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1.
Anticancer Res ; 41(4): 2117-2122, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33813422

RESUMEN

BACKGROUND/AIM: Stanniocalcin2 (STC2) is associated with proliferation, invasion, and metastasis in various cancers. We examined the clinical significance of STC2 mRNA expression in patients with colorectal cancer (CRC). PATIENTS AND METHODS: Relative expression levels of STC2 mRNA in CRC tissues and corresponding normal mucosa obtained from 202 patients were measured using quantitative real-time reverse transcriptase-polymerase chain reaction. RESULTS: Expression of STC2 mRNA was higher in the cancer tissue than in the adjacent normal mucosa. STC2 mRNA expression in cancer tissues was associated with tumour size, liver metastasis, venous invasion, and lymph node metastasis. High expression of STC2 mRNA was significantly associated with poorer postoperative survival (p=0.0003). Multivariate analysis showed that high expression of STC2 mRNA was an independent predictor of postoperative survival. CONCLUSION: High expression of STC2 mRNA in CRC tissue may be a useful prognostic marker in patients with CRC.


Asunto(s)
Adenocarcinoma/diagnóstico , Biomarcadores de Tumor/genética , Neoplasias Colorrectales/diagnóstico , Glicoproteínas/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Adenocarcinoma/genética , Adenocarcinoma/mortalidad , Adenocarcinoma/cirugía , Anciano , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/cirugía , Femenino , Regulación Neoplásica de la Expresión Génica , Glicoproteínas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Metástasis Linfática , Masculino , Persona de Mediana Edad , Periodo Posoperatorio , Pronóstico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Supervivencia , Resultado del Tratamiento
2.
Nat Commun ; 12(1): 1547, 2021 03 11.
Artículo en Inglés | MEDLINE | ID: mdl-33707436

RESUMEN

Hypertension, exercise, and pregnancy are common triggers of cardiac remodeling, which occurs primarily through the hypertrophy of individual cardiomyocytes. During hypertrophy, stress-induced signal transduction increases cardiomyocyte transcription and translation, which promotes the addition of new contractile units through poorly understood mechanisms. The cardiomyocyte microtubule network is also implicated in hypertrophy, but via an unknown role. Here, we show that microtubules are indispensable for cardiac growth via spatiotemporal control of the translational machinery. We find that the microtubule motor Kinesin-1 distributes mRNAs and ribosomes along microtubule tracks to discrete domains within the cardiomyocyte. Upon hypertrophic stimulation, microtubules redistribute mRNAs and new protein synthesis to sites of growth at the cell periphery. If the microtubule network is disrupted, mRNAs and ribosomes collapse around the nucleus, which results in mislocalized protein synthesis, the rapid degradation of new proteins, and a failure of growth, despite normally increased translation rates. Together, these data indicate that mRNAs and ribosomes are actively transported to specific sites to facilitate local translation and assembly of contractile units, and suggest that properly localized translation - and not simply translation rate - is a critical determinant of cardiac hypertrophy. In this work, we find that microtubule based-transport is essential to couple augmented transcription and translation to productive cardiomyocyte growth during cardiac stress.


Asunto(s)
Cardiomegalia/patología , Microtúbulos/metabolismo , Miocitos Cardíacos/patología , Biosíntesis de Proteínas/fisiología , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Animales , Remodelación Atrial/fisiología , Transporte Biológico/fisiología , Células Cultivadas , Humanos , Cinesina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Transducción de Señal/fisiología , Remodelación Ventricular/fisiología
3.
Medicine (Baltimore) ; 100(12): e25093, 2021 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-33761671

RESUMEN

ABSTRACT: Based on the Thompson classification of intervertebral discs (IVDs), we systematically analyzed gene expression differences between severely degenerated and mildly degenerated IVDs and explored the underlying molecular mechanisms using bioinformatics methods and multichip integration. We used multiomics analysis, includes mRNA microarray and methylation chips, to explore the genetic network and mechanisms of lumbar disc herniation (LDH). Subsequently, the Combat function of the R language SVA package was applied to eliminate heterogeneity between the gene expression data. And the protein-protein interaction (PPI) network, gene ontology (GO), and molecular pathways were used to constructs the mechanisms network. Consequently, we obtained 149 differentially expressed genes. Related molecular pathways are the following: ribosome activity, oxidative phosphorylation, extracellular matrix response. Besides, through PPI network analysis, genes with higher connectivity such as UBA52, RPLP0, RPL3, RPLP2, and RPL27 were also identified, suggesting that they play important regulatory roles in the complex network associated with LDH. Additionally, cg12556991 (RPL27) and cg06852319 (RPLP0) were found to be LDH-related candidate DNA methylation modification sites in the IVDs tissue of LDH patients. In conclusions, ribosome activity, oxidative phosphorylation, and extracellular matrix response may be potential molecular mechanisms underlying LDH, while hub genes involved in UBA52, RPLP0, RPL3, RPLP2, and RPL27, and candidate DNA methylation modification sites of cg12556991and cg06852319 are likely key regulators in the development of LDH.


Asunto(s)
Metilación de ADN/genética , Matriz Extracelular/genética , Desplazamiento del Disco Intervertebral/genética , Fosforilación/genética , Proteínas Ribosómicas/genética , Biología Computacional , Expresión Génica/genética , Perfilación de la Expresión Génica , Ontología de Genes , Redes Reguladoras de Genes/genética , Humanos , Vértebras Lumbares/metabolismo , Análisis por Micromatrices , Mapas de Interacción de Proteínas/genética , ARN Mensajero/metabolismo
4.
Nat Commun ; 12(1): 1475, 2021 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-33674569

RESUMEN

Accurate measurements of promoter activities are crucial for predictably building genetic systems. Here we report a method to simultaneously count plasmid DNA, RNA transcripts, and protein expression in single living bacteria. From these data, the activity of a promoter in units of RNAP/s can be inferred. This work facilitates the reporting of promoters in absolute units, the variability in their activity across a population, and their quantitative toll on cellular resources, all of which provide critical insights for cellular engineering.


Asunto(s)
Bacterias/genética , Variaciones en el Número de Copia de ADN , Plásmidos , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Transcripción Genética
5.
Nat Commun ; 12(1): 1464, 2021 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-33674610

RESUMEN

The interpretation of high throughput sequencing data is limited by our incomplete functional understanding of coding and non-coding transcripts. Reliably predicting the function of such transcripts can overcome this limitation. Here we report the use of a consensus independent component analysis and guilt-by-association approach to predict over 23,000 functional groups comprised of over 55,000 coding and non-coding transcripts using publicly available transcriptomic profiles. We show that, compared to using Principal Component Analysis, Independent Component Analysis-derived transcriptional components enable more confident functionality predictions, improve predictions when new members are added to the gene sets, and are less affected by gene multi-functionality. Predictions generated using human or mouse transcriptomic data are made available for exploration in a publicly available web portal.


Asunto(s)
Perfilación de la Expresión Génica/métodos , Transcriptoma , Animales , Biología Computacional , Técnicas de Inactivación de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , ARN Mensajero/metabolismo
6.
Nat Commun ; 12(1): 1716, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741917

RESUMEN

Cancer stem cells (CSCs) are a small but critical cell population for cancer biology since they display inherent resistance to standard therapies and give rise to metastases. Despite accruing evidence establishing a link between deregulation of epitranscriptome-related players and tumorigenic process, the role of messenger RNA (mRNA) modifications in the regulation of CSC properties remains poorly understood. Here, we show that the cytoplasmic pool of fat mass and obesity-associated protein (FTO) impedes CSC abilities in colorectal cancer through its N6,2'-O-dimethyladenosine (m6Am) demethylase activity. While m6Am is strategically located next to the m7G-mRNA cap, its biological function is not well understood and has not been addressed in cancer. Low FTO expression in patient-derived cell lines elevates m6Am level in mRNA which results in enhanced in vivo tumorigenicity and chemoresistance. Inhibition of the nuclear m6Am methyltransferase, PCIF1/CAPAM, fully reverses this phenotype, stressing the role of m6Am modification in stem-like properties acquisition. FTO-mediated regulation of m6Am marking constitutes a reversible pathway controlling CSC abilities. Altogether, our findings bring to light the first biological function of the m6Am modification and its potential adverse consequences for colorectal cancer management.


Asunto(s)
Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Neoplasias Colorrectales/metabolismo , Citoplasma/metabolismo , Desmetilación , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Línea Celular Tumoral , Núcleo Celular/metabolismo , Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Humanos , Metiltransferasas/metabolismo , Proteínas Nucleares/metabolismo , ARN Mensajero/metabolismo
7.
Nat Commun ; 12(1): 1515, 2021 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-33750777

RESUMEN

Ribosome-profiling has uncovered pervasive translation in non-canonical open reading frames, however the biological significance of this phenomenon remains unclear. Using genetic variation from 71,702 human genomes, we assess patterns of selection in translated upstream open reading frames (uORFs) in 5'UTRs. We show that uORF variants introducing new stop codons, or strengthening existing stop codons, are under strong negative selection comparable to protein-coding missense variants. Using these variants, we map and validate gene-disease associations in two independent biobanks containing exome sequencing from 10,900 and 32,268 individuals, respectively, and elucidate their impact on protein expression in human cells. Our results suggest translation disrupting mechanisms relating uORF variation to reduced protein expression, and demonstrate that translation at uORFs is genetically constrained in 50% of human genes.


Asunto(s)
Enfermedad/genética , Sistemas de Lectura Abierta , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regiones no Traducidas 5' , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Estudios de Asociación Genética , Genoma Humano , Humanos , Fenotipo , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas/genética , Receptor EphB2
8.
Nat Commun ; 12(1): 1537, 2021 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-33750804

RESUMEN

Quaking RNA binding protein (QKI) is essential for oligodendrocyte development as myelination requires myelin basic protein mRNA regulation and localization by the cytoplasmic isoforms (e.g., QKI-6). QKI-6 is also highly expressed in astrocytes, which were recently demonstrated to have regulated mRNA localization. Here, we define the targets of QKI in the mouse brain via CLIPseq and we show that QKI-6 binds 3'UTRs of a subset of astrocytic mRNAs. Binding is also enriched near stop codons, mediated partially by QKI-binding motifs (QBMs), yet spreads to adjacent sequences. Using a viral approach for mosaic, astrocyte-specific gene mutation with simultaneous translating RNA sequencing (CRISPR-TRAPseq), we profile ribosome associated mRNA from QKI-null astrocytes in the mouse brain. This demonstrates a role for QKI in stabilizing CLIP-defined direct targets in astrocytes in vivo and further shows that QKI mutation disrupts the transcriptional changes for a discrete subset of genes associated with astrocyte maturation.


Asunto(s)
Astrocitos/metabolismo , Encéfalo/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Animales , Secuencia de Bases , Citoplasma/metabolismo , Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/metabolismo , Isoformas de Proteínas , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN , Transcriptoma
9.
Nat Commun ; 12(1): 1458, 2021 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-33674589

RESUMEN

Epitranscriptomic modifications can impact behavior. Here, we used Drosophila melanogaster to study N6-methyladenosine (m6A), the most abundant modification of mRNA. Proteomic and functional analyses confirm its nuclear (Ythdc1) and cytoplasmic (Ythdf) YTH domain proteins as major m6A binders. Assays of short term memory in m6A mutants reveal neural-autonomous requirements of m6A writers working via Ythdf, but not Ythdc1. Furthermore, m6A/Ythdf operate specifically via the mushroom body, the center for associative learning. We map m6A from wild-type and Mettl3 mutant heads, allowing robust discrimination of Mettl3-dependent m6A sites that are highly enriched in 5' UTRs. Genomic analyses indicate that Drosophila m6A is preferentially deposited on genes with low translational efficiency and that m6A does not affect RNA stability. Nevertheless, functional tests indicate a role for m6A/Ythdf in translational activation. Altogether, our molecular genetic analyses and tissue-specific m6A maps reveal selective behavioral and regulatory defects for the Drosophila Mettl3/Ythdf pathway.


Asunto(s)
Adenosina/análogos & derivados , Adenosina/metabolismo , Drosophila melanogaster/fisiología , Aprendizaje/fisiología , Memoria/fisiología , Regiones no Traducidas 5' , Adenosina/genética , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Femenino , Proteínas Nucleares/metabolismo , Proteómica , Estabilidad del ARN , ARN Mensajero/metabolismo
10.
Phys Rev Lett ; 126(7): 078101, 2021 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-33666486

RESUMEN

Gene expression is a stochastic process. Despite the increase of protein numbers in growing cells, the protein concentrations are often found to be confined within small ranges throughout the cell cycle. Generally, the noise in protein concentration can be decomposed into an intrinsic and an extrinsic component, where the former vanishes for high expression levels. Considering the time trajectory of protein concentration as a random walker in the concentration space, an effective restoring force (with a corresponding "spring constant") must exist to prevent the divergence of concentration due to random fluctuations. In this work, we prove that the magnitude of the effective spring constant is directly related to the fraction of intrinsic noise in the total protein concentration noise. We show that one can infer the magnitude of intrinsic, extrinsic, and measurement noises of gene expression solely based on time-resolved data of protein concentration, without any a priori knowledge of the underlying gene expression dynamics. We apply this method to experimental data of single-cell bacterial gene expression. The results allow us to estimate the average copy numbers and the translation burst parameters of the studied proteins.


Asunto(s)
Modelos Genéticos , Procesos de Crecimiento Celular/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Distribución de Poisson , Proteínas/genética , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo
11.
Nat Commun ; 12(1): 1361, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33649327

RESUMEN

Sperm contributes diverse RNAs to the zygote. While sperm small RNAs have been shown to impact offspring phenotypes, our knowledge of the sperm transcriptome, especially the composition of long RNAs, has been limited by the lack of sensitive, high-throughput experimental techniques that can distinguish intact RNAs from fragmented RNAs, known to abound in sperm. Here, we integrate single-molecule long-read sequencing with short-read sequencing to detect sperm intact RNAs (spiRNAs). We identify 3440 spiRNA species in mice and 4100 in humans. The spiRNA profile consists of both mRNAs and long non-coding RNAs, is evolutionarily conserved between mice and humans, and displays an enrichment in mRNAs encoding for ribosome. In sum, we characterize the landscape of intact long RNAs in sperm, paving the way for future studies on their biogenesis and functions. Our experimental and bioinformatics approaches can be applied to other tissues and organisms to detect intact transcripts.


Asunto(s)
Secuencia Conservada/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN/genética , Imagen Individual de Molécula , Espermatozoides/metabolismo , Animales , Evolución Molecular , Ontología de Genes , Humanos , Masculino , Ratones Endogámicos C57BL , ARN/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Testículo/metabolismo , Transcriptoma/genética
12.
Nat Commun ; 12(1): 1368, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33649334

RESUMEN

The homeostasis of the gut epithelium relies upon continuous renewal and proliferation of crypt-resident intestinal epithelial stem cells (IESCs). Wnt/ß-catenin signaling is required for IESC maintenance, however, it remains unclear how this pathway selectively governs the identity and proliferative decisions of IESCs. Here, we took advantage of knock-in mice harboring transgenic ß-catenin alleles with mutations that specifically impair the recruitment of N- or C-terminal transcriptional co-factors. We show that C-terminally-recruited transcriptional co-factors of ß-catenin act as all-or-nothing regulators of Wnt-target gene expression. Blocking their interactions with ß-catenin rapidly induces loss of IESCs and intestinal homeostasis. Conversely, N-terminally recruited co-factors fine-tune ß-catenin's transcriptional output to ensure proper self-renewal and proliferative behaviour of IESCs. Impairment of N-terminal interactions triggers transient hyperproliferation of IESCs, eventually resulting in exhaustion of the self-renewing stem cell pool. IESC mis-differentiation, accompanied by unfolded protein response stress and immune infiltration, results in a process resembling aberrant "villisation" of intestinal crypts. Our data suggest that IESC-specific Wnt/ß-catenin output requires selective modulation of gene expression by transcriptional co-factors.


Asunto(s)
Mucosa Intestinal/citología , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , beta Catenina/química , beta Catenina/metabolismo , Algoritmos , Animales , Secuencia de Bases , Diferenciación Celular , Proliferación Celular , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Homeostasis , Hiperplasia , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Proteínas Mutantes/metabolismo , Mutación/genética , Organoides/metabolismo , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal
13.
Nat Commun ; 12(1): 1352, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33649340

RESUMEN

Local translation allows for a spatial control of gene expression. Here, we use high-throughput smFISH to screen centrosomal protein-coding genes, and we describe 8 human mRNAs accumulating at centrosomes. These mRNAs localize at different stages during cell cycle with a remarkable choreography, indicating a finely regulated translational program at centrosomes. Interestingly, drug treatments and reporter analyses reveal a common translation-dependent localization mechanism requiring the nascent protein. Using ASPM and NUMA1 as models, single mRNA and polysome imaging reveals active movements of endogenous polysomes towards the centrosome at the onset of mitosis, when these mRNAs start localizing. ASPM polysomes associate with microtubules and localize by either motor-driven transport or microtubule pulling. Remarkably, the Drosophila orthologs of the human centrosomal mRNAs also localize to centrosomes and also require translation. These data identify a conserved family of centrosomal mRNAs that localize by active polysome transport mediated by nascent proteins.


Asunto(s)
Centrosoma/metabolismo , Polirribosomas/metabolismo , Transporte de ARN , Animales , Proteínas de Ciclo Celular/metabolismo , Centrosoma/efectos de los fármacos , Cicloheximida/farmacología , Drosophila/genética , Células HeLa , Humanos , Mitosis/efectos de los fármacos , Sistemas de Lectura Abierta/genética , Polirribosomas/efectos de los fármacos , Puromicina/farmacología , Transporte de ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo
14.
Nat Commun ; 12(1): 1351, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33649372

RESUMEN

Exon junction complexes (EJCs) mark untranslated spliced mRNAs and are crucial for the mRNA lifecycle. An imbalance in EJC dosage alters mouse neural stem cell (mNSC) division and is linked to human neurodevelopmental disorders. In quiescent mNSC and immortalized human retinal pigment epithelial (RPE1) cells, centrioles form a basal body for ciliogenesis. Here, we report that EJCs accumulate at basal bodies of mNSC or RPE1 cells and decline when these cells differentiate or resume growth. A high-throughput smFISH screen identifies two transcripts accumulating at centrosomes in quiescent cells, NIN and BICD2. In contrast to BICD2, the localization of NIN transcripts is EJC-dependent. NIN mRNA encodes a core component of centrosomes required for microtubule nucleation and anchoring. We find that EJC down-regulation impairs both pericentriolar material organization and ciliogenesis. An EJC-dependent mRNA trafficking towards centrosome and basal bodies might contribute to proper mNSC division and brain development.


Asunto(s)
Centrosoma/metabolismo , Cilios/metabolismo , Exones/genética , Transporte de ARN , ARN Mensajero/metabolismo , Animales , Autoantígenos/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Proteínas del Citoesqueleto/metabolismo , ARN Helicasas DEAD-box/metabolismo , Factor 4A Eucariótico de Iniciación/metabolismo , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Células-Madre Neurales/metabolismo , Proteínas Nucleares/metabolismo , Biosíntesis de Proteínas , Proteínas de Unión al ARN/metabolismo
15.
Nat Commun ; 12(1): 1451, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33649316

RESUMEN

Proprioceptive feedback mainly derives from groups Ia and II muscle spindle (MS) afferents and group Ib Golgi tendon organ (GTO) afferents, but the molecular correlates of these three afferent subtypes remain unknown. We performed single cell RNA sequencing of genetically identified adult proprioceptors and uncovered five molecularly distinct neuronal clusters. Validation of cluster-specific transcripts in dorsal root ganglia and skeletal muscle demonstrates that two of these clusters correspond to group Ia MS afferents and group Ib GTO afferent proprioceptors, respectively, and suggest that the remaining clusters could represent group II MS afferents. Lineage analysis between proprioceptor transcriptomes at different developmental stages provides evidence that proprioceptor subtype identities emerge late in development. Together, our data provide comprehensive molecular signatures for groups Ia and II MS afferents and group Ib GTO afferents, enabling genetic interrogation of the role of individual proprioceptor subtypes in regulating motor output.


Asunto(s)
Mecanorreceptores/metabolismo , Husos Musculares/metabolismo , Neuronas Aferentes/metabolismo , Animales , Calbindina 2/metabolismo , Fenómenos Electrofisiológicos , Canales Iónicos/metabolismo , Ratones Transgénicos , Neuronas/metabolismo , Propiocepción , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Neurotransmisores/metabolismo , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Transcriptoma/genética
16.
Methods Mol Biol ; 2265: 487-512, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704736

RESUMEN

MicroRNAs (miRNAs) can regulate the expression of potentially every transcript in the cell, and the definition of miRNA-target interactions is crucial to understand their role in all biological processes. However, the identification of the miRNAs that target a specific mRNA remains a challenge. Here, we describe an innovative method called miR-CATCHv2.0 for the high-throughput identification of the miRNA species bound to an RNA of interest. We also describe how this method can overcome the limitations of the current computational and experimental methods available in this field.


Asunto(s)
Biología Computacional , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Melanoma , MicroARNs , ARN Mensajero , Línea Celular Tumoral , Humanos , Melanoma/genética , Melanoma/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo
17.
Nat Commun ; 12(1): 1375, 2021 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-33654095

RESUMEN

Cellular adaptation to hypoxia is a hallmark of cancer, but the relative contribution of hypoxia-inducible factors (HIFs) versus other oxygen sensors to tumorigenesis is unclear. We employ a multi-omics pipeline including measurements of nascent RNA to characterize transcriptional changes upon acute hypoxia. We identify an immediate early transcriptional response that is strongly dependent on HIF1A and the kinase activity of its cofactor CDK8, includes indirect repression of MYC targets, and is highly conserved across cancer types. HIF1A drives this acute response via conserved high-occupancy enhancers. Genetic screen data indicates that, in normoxia, HIF1A displays strong cell-autonomous tumor suppressive effects through a gene module mediating mTOR inhibition. Conversely, in advanced malignancies, expression of a module of HIF1A targets involved in collagen remodeling is associated with poor prognosis across diverse cancer types. In this work, we provide a valuable resource for investigating context-dependent roles of HIF1A and its targets in cancer biology.


Asunto(s)
Redes Reguladoras de Genes , Genes Supresores de Tumor , Genómica , Hipoxia/genética , Oncogenes , Línea Celular Tumoral , Supervivencia Celular , Quinasa 8 Dependiente de Ciclina/metabolismo , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Genoma Humano , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias/genética , Neoplasias/patología , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Transcripción Genética , Activación Transcripcional/genética , Regulación hacia Arriba/genética
18.
Chem Biol Interact ; 338: 109428, 2021 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-33647240

RESUMEN

Camostat mesylate, a potent inhibitor of the human transmembrane protease, serine 2 (TMPRSS2), is currently under investigation for its effectiveness in COVID-19 patients. For its safe application, the risks of camostat mesylate to induce pharmacokinetic drug-drug interactions with co-administered drugs should be known. We therefore tested in vitro the potential inhibition of important efflux (P-glycoprotein (P-gp, ABCB1), breast cancer resistance protein (BCRP, ABCG2)), and uptake transporters (organic anion transporting polypeptides OATP1B1, OATP1B3, OATP2B1) by camostat mesylate and its active metabolite 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA). Transporter inhibition was evaluated using fluorescent probe substrates in transporter over-expressing cell lines and compared to the respective parental cell lines. Moreover, possible mRNA induction of pharmacokinetically relevant genes regulated by the nuclear pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR) was analysed in LS180 cells by quantitative real-time PCR. The results of our study for the first time demonstrated that camostat mesylate and GBPA do not relevantly inhibit P-gp, BCRP, OATP1B1 or OATP1B3. Only OATP2B1 was profoundly inhibited by GBPA with an IC50 of 11 µM. Induction experiments in LS180 cells excluded induction of PXR-regulated genes such as cytochrome P450 3A4 (CYP3A4) and ABCB1 and AhR-regulated genes such as CYP1A1 and CYP1A2 by camostat mesylate and GBPA. Together with the summary of product characteristics of camostat mesylate indicating no inhibition of CYP1A2, 2C9, 2C19, 2D6, and 3A4 in vitro, our data suggest a low potential of camostat mesylate to act as a perpetrator in pharmacokinetic drug-drug interactions. Only inhibition of OATP2B1 by GBPA warrants further investigation.


Asunto(s)
Interacciones Farmacológicas , Ésteres/metabolismo , Guanidinas/metabolismo , Inhibidores de Serina Proteinasa/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/antagonistas & inhibidores , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Ésteres/química , Ésteres/farmacología , Guanidinas/química , Guanidinas/farmacología , Humanos , Transportadores de Anión Orgánico/antagonistas & inhibidores , Transportadores de Anión Orgánico/metabolismo , Receptor X de Pregnano/genética , Receptor X de Pregnano/metabolismo , ARN Mensajero/metabolismo , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología
19.
Int J Nanomedicine ; 16: 1405-1422, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33658780

RESUMEN

Aim: Iridoid glycosides (IG) as the major active fraction of Syringa oblata Lindl. has a proven anti-inflammatory effect for ulcerative colitis (UC). However, its current commercial formulations are hampered by low bioavailability and unable to reach inflamed colon. To overcome the limitation, dual functional IG-loaded nanoparticles (DFNPs) were prepared to increase the residence time of IG in colon. The protective mechanism of DFNPs on DSS-induced colonic injury was evaluated in rats. Materials and Methods: We prepared DFNPs using the oil-in-water emulsion method. PLGA was selected as sustained-release polymer, and ES100 and EL30D-55 as pH-responsive polymers. The morphology and size distribution of NPs were measured by SEM and DLS technique. To evaluate colon targeting of DFNPs, DiR, was encapsulated as a fluorescent probe into NPs. Fluorescent distribution of NPs were investigated. The therapeutic potential and in vivo transportation of NPs in gastrointestinal tract were evaluated in a colitis model. Results: SEM images and zeta data indicated the successful preparation of DFNPs. This formulation exhibited high loading capacity. Drug release results suggested DFNPs released less than 20% at the first 6 h in simulated gastric fluid (pH1.2) and simulated small intestine fluid (pH6.8). A high amount of 84.7% sustained release from NPs in simulated colonic fluid (pH7.4) was beyond 24 h. DiR-loaded NPs demonstrated a much higher colon accumulation, suggesting effective targeting due to functionalization with pH and time-dependent polymers. DFNPs could significantly ameliorate the colonic damage by reducing DAI, macroscopic score, histological damage and cell apoptosis. Our results also proved that the potent anti-inflammatory effect of DFNPs is contributed by decrease of NADPH, gene expression of COX-2 and MMP-9 and the production of TNF-α, IL-17, IL-23 and PGE2. Conclusion: We confirm that DFNPs exert protective effects through inhibiting the inflammatory response, which could be developed as a potential colon-targeted system.


Asunto(s)
Colitis Ulcerosa/tratamiento farmacológico , Colon/patología , Glicósidos Iridoides/uso terapéutico , Nanopartículas/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Ácidos Polimetacrílicos/química , Animales , Apoptosis/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/genética , Colitis Ulcerosa/patología , Colon/efectos de los fármacos , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Sulfato de Dextran , Liberación de Fármacos , Fluorescencia , Concentración de Iones de Hidrógeno , Glicósidos Iridoides/sangre , Glicósidos Iridoides/farmacocinética , Glicósidos Iridoides/farmacología , Masculino , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones Endogámicos ICR , NADPH Oxidasas/metabolismo , Nanopartículas/ultraestructura , Tamaño de la Partícula , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Distribución Tisular/efectos de los fármacos
20.
J Vis Exp ; (168)2021 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-33720114

RESUMEN

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5' extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.


Asunto(s)
Aptámeros de Nucleótidos/metabolismo , Cromatografía de Afinidad , Bacterias Grampositivas/genética , Análisis de Secuencia de ARN , Secuencia de Bases , Tampones (Química) , Fraccionamiento Celular , Análisis de Datos , Regulación Bacteriana de la Expresión Génica , Humanos , Plásmidos/genética , ARN Bacteriano/genética , ARN Bacteriano/aislamiento & purificación , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/genética , Reproducibilidad de los Resultados , Staphylococcus aureus/genética
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