrophil functions by CoV-infected lung cells. Induced pluripotent stem cell (iPSC)-derived alveolar epithelial type II cells (iAECIIs) exhibiting specific biomarkers and phenotypes were employed as an experimental model for CoV infection. After infection, the detection of dsRNA, S, and N proteins validated the infection of iAECIIs with HCoV-229E. The culture medium conditioned by the infected iAECIIs promoted the migration of neutrophils as well as their adhesion to the infected iAECIIs. Cytokine array revealed the elevated secretion of cytokines associated with chemotaxis and adhesion into the conditioned media from the infected iAECIIs. The importance of IL-8 secretion and ICAM-1 expression for neutrophil migration and adhesion, respectively, was demonstrated by using neutralizing antibodies. Moreover, next-generation sequencing analysis of the transcriptome revealed the upregulation of genes associated with cytokine signaling. To summarize, we established an in vitro model of CoV infection that can be applied for the study of the immune system perturbations during severe coronaviral disease.
Asunto(s)
Células Epiteliales Alveolares , Células Madre Pluripotentes Inducidas , Neutrófilos , Humanos , Neutrófilos/inmunología , Neutrófilos/virología , Células Madre Pluripotentes Inducidas/virología , Células Epiteliales Alveolares/virología , COVID-19/virología , COVID-19/inmunología , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , SARS-CoV-2/patogenicidad , SARS-CoV-2/fisiología , SARS-CoV-2/inmunología , Interleucina-8/genética , Interleucina-8/metabolismoRESUMEN
The blood supply in the retina ensures photoreceptor function and maintains regular vision. Leber's hereditary optic neuropathy (LHON), caused by the mitochondrial DNA mutations that deteriorate complex I activity, is characterized by progressive vision loss. Although some reports indicated retinal vasculature abnormalities as one of the comorbidities in LHON, the paracrine influence of LHON-affected retinal ganglion cells (RGCs) on vascular endothelial cell physiology remains unclear. To address this, we established an in vitro model of mitochondrial complex I deficiency using induced pluripotent stem cell-derived RGCs (iPSC-RGCs) treated with a mitochondrial complex I inhibitor rotenone (Rot) to recapitulate LHON pathologies. The secretomes from Rot-treated iPSC-RGCs (Rot-iPSC-RGCs) were collected, and their treatment effect on human umbilical vein endothelial cells (HUVECs) was studied. Rot induced LHON-like characteristics in iPSC-RGCs, including decreased mitochondrial complex I activity and membrane potential, and increased mitochondrial reactive oxygen species (ROS) and apoptosis, leading to mitochondrial dysfunction. When HUVECs were exposed to conditioned media (CM) from Rot-iPSC-RGCs, the angiogenesis of HUVECs was suppressed compared to those treated with CM from control iPSC-RGCs (Ctrl-iPSC-RGCs). Angiogenesis-related proteins were altered in the secretomes from Rot-iPSC-RGC-derived CM, particularly angiopoietin, MMP-9, uPA, collagen XVIII, and VEGF were reduced. Notably, GeneMANIA analysis indicated that VEGFA emerged as the pivotal angiogenesis-related protein among the identified proteins secreted by health iPSC-RGCs but reduced in the secretomes from Rot-iPSC-RGCs. Quantitative real-time PCR and western blots confirmed the reduction of VEGFA at both transcription and translation levels, respectively. Our study reveals that Rot-iPSC-RGCs establish a microenvironment to diminish the angiogenic potential of vascular cells nearby, shedding light on the paracrine regulation of LHON-affected RGCs on retinal vasculature.
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Células Endoteliales de la Vena Umbilical Humana , Células Madre Pluripotentes Inducidas , Atrofia Óptica Hereditaria de Leber , Células Ganglionares de la Retina , Humanos , Atrofia Óptica Hereditaria de Leber/metabolismo , Atrofia Óptica Hereditaria de Leber/patología , Atrofia Óptica Hereditaria de Leber/genética , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/patología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Fenotipo , Especies Reactivas de Oxígeno/metabolismo , Rotenona/farmacología , Medios de Cultivo Condicionados/farmacología , Apoptosis/efectos de los fármacos , Complejo I de Transporte de Electrón/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Neovascularización Patológica/metabolismo , AngiogénesisRESUMEN
Mesenchymal stem cells (MSCs) are multipotent cells derived from adult human tissues that have the ability to proliferate in vitro and maintain their multipotency, making them attractive cell sources for regenerative medicine. However, MSCs reportedly show limited proliferative capacity with inconsistent therapeutic outcomes due to their heterogeneous nature. On the other hand, induced pluripotent stem cells (iPSC) have emerged as an alternative source for the production of various specialized cell types via their ability to differentiate from all three primary germ layers, leading to applications in regenerative medicine, disease modeling, and drug therapy. Notably, iPSCs can differentiate into MSCs in monolayer, commonly referred to as induced mesenchymal stem cells (iMSCs). These cells show superior therapeutic qualities compared with adult MSCs as the applications of the latter are restricted by passage number and autoimmune rejection when applied in tissue regeneration trials. Furthermore, increasing evidence shows that the therapeutic properties of stem cells are a consequence of the paracrine effects mediated by their secretome such as from exosomes, a type of extracellular vesicle secreted by most cell types. Several studies that investigated the potential of exosomes in regenerative medicine and therapy have revealed promising results. Therefore, this review focuses on the recent findings of exosomes secreted from iMSCs as a potential noncell-based therapy.
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Exosomas , Células Madre Pluripotentes Inducidas , Células Madre Mesenquimatosas , Adulto , Humanos , Diferenciación Celular , Exosomas/metabolismo , Células Madre Mesenquimatosas/metabolismoRESUMEN
Radiotherapy (RT) is currently only used in children with high-risk neuroblastoma (NB) due to concerns of long-term side effects as well as lack of effective adjuvant. Calreticulin (CALR) has served distinct physiological roles in cancer malignancies; nonetheless, impact of radiation on chaperones and molecular roles they play remains largely unknown. In present study, we systemically analyzed correlation between CALR and NB cells of different malignancies to investigate potential role of CALR in mediating radioresistance of NB. Our data revealed that more malignant NB cells are correlated to lower CALR expression, greater radioresistance, and elevated stemness as indicated by colony- and neurospheroid-forming abilities and vice versa. Of note, manipulating CALR expression in NB cells of varying endogenous CALR expression manifested changes in not only stemness but also radioresistant properties of those NB cells. Further, CALR overexpression resulted in greatly enhanced ROS and led to increased secretion of proinflammatory cytokines. Importantly, growth of NB tumors was significantly hampered by CALR overexpression and was synergistically ablated when RT was also administered. Collectively, our current study unraveled a new notion of utilizing CALR expression in malignant NB to diminish cancer stemness and mitigate radioresistance to achieve favorable therapeutic outcome for NB.
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Calreticulina , Neuroblastoma , Niño , Humanos , Adyuvantes Inmunológicos , Calreticulina/genética , Calreticulina/metabolismo , Línea Celular Tumoral , Neuroblastoma/patología , Neuroblastoma/radioterapia , Tolerancia a RadiaciónRESUMEN
N6-methyladenosine (m6A), one of the most prevalent mRNA modifications in eukaryotes, plays a critical role in modulating both biological and pathological processes. However, it is unknown whether mutant p53 neomorphic oncogenic functions exploit dysregulation of m6A epitranscriptomic networks. Here, we investigate Li-Fraumeni syndrome (LFS)-associated neoplastic transformation driven by mutant p53 in iPSC-derived astrocytes, the cell-of-origin of gliomas. We find that mutant p53 but not wild-type (WT) p53 physically interacts with SVIL to recruit the H3K4me3 methyltransferase MLL1 to activate the expression of m6A reader YTHDF2, culminating in an oncogenic phenotype. Aberrant YTHDF2 upregulation markedly hampers expression of multiple m6A-marked tumor-suppressing transcripts, including CDKN2B and SPOCK2, and induces oncogenic reprogramming. Mutant p53 neoplastic behaviors are significantly impaired by genetic depletion of YTHDF2 or by pharmacological inhibition using MLL1 complex inhibitors. Our study reveals how mutant p53 hijacks epigenetic and epitranscriptomic machinery to initiate gliomagenesis and suggests potential treatment strategies for LFS gliomas.
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Glioma , Síndrome de Li-Fraumeni , Humanos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Síndrome de Li-Fraumeni/genética , Transformación Celular Neoplásica/genética , Glioma/genética , Proteoglicanos/metabolismoRESUMEN
MicroRNA-200c (miR200c) is emerging as an important regulator of tumourigenicity and cancer metastasis with a strong capacity for inducing epithelial-mesenchymal transitions. However, the role of miR200c in head and neck squamous cell carcinoma (HNSCC) and HNSCC-associated cancer stem cells (HNSCC-CSCs) is unknown. In this study, the expression of miR200c in the regional metastatic lymph node of HNSCC tissues was significantly decreased, but BMI1 expression was increased as compared to parental tumours. Importantly, site-directed mutagenesis with a luciferase reporter assay showed that miR200c targeted the 3' UTR of BMI1 in HNSCC cells. Isolated HNSCC-derived ALDH1(+) /CD44(+) cells displayed CSC-like tumour initiating and radio-resistant properties. The expression levels of miR200c were significantly down-regulated while BMI1 was increased in HNSCC-ALDH1(+) /CD44(+) compared to the other subsets of HNSCC cells. Furthermore, increased miR200c expression or knockdown of BMI1 could significantly inhibit the malignant CSC-like properties of ALDH1(+) /CD44(+) cells. miR200c over-expression further down-regulated the expressions of ZEB1, Snail and N-cadherin, but up-regulated E-cadherin expression in ALDH1(+) /CD44(+) cells. Finally, a xenotransplantion study confirmed that over-expression of miR200c or BMI1 knockdown effectively inhibited the lung metastatic ability and prolonged the survival rate of ALDH1(+) /CD44(+) -transplanted mice. In summary, miR200c negatively modulates the expression of BMI1 but also significantly inhibits the metastatic capability of epithelial-mesenchymal transitions in malignant HNSCC by reducing the expression of BMI1/ZEB1. Restoration of miR200c in HNSCC and CSCs may be a promising therapeutic approach.