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1.
Anticancer Res ; 44(6): 2445-2451, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38821626

RESUMO

BACKGROUND/AIM: Non-small cell lung cancer (NSCLC) is the deadliest form of cancer worldwide. Understanding the mechanisms of lung cancer development is vital for targeted therapy advancements. This article explores the little-known role of the guanylate kinase-associated protein (GKAP), encoded by the Disks large-associated protein 1 (DLGAP1) gene, in NSCLC along with assessing microRNA-30a-5p's influence on DLGAP1 gene expression in the A549 cell line. MATERIALS AND METHODS: Experiments were conducted on A549 cells transfected with synthetic oligonucleotides. The luciferase assay was employed to confirm the binding site of miR-30a-5p to the 3'UTR of DLGAP1 mRNA. The role of miRNA-30a-5p mimic in regulating potential target gene expression at the protein and mRNA levels was studied by performing RT-qPCR and western blot analyses. The effects of DLGAP1 knockdown and miRNA-30a-5p mimic on cell viability and the cell cycle were evaluated using the MTT test and flow cytometry with annexin/iodide cell staining. RESULTS: The luciferase assay indicated that miR-30a-5p has the ability to bind to the 3'UTR of DLGAP1 mRNA. RT-qPCR revealed that the overexpression of miR-30a-5p down-regulates DLGAP1 mRNA. Western blot analysis indicated that miR-30a-5p slightly reduces the level of the GKAP protein. Knockdown of DLGAP1 with synthetic oligonucleotides, as well as transfection with a miR-30a-5p mimic, significantly attenuates cell proliferation and increases the number of cells in the early and late stages of apoptosis. CONCLUSION: Our findings reveal the antiproliferative effect of miR-30a-5p and DLGAP1 gene knockdown on A549 cancer cells, implying that these elements could be considered as therapeutic targets for personalized medicine in NSCLC patients.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares , MicroRNAs , Humanos , Regiões 3' não Traduzidas/genética , Células A549 , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , MicroRNAs/genética , Proteínas Associadas SAP90-PSD95/genética
2.
Artigo em Inglês | MEDLINE | ID: mdl-38808484

RESUMO

BACKGROUND: Grip strength is a robust indicator of overall health, is moderately heritable, and predicts longevity in older adults. METHODS: Using genome-wide linkage analysis, we identified a novel locus on chromosome 18p (mega-basepair region: 3.4-4.0) linked to grip strength in 3 755 individuals from 582 families aged 64 ±â€…12 years (range 30-110 years; 55% women). There were 26 families that contributed to the linkage peak (cumulative logarithm of the odds [LOD] score = 10.94), with 6 families (119 individuals) accounting for most of the linkage signal (LOD = 6.4). In these 6 families, using whole genome sequencing data, we performed association analyses between the 7 312 single nucleotide (SNVs) and insertion deletion (INDELs) variants in the linkage region and grip strength. Models were adjusted for age, age2, sex, height, field center, and population substructure. RESULTS: We found significant associations between genetic variants (8 SNVs and 4 INDELs, p < 5 × 10-5) in the Disks Large-associated Protein 1 (DLGAP1) gene and grip strength. Haplotypes constructed using these variants explained up to 98.1% of the LOD score. Finally, RNAseq data showed that these variants were significantly associated with the expression of nearby Myosin Light Chain 12A (MYL12A), Structural Maintenance of Chromosomes Flexible Hinge Domain Containing 1 (SMCHD1), Erythrocyte Membrane Protein Band 4.1 Like 3 (EPB41L3) genes (p < .0004). CONCLUSIONS: The DLGAP1 gene plays an important role in the postsynaptic density of neurons; thus, it is both a novel positional and biological candidate gene for follow-up studies aimed at uncovering genetic determinants of muscle strength.


Assuntos
Estudo de Associação Genômica Ampla , Força da Mão , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ligação Genética/genética , Força da Mão/fisiologia , Longevidade/genética , Força Muscular/genética , Força Muscular/fisiologia , Polimorfismo de Nucleotídeo Único , Proteínas Associadas SAP90-PSD95/genética
3.
Cells ; 11(23)2022 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-36497075

RESUMO

Excitatory (glutamatergic) synaptic transmission underlies many aspects of brain activity and the genesis of normal human behavior. The postsynaptic scaffolding proteins SAP90/PSD-95-associated proteins (SAPAPs), which are abundant components of the postsynaptic density (PSD) at excitatory synapses, play critical roles in synaptic structure, formation, development, plasticity, and signaling. The convergence of human genetic data with recent in vitro and in vivo animal model data indicates that mutations in the genes encoding SAPAP1-4 are associated with neurological and psychiatric disorders, and that dysfunction of SAPAP scaffolding proteins may contribute to the pathogenesis of various neuropsychiatric disorders, such as schizophrenia, autism spectrum disorders, obsessive compulsive disorders, Alzheimer's disease, and bipolar disorder. Here, we review recent major genetic, epigenetic, molecular, behavioral, electrophysiological, and circuitry studies that have advanced our knowledge by clarifying the roles of SAPAP proteins at the synapses, providing new insights into the mechanistic links to neurodevelopmental and neuropsychiatric disorders.


Assuntos
Transtornos Mentais , Proteínas Associadas SAP90-PSD95 , Animais , Humanos , Proteínas Associadas SAP90-PSD95/química , Proteínas Associadas SAP90-PSD95/metabolismo , Sinapses/metabolismo , Transtornos Mentais/metabolismo
4.
Nat Commun ; 13(1): 2746, 2022 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-35585091

RESUMO

Subcortical heterotopias are malformations associated with epilepsy and intellectual disability, characterized by the presence of ectopic neurons in the white matter. Mouse and human heterotopia mutations were identified in the microtubule-binding protein Echinoderm microtubule-associated protein-like 1, EML1. Further exploring pathological mechanisms, we identified a patient with an EML1-like phenotype and a novel genetic variation in DLGAP4. The protein belongs to a membrane-associated guanylate kinase family known to function in glutamate synapses. We showed that DLGAP4 is strongly expressed in the mouse ventricular zone (VZ) from early corticogenesis, and interacts with key VZ proteins including EML1. In utero electroporation of Dlgap4 knockdown (KD) and overexpression constructs revealed a ventricular surface phenotype including changes in progenitor cell dynamics, morphology, proliferation and neuronal migration defects. The Dlgap4 KD phenotype was rescued by wild-type but not mutant DLGAP4. Dlgap4 is required for the organization of radial glial cell adherens junction components and actin cytoskeleton dynamics at the apical domain, as well as during neuronal migration. Finally, Dlgap4 heterozygous knockout (KO) mice also show developmental defects in the dorsal telencephalon. We hence identify a synapse-related scaffold protein with pleiotropic functions, influencing the integrity of the developing cerebral cortex.


Assuntos
Lissencefalias Clássicas e Heterotopias Subcorticais em Banda , Proteínas Associadas SAP90-PSD95/metabolismo , Animais , Movimento Celular/genética , Córtex Cerebral/metabolismo , Lissencefalias Clássicas e Heterotopias Subcorticais em Banda/metabolismo , Lissencefalias Clássicas e Heterotopias Subcorticais em Banda/patologia , Humanos , Camundongos , Camundongos Knockout , Neurogênese/genética , Neurônios/fisiologia
5.
Mol Biol Rep ; 49(5): 3939-3947, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35449318

RESUMO

BACKGROUND: Tamoxifen is a first-line endocrine agent and is often used to treat estrogen receptor-positive (ER+) breast cancer. Unfortunately, approximately 30-40% of patients who received tamoxifen therapy experience recurrence or progression to a fatal advanced stage due to tamoxifen resistance. However, the mechanisms of tamoxifen resistance remain unclear. METHODS: The expression of lncRNA DLGAP1 antisense RNA 2 (DLGAP1-AS2) was detected by qPCR. The effect of DLGAP1-AS2 on tamoxifen resistance was evaluated by MTT, colony formation, TUNEL and flow cytometric assays. The mechanisms by which DLGAP1-AS2 regulates tamoxifen resistance were investigated through qPCR, RNA pull-down assays and RNA immunoprecipitation (RIP) assays. RESULTS: Our results showed that DLGAP1-AS2 is significantly upregulated in breast cancer and that tamoxifen can induce DLGAP1-AS2 expression. Further investigation suggested that upregulation of DLGAP1-AS2 can increase cell viability and inhibit apoptosis, while downregulation of DLGAP1-AS2 results in the opposite effects. Mechanistically, DLGAP1-AS2 can bind to the AFF3 protein to inhibit its degradation, which further promotes ER signalling. CONCLUSIONS: Our research clarified that DLGAP1-AS2 promotes ER signalling to induce tamoxifen resistance and that targeting DLGAP1-AS2 might be a promising strategy to overcome tamoxifen resistance in breast cancer.


Assuntos
Neoplasias da Mama , Resistencia a Medicamentos Antineoplásicos , RNA Longo não Codificante , Proteínas Associadas SAP90-PSD95/genética , Tamoxifeno/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , RNA Antissenso/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo
6.
J Mol Histol ; 53(2): 321-332, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34739656

RESUMO

Circular RNAs (circRNAs) have been reported to play vital regulatory roles in human diseases. However, the functions of circRNAs in ischemic stroke (IS) are limited. In this study, we aimed to explore the functions and mechanisms of circRNA DLG associated protein 4 (circDLGAP4) in IS development. Oxygen-glucose deprivation (OGD)-treated HCN-2 cells were used to mimic IS environment in vitro. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was used to detect the levels of circDLGAP4, microRNA-503-3p (miR-503-3p) and neuronal growth regulator 1 (NEGR1) mRNA. RNase R assay was conducted to analyze the stability of circDLGAP4. Cell Counting Kit-8 (CCK-8) assay and flow cytometry analysis were adopted for cell viability and death, respectively. Western blot assay was performed for protein levels. Enzyme-linked immunosorbent assay (ELISA) kits were used to examine the concentrations of inflammatory cytokines. Dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull-down assay were employed to analyze the relationships among circDLGAP4, miR-503-3p and NEGR1. CircDLGAP4 level was declined in HCN-2 cells after OGD treatment. CircDLGAP4 overexpression promoted cell viability and suppressed cell death and inflammatory cytokine concentrations in OGD-treated HCN-2 cells. CircDLGAP4 acted as the sponge for miR-503-3p and the impacts of circDLGAP4 overexpression on cell viability, death and inflammation in OGD-treated HCN-2 cells were reversed by miR-503-3p elevation. Furthermore, NEGR1 was the target gene of miR-503-3p. MiR-503-3p inhibition ameliorated OGD-induced HCN-2 cell impairments, but NEGR1 knockdown abolished the effects. CircDLGAP4 alleviated OGD-induced HCN-2 cell damage by regulating miR-503-3p/NEGR1 axis.


Assuntos
Moléculas de Adesão Celular Neuronais , Proteínas Ligadas por GPI , MicroRNAs , RNA Circular , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas Ligadas por GPI/metabolismo , Glucose/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Neurônios/metabolismo , Oxigênio/metabolismo , RNA Circular/genética , Proteínas Associadas SAP90-PSD95
7.
Bioengineered ; 12(2): 10935-10944, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34866525

RESUMO

Chemotherapy resistance is identified as an obstacle for breast cancer (BC) therapy, and, besides, increasing evidence indicates that long-noncoding RNAs (lncRNAs) participate in the regulation of BC adriamycin (ADR) resistance. Here, our work shows that lncRNA DLGAP1 antisense RNA 1 (DLGAP1-AS1) is up-regulated in ADR-resistant BC cells (MCF-7/ADR). Clinically, higher DLGAP1-AS1 expression was closely correlated to poorer clinical prognosis. Results showed that DLGAP1-AS1 promoted the ADR IC50 and proliferation of ADR-resistant cells. Moreover, N6-methyladenosine (m6A) methyltransferase WT1 associated protein (WTAP) binds to the m6A modified site of DLGAP1-AS1 and motivates its stability. Mechanistically, DLGAP1-AS1 sponged miR-299-3p through 3'-untranslated region (3'-UTR) binding, which in turn relieved the repression of WTAP and thus upregulated WTAP expression. In conclusion, above findings conclude that lncRNA DLGAP1-AS1 promotes BC ADR-resistance through WTAP/DLGAP1-AS1/miR-299-3p feedback loop.


Assuntos
Adenosina/análogos & derivados , Neoplasias da Mama/genética , Proteínas de Ciclo Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , MicroRNAs/genética , Fatores de Processamento de RNA/genética , RNA Longo não Codificante/genética , Proteínas Associadas SAP90-PSD95/genética , Regiões 3' não Traduzidas/genética , Adenosina/metabolismo , Animais , Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células MCF-7 , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Regulação para Cima/genética
8.
Sci Rep ; 11(1): 19497, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34593835

RESUMO

Numerous genetic variants associated with hypertension and blood pressure are known, but there is a paucity of evidence from genetic studies of resistant hypertension, especially in Asian populations. To identify novel genetic loci associated with resistant hypertension in the Japanese population, we conducted a genome-wide association study with 2705 resistant hypertension cases and 21,296 mild hypertension controls, all from BioBank Japan. We identified one novel susceptibility candidate locus, rs1442386 on chromosome 18p11.3 (DLGAP1), achieving genome-wide significance (odds ratio (95% CI) = 0.85 (0.81-0.90), P = 3.75 × 10-8) and 18 loci showing suggestive association, including rs62525059 of 8q24.3 (CYP11B2) and rs3774427 of 3p21.1 (CACNA1D). We further detected biological processes associated with resistant hypertension, including chemical synaptic transmission, regulation of transmembrane transport, neuron development and neurological system processes, highlighting the importance of the nervous system. This study provides insights into the etiology of resistant hypertension in the Japanese population.


Assuntos
Loci Gênicos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Hipertensão/epidemiologia , Hipertensão/genética , Proteínas Associadas SAP90-PSD95/genética , Alelos , Estudos de Casos e Controles , Biologia Computacional/métodos , Genótipo , Humanos , Japão/epidemiologia , Anotação de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Vigilância da População , Locos de Características Quantitativas
9.
BMC Pulm Med ; 21(1): 277, 2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34454450

RESUMO

BACKGROUND: LncRNA DLGAP1-AS2 plays an oncogenic role in glioma, while its role in other cancers is unknown. This study aimed to study the role of DLGAP1-AS2 in non-small cell lung cancer (NSCLC). METHODS: Expression of DLGAP1-AS2 in NSCLC and paired non-tumor tissues from 64 NSCLC patients and the prognostic value of DLGAP1-AS2 for NSCLC were analyzed by performing a 5-year follow-up study. The interaction between DLGAP1-AS2 and miR-503 was confirmed by dual luciferase reporter assay, and their relationship was explored in NSCLC cells transfected with DLGAP1-AS2 expression vector or miR-503 mimic. The roles of DLGAP1-AS2 and miR-503 in regulating cyclin D1 expression were analyzed by RT-qPCR and Western blot. Cell proliferation was analyzed by CCK-8 assay. RESULTS: DLGAP1-AS2 was upregulated in NSCLC and predicted poor survival. Interaction between DLGAP1-AS2 and miR-503 was confirmed by dual luciferase activity assay. Overexpression experiments showed that DLGAP1-AS2 and miR-503 overexpression failed to significantly affect the expression of each other. Interestingly, DLGAP1-AS2 overexpression upregulated cyclin D1, a target of miR-503, increased cell proliferation and reduced the effects of miR-503 overexpression on cyclin D1 expression and cell proliferation. CONCLUSIONS: DLGAP1-AS2 may regulate miR-503/cyclin D1 to promote cell proliferation in NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Ciclina D1/metabolismo , Neoplasias Pulmonares/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , Proteínas Associadas SAP90-PSD95/metabolismo , Idoso , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Ciclina D1/genética , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Prognóstico , Proteínas Associadas SAP90-PSD95/genética
10.
J Mol Neurosci ; 71(10): 2126-2133, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34273101

RESUMO

CircRNAs belong to a novel class of noncoding RNAs that are generated by exons of genes by alternative mRNA splicing and involved in pathophysiological processes of ischemic stroke by regulating neuro-inflammation. A total of 982 patients were enrolled in our study for stroke recovery analysis. The aim of our study was to first explore the association between the inflammation-related circRNA polymorphism and functional outcome 3 months after ischemic stroke by using multivariate logistic regression model. Next, we further investigated the role of circRNA polymorphism in predicting stroke recurrence by using Cox proportional hazard regression model. Five circRNA polymorphisms were genotyped by using polymerase chain reaction and ligation detection reaction method. We identified circ-STAT3 (signal transducer and activator of transcription) rs2293152 GG genotype to be associated with poorer recovery 90 days after stroke (OR = 1.452, 95% CI: 1.165-4.362, p = 0.016). After adjusting for confound factors, the association for rs2293152 with 3 months outcome after IS was stronger, suggesting a mechanism that rs2293152 is an independent risk factor for stroke recovery (OR = 2.255, 95% CI: 1.034-2.038, p = 0.031). However, no other circRNA polymorphisms (circ-DLGAP4 rs41274714, circ-TRAF2 rs10870141, circ-ITCH rs10485505, rs4911154) were associated with functional outcome 3 months after stroke in any genetic models. Subgroup analysis revealed that the negative effect of rs2293152 GG genotype was greater in female and older patients, subjects with history of hypertension. Additionally, all the circRNA polymorphisms were not correlated with recurrent risk of ischemic stroke. Our results indicated that circ-STAT3 might be a novel biomarker for predicting functional outcome after stroke and an important contributor to the ischemic stroke recovery.


Assuntos
AVC Isquêmico/genética , Polimorfismo de Nucleotídeo Único , RNA Circular/genética , Biomarcadores/metabolismo , Feminino , Humanos , AVC Isquêmico/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas Repressoras/genética , Proteínas Associadas SAP90-PSD95/genética , Fator de Transcrição STAT3/genética , Fator 2 Associado a Receptor de TNF/genética , Ubiquitina-Proteína Ligases/genética
11.
PLoS Genet ; 17(5): e1009506, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33956822

RESUMO

Identifying the molecular underpinnings of the neural specializations that underlie human cognitive and behavioral traits has long been of considerable interest. Much research on human-specific changes in gene expression and epigenetic marks has focused on the prefrontal cortex, a brain structure distinguished by its role in executive functions. The cerebellum shows expansion in great apes and is gaining increasing attention for its role in motor skills and cognitive processing, including language. However, relatively few molecular studies of the cerebellum in a comparative evolutionary context have been conducted. Here, we identify human-specific methylation in the lateral cerebellum relative to the dorsolateral prefrontal cortex, in a comparative study with chimpanzees (Pan troglodytes) and rhesus macaques (Macaca mulatta). Specifically, we profiled genome-wide methylation levels in the three species for each of the two brain structures and identified human-specific differentially methylated genomic regions unique to each structure. We further identified which differentially methylated regions (DMRs) overlap likely regulatory elements and determined whether associated genes show corresponding species differences in gene expression. We found greater human-specific methylation in the cerebellum than the dorsolateral prefrontal cortex, with differentially methylated regions overlapping genes involved in several conditions or processes relevant to human neurobiology, including synaptic plasticity, lipid metabolism, neuroinflammation and neurodegeneration, and neurodevelopment, including developmental disorders. Moreover, our results show some overlap with those of previous studies focused on the neocortex, indicating that such results may be common to multiple brain structures. These findings further our understanding of the cerebellum in human brain evolution.


Assuntos
Cerebelo/metabolismo , Metilação de DNA , Epigênese Genética , Proteínas ADAM , Animais , Autoantígenos , Proteínas de Transporte , Chade , Ilhas de CpG , Feminino , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Macaca mulatta/genética , Masculino , Proteínas dos Microfilamentos , Proteínas do Tecido Nervoso , Pan troglodytes/genética , Fosfoinositídeo Fosfolipase C , Proteínas Serina-Treonina Quinases , Proteínas , Proteínas Associadas SAP90-PSD95 , Especificidade da Espécie , Sítio de Iniciação de Transcrição
12.
Genes Brain Behav ; 20(1): e12723, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33347690

RESUMO

The postsynaptic terminal of vertebrate excitatory synapses contains a highly conserved multiprotein complex that comprises neurotransmitter receptors, cell-adhesion molecules, scaffold proteins and enzymes, which are essential for brain signalling and plasticity underlying behaviour. Increasingly, mutations in genes that encode postsynaptic proteins belonging to the PSD-95 protein complex, continue to be identified in neurodevelopmental disorders (NDDs) such as autism spectrum disorder, intellectual disability and epilepsy. These disorders are highly heterogeneous, sharing genetic aetiology and comorbid cognitive and behavioural symptoms. Here, by using genetically engineered mice and innovative touchscreen-based cognitive testing, we sought to investigate whether loss-of-function mutations in genes encoding key interactors of the PSD-95 protein complex display shared phenotypes in associative learning, updating of learned associations and reaction times. Our genetic dissection of mice with loss-of-function mutations in Syngap1, Nlgn3, Dlgap1, Dlgap2 and Shank2 showed that distinct components of the PSD-95 protein complex differentially regulate learning, cognitive flexibility and reaction times in cognitive processing. These data provide insights for understanding how human mutations in these genes lead to the manifestation of diverse and complex phenotypes in NDDs.


Assuntos
Aprendizagem , Mutação com Perda de Função , Proteínas do Tecido Nervoso/genética , Animais , Moléculas de Adesão Celular Neuronais/genética , Feminino , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Tempo de Reação , Proteínas Associadas SAP90-PSD95/genética , Proteínas Ativadoras de ras GTPase/genética
13.
Cell Res ; 31(1): 37-51, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33235361

RESUMO

Ca2+/calmodulin-dependent kinase IIα (CaMKIIα) is essential for synaptic plasticity and learning by decoding synaptic Ca2+ oscillations. Despite decades of extensive research, new mechanisms underlying CaMKIIα's function in synapses are still being discovered. Here, we discover that Shank3 is a specific binding partner for autoinhibited CaMKIIα. We demonstrate that Shank3 and GluN2B, via combined actions of Ca2+ and phosphatases, reciprocally bind to CaMKIIα. Under basal condition, CaMKIIα is recruited to the Shank3 subcompartment of postsynaptic density (PSD) via phase separation. Rise of Ca2+ concentration induces GluN2B-mediated recruitment of active CaMKIIα and formation of the CaMKIIα/GluN2B/PSD-95 condensates, which are autonomously dispersed upon Ca2+ removal. Protein phosphatases control the Ca2+-dependent shuttling of CaMKIIα between the two PSD subcompartments and PSD condensate formation. Activation of CaMKIIα further enlarges the PSD assembly and induces structural LTP. Thus, Ca2+-induced and phosphatase-checked shuttling of CaMKIIα between distinct PSD nano-domains can regulate phase separation-mediated PSD assembly and synaptic plasticity.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Plasticidade Neuronal/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Animais , Sítios de Ligação , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína 4 Homóloga a Disks-Large/genética , Proteína 4 Homóloga a Disks-Large/metabolismo , Células HEK293 , Humanos , Camundongos , Simulação de Acoplamento Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Fosforilação , Ligação Proteica , Ratos , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Associadas SAP90-PSD95/metabolismo
14.
Cell Death Dis ; 11(11): 1008, 2020 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-33230102

RESUMO

Diabetic kidney disease (DKD) is closely associated with the high risk of cardiovascular disease and mortality. Exosomal circRNAs can exert significant roles in the pathology of various diseases. Nevertheless, the role of exosomal circRNAs in DKD progression remains barely known. Circular RNA DLGAP4 has been reported to be in involved in acute ischemic stroke. In our study, we found exosomal circ_DLGAP4 was increased in the exosomes isolated from HG-treated mesangial cells (MCs), DKD patients, and DKD rat models compared with the corresponding normal subjects. Then, we observed that exo-circ_DLGAP4 significantly promoted proliferation and fibrosis of MCs cells. Moreover, to study the underlying mechanism of circ_DLGAP4 in regulating DKD, bioinformatics method was consulted and miR-143 was predicted as its target. The direct correlation between miR-143 and circ_DLGAP4 was validated in MCs. MCs proliferation and fibrosis were increased by circ_DLGAP4, which could be decreased by mimic-miR-143. Next, elevated expression of Erb-b2 receptor tyrosine kinase 3 (ERBB3) is involved in various diseases. However, the function of ERBB3 in DKD development remains poorly known. Next, ERBB3 was predicted as the downstream target for miR-143. It was displayed that circ_DLGAP4 promoted proliferation and fibrosis of MCs by sponging miR-143 and regulating ERBB3/NF-κB/MMP-2 axis. Meanwhile, the loss of exo-circ_DLGAP4 induced miR-143 and repressed ERBB3/NF-κB/MMP-2 expression in MCs. Subsequently, in vivo assays were performed and it was proved that overexpression of circ_DLGAP4 markedly promoted DKD progression in vivo via modulating miR-143/ERBB3/NF-κB/MMP-2. In conclusion, we indicated that exosomal circ_DLGAP4 could prove a novel insight for DKD development.


Assuntos
Nefropatias Diabéticas/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , MicroRNAs/metabolismo , RNA Circular/metabolismo , Receptor ErbB-3/metabolismo , Proteínas Associadas SAP90-PSD95/genética , Animais , Proliferação de Células/fisiologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Progressão da Doença , Exossomos/genética , Exossomos/metabolismo , Humanos , Metaloproteinase 2 da Matriz/genética , Camundongos , MicroRNAs/genética , RNA Circular/genética , Receptor ErbB-3/genética , Transdução de Sinais , Transfecção
15.
Lipids Health Dis ; 19(1): 39, 2020 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-32169116

RESUMO

BACKGROUND: Obesity is a metabolic imbalance characterized by excessive deposition of white fat. The browning of white fat can effectively treat obesity and related diseases. Although Dlgap1 (Discs, Large (Drosophila) Homolog-Associated Protein 1) is suspected to have an effect on this process, no empirical evidence is available. METHODS: To understand the role of Dlgap1, we cultured white and brown fat cells, then performed overexpression and knockout experiments. RESULTS: We found that Dlgap1 overexpression in brown adipocytes inhibits brown-fat-related gene expression, promotes white-fat-related genes, while also increasing brown-adipocyte proliferation and apoptosis. However, the gene overexpression has no effect on brown adipocyte maturation. Knocking out Dlgap1 in white fat cells promotes the expression and inhibition of brown-fat-related and white-fat-related genes, respectively. Additionally, the knockout inhibits white fat cell proliferation and apoptosis, while also promoting their maturation. CONCLUSIONS: Dlgap1 negatively regulates the browning of white adipocytes by influencing cell proliferation and apoptosis.


Assuntos
Adipócitos Marrons/metabolismo , Adipócitos Brancos/metabolismo , Apoptose/fisiologia , Proliferação de Células/fisiologia , Proteínas Associadas SAP90-PSD95/metabolismo , Adipócitos Marrons/citologia , Adipócitos Brancos/citologia , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/citologia , Tecido Adiposo Branco/metabolismo , Animais , Apoptose/genética , Western Blotting , Proliferação de Células/genética , Camundongos , Proteínas Associadas SAP90-PSD95/genética
16.
J Biochem ; 167(4): 411-418, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31899508

RESUMO

LncRNA DLGAP1 antisense RNA 2 (DLGAP1-AS2) is one kind cytoplasmic long non-coding RNA; however, there is rarely little information about its function in physiological process. Here, we demonstrated that LncRNA DLGAP1-AS2 was up-regulated in glioma and was quite correlated with poor prognosis of glioma patients. Depletion of DLGAP1-AS2 in glioma cells could inhibit cell proliferation and cell migration, and induce cell apoptosis, resulting in the suppression of the progression of glioma consequently. Furthermore, knockdown of DLGAP1-AS2 inhibited the growth of xenograft glioma tumour in vivo as well. Finally, we verified Yes Associated Protein 1 (YAP1) was the downstream target of DLGAP1-AS2 and DLGAP1-AS2 modulated glioma cell proliferation, migration and apoptosis via regulating YAP1. Our study revealed novel mechanism about how did lncRNA DLGAP1-AS2 execute function in glioma and thus provided potential therapeutic interventions for the treatment of glioma.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas Associadas SAP90-PSD95/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima , Proteínas Adaptadoras de Transdução de Sinal/genética , Apoptose , Neoplasias Encefálicas/diagnóstico , Movimento Celular , Proliferação de Células , Glioma/diagnóstico , Humanos , RNA Longo não Codificante/genética , Proteínas Associadas SAP90-PSD95/genética , Fatores de Transcrição/genética , Proteínas de Sinalização YAP
17.
J Cell Biochem ; 121(2): 1953-1962, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31633236

RESUMO

Hepatocellular carcinoma (HCC) is most prevalent tumor in liver and one of the most fatal cancers in the world. Long noncoding RNAs (lncRNAs) have been accepted as important regulators in carcinomas. But there are still many lncRNAs including DLGAP1-AS1 unannotated in HCC. First of all, GEPIA suggested that DLGAP1-AS1 presented high expression in HCC tissue samples relative to the normal tissues. Besides, overexpression of DLGAP1-AS1 was also proved in HCC cell lines. Moreover, DLGAP1-AS1 knockdown efficiently suppressed cell proliferation in HCC. Interestingly, miR-486-5p was predicted and validated to interact with DLGAP1-AS1, while the level of miR-486-5p was significantly increased In HCC after DLGAP1-AS1 knockdown. Moreover, we uncovered that ectopic expression of miR-486-5p induced suppression on HCC cell proliferation and that miR-486-5p inhibition offset the effect of DLGAP1-AS1 silence on HCC cell proliferation and apoptosis. Furthermore, H3F3B was identified as target of miR-486-5p and was therefore positively regulated by DLGAP1-AS1 in HCC. Of note, H3F3B upregulation partly revived the declined cell proliferative capacity in response to DLGAP1-AS1 knockdown. To conclude, DLGAP1-AS1 exerted its oncogenic role in HCC via miR-486-5p/H3F3B axis. Our new findings provided novel theoretical basis for discovery of therapeutic targets of HCC.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , MicroRNAs/genética , RNA Antissenso/genética , RNA Longo não Codificante/genética , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Movimento Celular , Proliferação de Células , Histonas/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proteínas Associadas SAP90-PSD95/genética , Células Tumorais Cultivadas
18.
ACS Chem Biol ; 14(10): 2252-2263, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31525028

RESUMO

Protein-protein interactions (PPIs) are vital to all biological processes. These interactions are often dynamic, sometimes transient, typically occur over large topographically shallow protein surfaces, and can exhibit a broad range of affinities. Considerable progress has been made in determining PPI structures. However, given the above properties, understanding the key determinants of their thermodynamic stability remains a challenge in chemical biology. An improved ability to identify and engineer PPIs would advance understanding of biological mechanisms and mutant phenotypes and also provide a firmer foundation for inhibitor design. In silico prediction of PPI hot-spot amino acids using computational alanine scanning (CAS) offers a rapid approach for predicting key residues that drive protein-protein association. This can be applied to all known PPI structures; however there is a trade-off between throughput and accuracy. Here we describe a comparative analysis of multiple CAS methods, which highlights effective approaches to improve the accuracy of predicting hot-spot residues. Alongside this, we introduce a new method, BUDE Alanine Scanning, which can be applied to single structures from crystallography and to structural ensembles from NMR or molecular dynamics data. The comparative analyses facilitate accurate prediction of hot-spots that we validate experimentally with three diverse targets: NOXA-B/MCL-1 (an α-helix-mediated PPI), SIMS/SUMO, and GKAP/SHANK-PDZ (both ß-strand-mediated interactions). Finally, the approach is applied to the accurate prediction of hot-spot residues at a topographically novel Affimer/BCL-xL protein-protein interface.


Assuntos
Aminoácidos/química , Proteínas/metabolismo , Animais , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida/métodos , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Multimerização Proteica , Proteínas/química , Ratos , Proteínas Associadas SAP90-PSD95/química , Proteínas Associadas SAP90-PSD95/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo
19.
Oncogene ; 38(47): 7234-7248, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31477833

RESUMO

Glioblastoma (GBM) is the most common malignant primary brain tumor in adults. Currently, the prognosis of the patients with GBM is very poor and new molecular targets and treatment strategies are urgently needed to combat it. MicroRNA-148a (miR-148a) has been shown to be dysregulated in certain tumor types. However, the role of miR-148a in the pathogenesis of GBM is not fully understood. Here we comprehensively analyzed the roles of miR-148a, downstream DLGAP1, and their molecular pathways in GBM. We showed that miR-148a promote the proliferation and growth of GBM cells both in vitro and in vivo, and also induced the migration, invasion, and EMT (epithelial-mesenchymal transition) program of GBM cells by directly targeting DLGAP1. Furthermore, we identified 31 new miR-148a targets and found that miR-148a function was mainly involved in the cell adhesion signaling pathway and was associated with nervous system diseases. Our findings provide a new mechanism for miR-148a-mediated GBM cell invasion and reveal previously unreported targets of miR-148a as well as novel miR-148a-mediated regulatory networks in GBM. These results increase the understanding of the role of miR-148a in GBM and may lead to novel therapeutic strategies for GBM.


Assuntos
Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica/genética , Glioblastoma/patologia , MicroRNAs/genética , Proteínas Associadas SAP90-PSD95/biossíntese , Proteínas Associadas SAP90-PSD95/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Proliferação de Células/genética , Progressão da Doença , Transição Epitelial-Mesenquimal/genética , Glioblastoma/genética , Glioblastoma/metabolismo , Xenoenxertos , Humanos , Camundongos , MicroRNAs/metabolismo , Invasividade Neoplásica/genética
20.
Transl Psychiatry ; 9(1): 7, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30664629

RESUMO

In humans, genetic variants of DLGAP1-4 have been linked with neuropsychiatric conditions, including autism spectrum disorder (ASD). While these findings implicate the encoded postsynaptic proteins, SAPAP1-4, in the etiology of neuropsychiatric conditions, underlying neurobiological mechanisms are unknown. To assess the contribution of SAPAP4 to these disorders, we characterized SAPAP4-deficient mice. Our study reveals that the loss of SAPAP4 triggers profound behavioural abnormalities, including cognitive deficits combined with impaired vocal communication and social interaction, phenotypes reminiscent of ASD in humans. These behavioural alterations of SAPAP4-deficient mice are associated with dramatic changes in synapse morphology, function and plasticity, indicating that SAPAP4 is critical for the development of functional neuronal networks and that mutations in the corresponding human gene, DLGAP4, may cause deficits in social and cognitive functioning relevant to ASD-like neurodevelopmental disorders.


Assuntos
Transtorno do Espectro Autista/genética , Disfunção Cognitiva/genética , Proteínas do Tecido Nervoso/genética , Proteínas Associadas SAP90-PSD95/genética , Animais , Comportamento Animal , Modelos Animais de Doenças , Feminino , Relações Interpessoais , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Comportamento Social , Sinapses/metabolismo
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