RESUMEN
MicroRNAs (miRNAs) play crucial regulatory roles in controlling immune responses, but their dynamic expression mechanisms are poorly understood. Here, we firstly confirm that the conserved miRNA miR-210 negatively regulates innate immune responses of Drosophila and human via targeting Toll and TLR6, respectively. Secondly, our findings demonstrate that the expression of miR-210 is dynamically regulated by NF-κB factor Dorsal in immune response of Drosophila Toll pathway. Thirdly, we find that Dorsal-mediated transcriptional inhibition of miR-210 is dependent on the transcriptional repressor Su(Hw). Mechanistically, Dorsal interacts with Su(Hw) to modulate cooperatively the dynamic expression of miR-210 in a time- and dose-dependent manner, thereby controlling the strength of Drosophila Toll immune response and maintaining immune homeostasis. Fourthly, we reveal a similar mechanism in human cells, where NF-κB/RelA cooperates with E4F1 to regulate the dynamic expression of hsa-miR-210 in the TLR immune response. Overall, our study reveals a conservative regulatory mechanism that maintains animal innate immune homeostasis and provides new insights into the dynamic regulation of miRNA expression in immune response.
Asunto(s)
Proteínas de Drosophila , Inmunidad Innata , MicroARNs , Factores de Transcripción , MicroARNs/genética , MicroARNs/metabolismo , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Humanos , Inmunidad Innata/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Regulación de la Expresión Génica , Drosophila melanogaster/genética , Drosophila melanogaster/inmunología , FN-kappa B/metabolismo , Receptor Toll-Like 6/genética , Receptor Toll-Like 6/metabolismo , Factor de Transcripción ReIA/metabolismo , Factor de Transcripción ReIA/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Transducción de Señal , Línea Celular , Drosophila/genética , Drosophila/inmunología , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Proteínas Nucleares , FosfoproteínasRESUMEN
AIM: The fragile X mental retardation protein (FMRP), the product of the FMR1 gene, is responsible for the fragile X syndrome (FXS). FMRP regulates miRNA expression and is involved in miRNA-mediated gene silencing. However, the question of whether FMRP is, in turn, regulated by miRNAs remains unanswered. MAIN METHODS: We detected the FMRP expression pattern by in situ hybridization. MiR-315 overexpression and knockout models were generated by germ-line transformation and ends-out homologous recombination, respectively. Western blotting and immunohistochemistry were used to detect Drosophila FMRP (dFMRP) and a Luciferase reporter assay was used to confirm the regulation of dfmr1 mRNA by mir-315. Synaptic structural quantification and electrophysiological methods were used to compare synaptic functions among groups. KEY FINDINGS: Here, we determined that the transcription product of dFMR1, the Drosophila homologue of FMR1, is a direct target of miR-315. MiR-315 is mainly expressed in the nervous system of Drosophila. Flies overexpressing miR-315 showed pupation defects and reduced hatching rates. A homozygous miR-315 knockout status is embryonic lethal in flies. These observations indicate that miR-315 is a key regulator of the Drosophila nervous system. Furthermore, computational prediction and cell-based luciferase and in vivo assays demonstrated that dfmr1 is directly targeted by miR-315. Lastly, using the neuromuscular junction as a model, we found that miR-315 regulates synaptic structure and transmission by targeting dfmr1. SIGNIFICANCE: These findings provide compelling evidence that miR-315 targets dfmr1 in the Drosophila nervous system, acting as a regulatory factor for the fine-tuned modulation of FMRP expression.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Regulación del Desarrollo de la Expresión Génica , Animales , Sistema Nervioso/embriología , Sistema Nervioso/metabolismo , Neurogénesis , Unión Neuromuscular/genética , Sinapsis/genéticaRESUMEN
Significant evidence from various sources suggests that structural alterations in mitochondrial function may play a role in both the pathogenesis of mood disorders and the therapeutic effects of available treatments. PGC-1α is a distinct transcriptional regulator designed to mediate the synchronous release of neurotransmitter in the brain and thereby to coordinate a number of gene expression pathways to promote mitochondrial biogenesis and oxidative phosphorylation. The role of PGC-1α in the context of affective disorder phenotypes and treatments has been suggested but not studied in depth. To further investigate the possible involvement of PGC-1α in affective disorders, we generated conditional PGC-1α null mice through transgenic expression of cre recombinase under the control of a Dlx5/6 promoter; cre-mediated excision events were limited to γ-amino-butyric-acid (GABA)-ergic specific neurons. We tested these mice in a battery of behavioral tests related to affective change including spontaneous activity, elevated plus maze, forced swim test, and tail suspension test. Results demonstrated that mice lacking PGC-1α in GABAergic neurons exhibited increased activity across tests that might be related to a mania-like phenotype. These results suggest possible relevance of PGC-1α to affective change, which corresponds with data connecting mitochondrial function and affective disorders and their treatment.
Asunto(s)
Trastorno Bipolar/genética , Hipercinesia/genética , Actividad Motora/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Animales , Trastorno Bipolar/metabolismo , Modelos Animales de Enfermedad , Neuronas GABAérgicas/metabolismo , Regulación de la Expresión Génica , Hipercinesia/metabolismo , Ratones , Ratones Noqueados , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Regiones Promotoras GenéticasRESUMEN
Synaptic vesicles (SVs) form distinct pools at synaptic terminals, and this well-regulated separation is necessary for normal neurotransmission. However, how the SV cluster, in particular synaptic compartments, maintains normal neurotransmitter release remains a mystery. The presynaptic protein Neurexin (NRX) plays a significant role in synaptic architecture and function, and some evidence suggests that NRX is associated with neurological disorders, including autism spectrum disorders. However, the role of NRX in SV clustering is unclear. Here, using the neuromuscular junction at the 2-3 instar stages of Drosophila larvae as a model and biochemical imaging and electrophysiology techniques, we demonstrate that Drosophila NRX (DNRX) plays critical roles in regulating synaptic terminal clustering and release of SVs. We found that DNRX controls the terminal clustering and release of SVs by stimulating presynaptic F-actin. Furthermore, our results indicate that DNRX functions through the scaffold protein Scribble and the GEF protein DPix to activate the small GTPase Ras-related C3 Botulinum toxin substrate 1 (Rac1). We observed a direct interaction between the C-terminal PDZ-binding motif of DNRX and the PDZ domains of Scribble and that Scribble bridges DNRX to DPix, forming a DNRX-Scribble-DPix complex that activates Rac1 and subsequently stimulates presynaptic F-actin assembly and SV clustering. Taken together, our work provides important insights into the function of DNRX in regulating SV clustering, which could help inform further research into pathological neurexin-mediated mechanisms in neurological disorders such as autism.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Citoesqueleto de Actina/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de la Membrana/metabolismo , Unión Neuromuscular/metabolismo , Vesículas Sinápticas/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Animales , Animales Modificados Genéticamente , Moléculas de Adhesión Celular Neuronal/química , Moléculas de Adhesión Celular Neuronal/genética , Proteínas de Drosophila/agonistas , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Fenómenos Electrofisiológicos , Eliminación de Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Larva/citología , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Mutación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Unión Neuromuscular/citología , Unión Neuromuscular/crecimiento & desarrollo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Unión al GTP rac/agonistas , Proteínas de Unión al GTP rac/química , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Despite great progress for two decades in microRNAs (miRNAs), the direct regulation of host gene by intragenic (mostly intronic) miRNA is conceptually plausible but evidence-limited. Here, we report that intronic miR-932 could target its host gene via binding with coding sequence (CDS) region rather than regular 3'UTR. The conserved miR-932 is embedded in the fourth intron of Drosophila neuroligin2 (dnlg2), which encodes a synaptic cell adhesion molecule, DNlg2. In silico analysis predicted two putative miR-932 target sites locate in the CDS region of dnlg2 instead of regular 3'-UTR miRNA binding sites. Employing luciferase reporter assay, we further proved that the miR-932 regulates expression of its host gene dnlg2 via the binding CDS region of dnlg2. Consistently, we observed miR-932 downregulated expression of dnlg2 in S2 cell, and the repression of dnlg2 by miR-932 at both protein and RNA level. Furthermore, we found CDS-located site1 is dominant for regulating expression of host dnlg2 by miR-932. In addition to providing thorough examination of one intronic miRNA targeting the CDS region of its host gene, our genome-wide analysis indicated that nearly half of fruitfly and human intronic miRNAs may target their own host gene at coding region. This study would be valuable in elucidating the regulation of intronic miRNA on host gene, and provide new information about the biological context of their genomic arrangements and functions.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Genes de Insecto , Intrones/genética , MicroARNs/metabolismo , Proteínas del Tejido Nervioso/genética , Sistemas de Lectura Abierta/genética , Animales , Secuencia de Bases , Moléculas de Adhesión Celular Neuronal/metabolismo , Línea Celular , Proteínas de Drosophila/genética , Regulación de la Expresión Génica , Genoma Humano , Cabeza , Humanos , Luciferasas/metabolismo , MicroARNs/genética , Proteínas del Tejido Nervioso/metabolismoRESUMEN
OBJECTIVE: To generate mice which are specific for peroxisomproliferator-activated receptor-γ coactivator-1(PGC-1α) knockout in the GABAergic interneuron. METHODS: Conditional mice specific for PGC-1αf/+ were introduced from the Jackson Laboratory, USA and initially inbred to obtain homozygote PGC-1α f/f mice. The PGC-1αf/f conditional mice were further crossed with Dlx5/6-Cre-IRES-EGFP transgenic mice to achieve specific knockout of PGC-1α in the GABAergic interneuron. RESULTS: The offspring with specific knockout PGC-1α gene were successful for the generation of GABAergic interneuron, with the resulting genotype being PGC-1α f/f;Dlx5/6-Cre-IRES-EGFP. CONCLUSION: The PGC-1α f/f;Dlx5/6-Cre-IRES-EGFP mice were obtained through a proper crossing strategy, which has provided a suitable platform for studying the function of PGC-1α in neuropsychiatric diseases.
Asunto(s)
Interneuronas/metabolismo , Enfermedades Neurodegenerativas/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Animales , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Inhibitors of Epidermal growth factor receptor tyrosine kinase (EGFR-TKIs) are producing impressive benefits to responsive types of cancers but challenged with drug resistances. FHND drugs are newly modified small molecule inhibitors based on the third-generation EGFR-TKI AZD9291 (Osimertinib) that are mainly for targeting the mutant-selective EGFR, particularly for the non-small cell lung cancer (NSCLC). Successful applications of EGFR-TKIs to other cancers are less certain, thus the present pre-clinical study aims to explore the anticancer effect and downstream targets of FHND in multiple myeloma (MM), which is an incurable hematological malignancy and reported to be insensitive to first/second generation EGFR-TKIs (Gefitinib/Afatinib). Cell-based assays revealed that FHND004 and FHND008 significantly inhibited MM cell proliferation and promoted apoptosis. The RNA-seq identified the involvement of the MAPK signaling pathway. The protein chip screened PDZ-binding kinase (PBK) as a potential drug target. The interaction between PBK and FHND004 was verified by molecular docking and microscale thermophoresis (MST) assay with site mutation (N124/D125). Moreover, the public clinical datasets showed high expression of PBK was associated with poor clinical outcomes. PBK overexpression evidently promoted the proliferation of two MM cell lines, whereas the FHND004 treatment significantly inhibited survival of 5TMM3VT cell-derived model mice and growth of patient-derived xenograft (PDX) tumors. The mechanistic study showed that FHND004 downregulated PBK expression, thus mediating ERK1/2 phosphorylation in the MAPK pathway. Our study not only demonstrates PBK as a promising novel target of FHND004 to inhibit MM cell proliferation, but also expands the EGFR kinase-independent direction for developing anti-myeloma therapy.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Quinasas de Proteína Quinasa Activadas por Mitógenos , Mieloma Múltiple , Humanos , Animales , Ratones , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Simulación del Acoplamiento Molecular , Resistencia a Antineoplásicos/genética , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/genética , Proliferación Celular , MutaciónRESUMEN
Organophosphate flame retardants (OPFRs) have been widely detected in multiple environment media and have many adverse effects with complex toxicity mechanisms. However, the early molecular responses to OPFRs have not been fully elucidated, thereby making it difficult to assess their risks accurately. In this work, we systematically explored the point of departure (POD) of biological pathways at genome-wide level perturbed by 14 OPFRs with three substituents (alkyl, halogen, and aryl) using a dose-dependent functional genomics approach in Saccharomyces cerevisiae at 24 h exposure. Firstly, our results demonstrated that the overall biological potency at gene level (PODDRG20) ranged from 0.013 to 35.079 µM for 14 OPFRs, especially the tributyl phosphate (TnBP) exhibited the strongest biological potency with the least PODDRG20. Secondly, we found that structural characteristics of carbon number and logKow were significantly negatively correlated with POD, and carbon number and logKow also significantly affected lipid metabolism associated processes. Thirdly, these early biological pathways of OPFRs toxification were found to be involved in lipid metabolism, oxidative stress, DNA damage, MAPK signaling pathway, and amino acid and carbohydrate metabolism, among which the lipid metabolism was the most sensitive molecular response perturbed by most OPFRs. More importantly, we identified one resistant mutant strain with knockout of ERG2 (YMR202W) gene participated in steroid biosynthesis pathway, which can serve as a key yeast strain of OPFRs toxification. Overall, our study demonstrated an effective platform for accurately assessing OPFRs risks and provided a basis for further green OPFRs development.
Asunto(s)
Retardadores de Llama , Genómica , Organofosfatos , Saccharomyces cerevisiae , Retardadores de Llama/toxicidad , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Organofosfatos/toxicidad , Relación Dosis-Respuesta a DrogaRESUMEN
Dose-dependent functional genomics approach has shown great advantage in identifying the molecular initiating event (MIE) of chemical toxification and yielding point of departure (POD) at genome-wide scale. However, POD variability and repeatability derived from experimental design (settings of dose, replicate number, and exposure time) has not been fully determined. In this work, we evaluated POD profiles perturbed by triclosan (TCS) using dose-dependent functional genomics approach in Saccharomyces cerevisiae at multiple time points (9 h, 24 h and 48 h). The full dataset (total 9 concentrations with 6 replicates per treatment) at 9 h was subsampled 484 times to generate subsets of 4 dose groups (Dose A - Dose D with varied concentration range and spacing) and 5 replicate numbers (2 reps - 6 reps). Firstly, given the accuracy of POD and the experimental cost, the POD profiles from 484 subsampled datasets demonstrated that the Dose C group (space narrow at high concentrations and wide dose range) with three replicates was best choice at both gene and pathway levels. Secondly, the variability of POD was found to be relatively robustness and stability across different experimental designs, but POD was more dependent on the dose range and interval than the number of replicates. Thirdly, MIE of TCS toxification was identified to be the glycerophospholipid metabolism pathway at all-time points, supporting the ability of our approach to accurately recognize MIE of chemical toxification at both short- and long-term exposure. Finally, we identified and validated 13 key mutant strains involved in MIE of TCS toxification, which could serve as biomarkers for TCS exposure. Taken together, our work evaluated the repeatability of dose-dependent functional genomics approach and the variability of POD and MIE of TCS toxification, which will benefit the experimental design for future dose-dependent functional genomics study.
Asunto(s)
Triclosán , GenómicaRESUMEN
Multiple genome studies have discovered that variation in deleted in colorectal carcinoma (Dcc) at transcription and translation level were associated with the occurrences of psychiatric disorders. Yet, little is known about the function of Dcc in schizophrenia (SCZ)-related behavioral abnormalities and the efficacy of antipsychotic drugs in vivo. Here, we used an animal model of prefrontal cortex-specific knockdown (KD) of Dcc in adult C57BL/6 mice to study the attention deficits and impaired locomotor activity. Our results supported a critical role of Dcc deletion in SCZ-related behaviors. Notably, olanzapine rescued the SCZ-related behaviors in the MK801-treated mice but not in the cortex-specific Dcc KD mice, indicating that Dcc play a critical in the mechanism of antipsychotic effects of olanzapine. Knockdown of Dcc in prefrontal cortex results in glutamatergic dysfunction, including defects in glutamine synthetase and postsynaptic maturation. As one of the major risk factors of the degree of antipsychotic response, Dcc deletion-induced glutamatergic dysfunction may be involved in the underlying mechanism of treatment resistance of olanzapine. Our findings identified Dcc deletion-mediated SCZ-related behavioral defects, which serve as a valuable animal model for study of SCZ and amenable to targeted investigations in mechanistic hypotheses of the mechanism underlying glutamatergic dysfunction-induced antipsychotic treatment resistance.
Asunto(s)
Antipsicóticos , Receptor DCC , Esquizofrenia , Animales , Ratones , Antipsicóticos/uso terapéutico , Receptor DCC/genética , Ratones Endogámicos C57BL , Olanzapina/farmacología , Fenotipo , Corteza Prefrontal , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/genéticaRESUMEN
Vascular endothelial dysfunction plays a central role in the most dreadful human diseases, including stroke, tumor metastasis, and the coronavirus disease 2019 (COVID-19). Strong evidence suggests that angiotensin II (Ang II)-induced mitochondrial dysfunction is essential for endothelial dysfunction pathogenesis. However, the precise molecular mechanisms remain obscure. Here, polymerase-interacting protein 2 (Poldip 2) was found in the endothelial mitochondrial matrix and no effects on Poldip 2 and NADPH oxidase 4 (NOX 4) expression treated by Ang II. Interestingly, we first found that Ang II-induced NOX 4 binds with Poldip 2 was dependent on cyclophilin D (CypD). CypD knockdown (KD) significantly inhibited the binding of NOX 4 to Poldip 2, and mitochondrial ROS generation in human umbilical vein endothelial cells (HUVECs). Similar results were also found in cyclosporin A (CsA) treated HUVECs. Our previous study suggested a crosstalk between extracellular regulated protein kinase (ERK) phosphorylation and CypD expression, and gallic acid (GA) inhibited mitochondrial dysfunction in neurons depending on regulating the ERK-CypD axis. Here, we confirmed that GA inhibited Ang II-induced NOX 4 activation and mitochondrial dysfunction via ERK/CypD/NOX 4/Poldip 2 pathway, which provide novel mechanistic insight into CypD act as a key regulator of the NOX 4/Poldip 2 axis in Ang II-induced endothelial mitochondrial dysfunction and GA might be beneficial in the treatment of wide variety of diseases, such as COVID-19, which is worthy further research.
Asunto(s)
COVID-19 , Enfermedades Vasculares , Humanos , NADPH Oxidasa 4/metabolismo , Angiotensina II/farmacología , Angiotensina II/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Peptidil-Prolil Isomerasa F/metabolismo , Peptidil-Prolil Isomerasa F/farmacología , NADPH Oxidasas/metabolismo , Estrés Oxidativo , Ácido Gálico/farmacología , COVID-19/metabolismo , Mitocondrias , Células Endoteliales de la Vena Umbilical HumanaRESUMEN
Non-protein target drugs, especially RNA-based gene therapies for treating hereditary diseases, have been recognized worldwide. As cancer is an insurmountable challenge, no miracle drug is currently available. With the advancements in the field of biopharmaceuticals, research on cancer therapy has gradually focused on non-protein target-targeted drugs, especially RNA therapeutics, including oligonucleotide drugs and mRNA vaccines. This review mainly summarizes the clinical research progress in RNA therapeutics and highlights that appropriate target selection and optimized delivery vehicles are key factors in increasing the effectiveness of cancer treatment in vivo.
Asunto(s)
Neoplasias , Humanos , Preparaciones Farmacéuticas , Neoplasias/tratamiento farmacológico , ARN , OligonucleótidosRESUMEN
Innate immunity defenses against infectious agent in all multicultural organisms. TNF-α is an important cytokine that can be stimulated by Lipopolysaccharide (LPS) to regulate the innate immunity. The lipopolysaccharide-induced TNF-α factor (LITAF) functions as a transcription factor for regulating the expression of TNF-α as well as various inflammatory cytokines in response to LPS stimulation. The physiological significance of LITAF gene in the innate immunity of various animals has recently been reported. However, no LITAF gene has yet been identified in amphioxus, which is the best available stand-in for the proximate invertebrate ancestor of the vertebrates. In this study, we identified and characterized an amphioxus LITAF gene (designated as AmphiLITAF). First, we identified the AmphiLITAF from the amphioxus and found that AmphiLITAF gene with ~1.6 kb in length has a 827bp cDNA transcription product which encodes a putative protein with 127 amino acids containing conserved LITAF-domain, and the deduced amino acid of AmphiLITAF shared 37-60% similarity with the LITAFs from other species; second, we uncovered the spatial distribution of the LITAF in different tissues, the expression level of AmphiLITAF mRNA was the highest in hepatic cecum and intestine, moderate in muscles, gills and gonad, and the lowest in notochord. Our findings provide an insight into the innate immune response in the amphioxus and the evolution of the LITAF family.
Asunto(s)
Cordados/genética , Cordados/inmunología , Evolución Molecular , Inmunidad Innata , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Secuencia de Aminoácidos , Animales , Cordados/clasificación , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Orden Génico , Datos de Secuencia Molecular , Proteínas Nucleares/química , Filogenia , Alineación de SecuenciaRESUMEN
Gram-negative bacteria-binding proteins (GNBPs) are important pattern recognition proteins (PRPs), which can initiate host defense in response to pathogen surface molecules. The roles of GNBP in innate immunity of arthropods and molluscs have recently been reported. However, the GNBP gene has not been characterized in the species of higher evolutionary status yet. In this study, we identified and characterized an amphioxus GNBP gene (designated as AmphiGNBP). First, we identified and cloned the AmphiGNBP and found that the AmphiGNBP encodes a putative protein with 558 amino acids, which contains a conserved ß-1, 3-glucan recognizing and binding domain. Second, we found that the AmphiGNBP encodes two extra WSC (cell Wall integrity and Stress response Component) domains, which are unique in AmphiGNBP protein. The two WSC domains of AmphiGNBP protein coupled with the expansion of amphioxus immunity repertoire might undergo intensive domain shuffling during the age of the Cambrian explosion. Finally, we found that the AmphiGNBP was mainly expressed in immune tissues, such as hepatic cecum and intestine, and the expression of AmphiGNBP was affected after LPS stimulation. In conclusion, our findings disclose the particularity and universality of AmphiGNBP and provide profound insights into the function and evolution of GNBP.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Cordados/genética , Cordados/inmunología , Evolución Molecular , Inmunidad Innata , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/química , Clonación Molecular , ADN Complementario/genética , ADN Complementario/metabolismo , Perfilación de la Expresión Génica , Lipopolisacáridos/inmunología , Datos de Secuencia Molecular , Especificidad de Órganos , Filogenia , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The IκB kinase (IKK) plays a crucial role in activation of the transcription factor nuclear factor kappa B (NF-κB) by phosphorylating the Inhibitory of NF-κB (IκB), which triggers the subsequent polyubiquitylation and degradation of IκBα through the 26S proteasome. In this study, we reported the cloning of an IKK homologue cDNA (designated as AmphiIKK) from amphioxus, Branchiostoma belcheri. The full-length cDNA consists of 3,087 bp with an ORF that encoded a predicted protein of 777 amino acid residues. The putative amphioxus IKK protein possesses the characteristic organization of the mammalian IKK proteins, which consists of a serine/threonine kinase domain, a leucine zipper motif and a putative helix-loop-helix motif. And the deduced amino acid of AmphiIKK shared 43.8-64.0 % similarity and 23.6-43.3 % identified with the IKKs from other species. Real-time PCR (RT-PCR) analysis indicated that AmphiIKK was ubiquitously expressed in all tissues and we also discovered that the expression of AmphiIKK was affected after lipopolysaccharides stimulation. Our findings help to highlight a potential important regulatory pathway in the innate immune response in the amphioxus and the evolution of the IKK family.
Asunto(s)
Cordados/genética , Quinasa I-kappa B/genética , Homología de Secuencia de Ácido Nucleico , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Quinasa I-kappa B/química , Lipopolisacáridos/farmacología , Datos de Secuencia Molecular , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Programas Informáticos , Factores de TiempoRESUMEN
AIM: To investigate whether asiatic acid (AA), a pentacyclic triterpene in Centella asiatica, exerted neuroprotective effects in vitro and in vivo, and to determine the underlying mechanisms. METHODS: Human neuroblastoma SH-SY5Y cells were used for in vitro study. Cell viability was determined with the MTT assay. Hoechst 33342 staining and flow cytometry were used to examine the apoptosis. The mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were measured using fluorescent dye. PGC-1α and Sirt1 levels were examined using Western blotting. Neonatal mice were given monosodium glutamate (2.5 mg/g) subcutaneously at the neck from postnatal day (PD) 7 to 13, and orally administered with AA on PD 14 daily for 30 d. The learning and memory of the mice were evaluated with the Morris water maze test. HE staining was used to analyze the pyramidal layer structure in the CA1 and CA3 regions. RESULTS: Pretreatment of SH-SY5Y cells with AA (0.1-100 nmol/L) attenuated toxicity induced by 10 mmol/L glutamate in a concentration-dependent manner. AA 10 nmol/L significantly decreased apoptotic cell death and reduced reactive oxygen species (ROS), stabilized the mitochondrial membrane potential (MMP), and promoted the expression of PGC-1α and Sirt1. In the mice models, oral administration of AA (100 mg/kg) significantly attenuated cognitive deficits in the Morris water maze test, and restored lipid peroxidation and glutathione and the activity of SOD in the hippocampus and cortex to the control levels. AA (50 and 100 mg/kg) also attenuated neuronal damage of the pyramidal layer in the CA1 and CA3 regions. CONCLUSION: AA attenuates glutamate-induced cognitive deficits of mice and protects SH-SY5Y cells against glutamate-induced apoptosis in vitro.
Asunto(s)
Apoptosis/efectos de los fármacos , Centella , Cognición/efectos de los fármacos , Demencia/prevención & control , Neuroblastoma/patología , Fármacos Neuroprotectores/farmacología , Nootrópicos/farmacología , Triterpenos Pentacíclicos/farmacología , Glutamato de Sodio , Animales , Animales Recién Nacidos , Conducta Animal/efectos de los fármacos , Western Blotting , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/patología , Región CA3 Hipocampal/efectos de los fármacos , Región CA3 Hipocampal/metabolismo , Región CA3 Hipocampal/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Centella/química , Demencia/inducido químicamente , Demencia/metabolismo , Demencia/patología , Demencia/psicología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Glutatión/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Peroxidación de Lípido/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Memoria/efectos de los fármacos , Ratones , Neuroblastoma/metabolismo , Fármacos Neuroprotectores/aislamiento & purificación , Nootrópicos/aislamiento & purificación , Estrés Oxidativo/efectos de los fármacos , Triterpenos Pentacíclicos/aislamiento & purificación , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Sirtuina 1/metabolismo , Superóxido Dismutasa/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismoRESUMEN
MicroRNA (miRNA) transcription is still not well understood until now. To increase the miRNA abundance, we stimulated miRNA transcription with CuSO(4) and knocked down Drosha enzyme using dsRNA in Drosophila S2 cells. The full length transcripts of bantam, miR-276a and miR-277, the 5'-end of miR-8, the 3'-end of miR-2b and miR-10 were obtained. We also conducted a series of miRNA promoter analysis to prove the reliability of RACE results. Luciferase-reporter assays proved that both bantam and miR-276a promoters successfully drove the expressions of downstream luciferase genes. The promoter activities were impaired by introducing one or multiple mutations at predicted transcription factor binding sites. Chromatin immunoprecipitation analysis confirmed that hypophosphorylated RNA polymerase II and transcription factor c-Myc physically bind at miRNA promoter. RNA interference of transcription factors Mad and Prd led to down-expression of bantam, miR-277 and miR-2b but not miR-276a, whereas RNAi of Dorsal had the opposite effect.
Asunto(s)
Drosophila melanogaster/metabolismo , Regulación de la Expresión Génica , MicroARNs/genética , MicroARNs/metabolismo , Regiones Promotoras Genéticas/genética , Transcripción Genética , Animales , Sitios de Unión , Inmunoprecipitación de Cromatina , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Luciferasas/genética , Luciferasas/metabolismo , MicroARNs/química , Interferencia de ARN , Análisis de Secuencia de ADN , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Schizophrenia is a chronic, serious and disabling mental disorder. Most patients can effectively control their condition through drug treatment, but there are still some patients who are difficult to gain benefits from drug treatment. Among them, the failure to respond to clozapine full-scale treatment is ultra-treatment-resistant schizophrenia. We generated induced pluripotent stem cells (iPSCs) from an ultra-treatment-resistant schizophrenia patient by electroporation of peripheral blood mononuclear cells (PBMCs) with episomal plasmids encoding OCT 4, SOX 2, NANOG, LIN 28, KLF 4 and MYC. The iPSCs demonstrated normal karyotype, expressed pluripotency markers and differentiated into the three germ layers in vivo.
Asunto(s)
Células Madre Pluripotentes Inducidas , Esquizofrenia , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares/metabolismo , Esquizofrenia/genética , Esquizofrenia/metabolismo , Esquizofrenia Resistente al Tratamiento , Factores de Transcripción/metabolismoRESUMEN
The present review aimed to outline different types of RNAs in cancer diagnostics and treatment, and to provide novel insights into their clinical applications. RNAs, including mRNA, long non-coding (lnc)RNA, circular (circ)RNA and micro (mi)RNA, are now increasingly utilized in the diagnosis and treatment of various cancers. Each aforementioned type of RNA possess their own unique characteristics and could be aberrantly expressed as diagnostic markers or therapeutic targets in different cancers. In addition to mRNAs, which have become a promising alternative in cancer diagnostics and therapy, the uses of lncRNA, circRNA and miRNA in predictive tumor diagnostics and therapy has rapidly increased in recent years. In the present review, the mechanisms of mRNA, lncRNA, circRNA and miRNA in regulating and participating in the development of different cancers were determined, and their potential capacity in cancer diagnostics and therapy were investigated. In addition, the present review analyzed the assoaciations between different RNAs and their subsequent potential in cancer prediction and treatment.
Asunto(s)
MicroARNs , Neoplasias , ARN Largo no Codificante , Humanos , MicroARNs/genética , Neoplasias/diagnóstico , Neoplasias/tratamiento farmacológico , Neoplasias/genética , ARN Circular , ARN Largo no Codificante/genética , ARN MensajeroRESUMEN
BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) as the rate-limiting enzyme in the pentose phosphate pathway (PPP) is well-established as an aberrantly expressed protein in numerous clinical diseases; however, its role in cancer, specifically in multiple myeloma (MM) remains elusive. METHODS: In this study, serum metabolites in 70 normal people and 70 newly diagnosed MM patients were analyzed using untargeted metabolomics and the results were verified using ELISA. The survival analysis of multiple clinical datasets was performed to identify a potential target gene in MM. The oncogenic role of G6PD was investigated using lentivirus-based overexpression or knockdown of G6PD using RNAi or an inhibitor in vitro, and in a xenograft mouse model in vivo. The mechanisms of induced Dexamethasone (Dexa)-resistance of G6PD were further explored using the above established MM cell lines in vitro. RESULTS: Based on the screening of potential genes, PPP was shown to be involved in the occurrence of MM, which was evidenced by the differential expression of serum metabolites of G6P and Dehydroepiandrosterone sulfate (DHEAS, the more stable sulfate ester form of an endogenously uncompetitive G6PD inhibitor known as DHEA). Elevated G6PD promoted MM cell proliferation. Mechanistically, high G6PD expression enhanced enzymatic generation of the antioxidant NADPH via the PPP and decreased the production of reactive oxygen species (ROS), thus inducing the proliferation and Dexa resistance in MM cells. Furthermore, canonical Wnt/ß-catenin signaling also participated in regulating G6PD-induced drug resistance and cellular redox levels of ROS. Intriguingly, DHEA treatment could enhance the sensitivity of MM cells to Dexa primarily through augmenting cellular oxidative stress. CONCLUSIONS: Our data demonstrate that G6PD enhances the generation of the enzymatic anti-oxidant NADPH and decreases ROS generation, thereby promoting resistance to Dexa-induced apoptosis via the enzymatic PPP and non-enzymatic Wnt/ß-catenin signaling pathway in MM. Targeting G6PD to harness cellular redox may serve as a promising novel strategy for the management of MM.