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1.
Nat Commun ; 10(1): 2983, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31278301

RESUMO

Ttriple-negative breast cancer (TNBC) is an aggressive and highly metastatic breast cancer subtype. Enhanced TNBC cell motility is a prerequisite of TNBC cell dissemination. Here, we apply an imaging-based RNAi phenotypic cell migration screen using two highly motile TNBC cell lines (Hs578T and MDA-MB-231) to provide a repository of signaling determinants that functionally drive TNBC cell motility. We have screened ~4,200 target genes individually and discovered 133 and 113 migratory modulators of Hs578T and MDA-MB-231, respectively, which are linked to signaling networks predictive for breast cancer progression. The splicing factors PRPF4B and BUD31 and the transcription factor BPTF are essential for cancer cell migration, amplified in human primary breast tumors and associated with metastasis-free survival. Depletion of PRPF4B, BUD31 and BPTF causes primarily down regulation of genes involved in focal adhesion and ECM-interaction pathways. PRPF4B is essential for TNBC metastasis formation in vivo, making PRPF4B a candidate for further drug development.


Assuntos
Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Serina-Treonina Quinases/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Linhagem Celular Tumoral , Estudos de Coortes , Conjuntos de Dados como Assunto , Intervalo Livre de Doença , Matriz Extracelular/metabolismo , Feminino , Adesões Focais/genética , Humanos , Microscopia Intravital , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Processamento de RNA/genética , RNA Interferente Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Transdução de Sinais/genética , Análise de Sobrevida , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/mortalidade
2.
Nat Commun ; 10(1): 2208, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101817

RESUMO

Cortical force generators connect epithelial polarity sites with astral microtubules, allowing dynein movement to orient the mitotic spindle as astral microtubules depolymerize. Complexes of the LGN and NuMA proteins, fundamental components of force generators, are recruited to the cortex by Gαi-subunits of heterotrimeric G-proteins. They associate with dynein/dynactin and activate the motor activity pulling on astral microtubules. The architecture of cortical force generators is unknown. Here we report the crystal structure of NuMA:LGN hetero-hexamers, and unveil their role in promoting the assembly of active cortical dynein/dynactin motors that are required in orchestrating oriented divisions in polarized cells. Our work elucidates the basis for the structural organization of essential spindle orientation motors.


Assuntos
Antígenos Nucleares/metabolismo , Polaridade Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Fuso Acromático/metabolismo , Antígenos Nucleares/química , Antígenos Nucleares/genética , Antígenos Nucleares/isolamento & purificação , Células CACO-2 , Cristalografia por Raios X , Complexo Dinactina/metabolismo , Dineínas/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Microtúbulos/metabolismo , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/isolamento & purificação , Ligação Proteica/fisiologia , Multimerização Proteica/fisiologia , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
3.
BMC Res Notes ; 12(1): 225, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30987672

RESUMO

OBJECTIVE: Delivery of constructs for silencing or over-expressing genes or their modified versions is a crucial step for studying neuronal cell biology. Therefore, efficient transfection is important for the success of these experimental techniques especially in post-mitotic cells like neurons. In this study, we have assessed the transfection rate, using a previously established protocol, in both primary cortical cultures and neuroblastoma cell lines. Transfection efficiencies in these preparations have not been systematically determined before. RESULTS: Transfection efficiencies obtained herein were (10-12%) for neuroblastoma, (5-12%) for primary astrocytes and (1.3-6%) for primary neurons. We also report on cell-type specific transfection efficiency of neurons and astrocytes within primary cortical cultures when applying cell-type selective transfection protocols. Previous estimations described in primary cortical or hippocampal cultures were either based on general observations or on data derived from unspecified number of biological and/or technical replicates. Also to the best of our knowledge, transfection efficiency of pure primary neuronal cultures or astrocytes cultured in the context of pure or mixed (neurons/astrocytes) population cultures have not been previously determined. The transfection strategy used herein represents a convenient, and a straightforward tool for targeted cell transfection that can be utilized in a variety of in vitro applications.


Assuntos
Astrócitos/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Plasmídeos/metabolismo , Transfecção/métodos , Animais , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Técnicas de Cocultura , Expressão Gênica , Genes Reporter , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lipídeos/química , Lipídeos/farmacologia , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Especificidade de Órgãos , Plasmídeos/química , Cultura Primária de Células
4.
Nat Commun ; 10(1): 1686, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30975996

RESUMO

Cohesin is a multiprotein ring that is responsible for cohesion of sister chromatids and formation of DNA loops to regulate gene expression. Genomic analyses have identified that the cohesin subunit STAG2 is frequently inactivated by mutations in cancer. However, the reason STAG2 mutations are selected during tumorigenesis and strategies for therapeutically targeting mutant cancer cells are largely unknown. Here we show that STAG2 is essential for DNA replication fork progression, whereby STAG2 inactivation in non-transformed cells leads to replication fork stalling and collapse with disruption of interaction between the cohesin ring and the replication machinery as well as failure to establish SMC3 acetylation. As a consequence, STAG2 mutation confers synthetic lethality with DNA double-strand break repair genes and increased sensitivity to select cytotoxic chemotherapeutic agents and PARP or ATR inhibitors. These studies identify a critical role for STAG2 in replication fork procession and elucidate a potential therapeutic strategy for cohesin-mutant cancers.


Assuntos
Antígenos Nucleares/metabolismo , Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Neoplasias/genética , Mutações Sintéticas Letais , Antígenos Nucleares/genética , Antineoplásicos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Carcinogênese/genética , Linhagem Celular Tumoral , Cromátides/metabolismo , Quebras de DNA de Cadeia Dupla , Replicação do DNA , Ensaios de Seleção de Medicamentos Antitumorais , Técnicas de Inativação de Genes , Humanos , Mutagênese , Neoplasias/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/metabolismo , Reparo de DNA por Recombinação
5.
Mol Cancer ; 18(1): 45, 2019 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-30922402

RESUMO

BACKGROUND: Circular RNAs (circRNAs) are a novel type of noncoding RNAs and play important roles in tumorigenesis, including gastric cancer (GC). However, the functions of most circRNAs remain poorly understood. In our study, we aimed to investigate the functions of a new circRNA circ-DONSON in GC progression. METHODS: The expression of circ-DONSON in gastric cancer tissues and adjacent normal tissues was analyzed by bioinformatics method, qRT-PCR, Northern blotting and in situ hybridization (ISH). The effects of circ-DONSON on GC cell proliferation, apoptosis, migration and invasion were measured by using CCK8, colony formation, EdU, immunofluorescence (IF), FACS and Transwell assays. qRT-PCR and Western blotting were utilized to validate how circ-DONSON regulates SOX4 expression. ChIP, DNA fluorescence in situ hybridization (DNA-FISH) and DNA accessibility assays were used to investigate how circ-DONSON regulates SOX4 transcription. The interaction between circ-DONSON and NURF complex was evaluated by mass spectrum, RNA immunoprecipitation (RIP), pulldown and EMSA assays. Xenograft mouse model was used to analyze the effect of circ-DONSON on GC growth in vivo. RESULTS: Elevated expression of circ-DONSON was observed in GC tissues and positively associated with advanced TNM stage and unfavorable prognosis. Silencing of circ-DONSON significantly suppressed the proliferation, migration and invasion of GC cells while promoting apoptosis. circ-DONSON was localized in the nucleus, recruited the NURF complex to SOX4 promoter and initiated its transcription. Silencing of the NURF complex subunit SNF2L, BPTF or RBBP4 similarly attenuated GC cell growth and increased apoptosis. circ-DONSON knockdown inhibited GC growth in vivo. CONCLUSION: circ-DONSON promotes GC progression through recruiting the NURF complex to initiate SOX4 expression.


Assuntos
Antígenos Nucleares/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , RNA/genética , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Fatores de Transcrição SOXC/metabolismo , Neoplasias Gástricas/patologia , Fatores de Transcrição/metabolismo , Animais , Antígenos Nucleares/genética , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Movimento Celular , Proliferação de Células , Proteínas de Ligação a DNA/genética , Progressão da Doença , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Proteínas do Tecido Nervoso/genética , Prognóstico , Proteína 4 de Ligação ao Retinoblastoma/genética , Fatores de Transcrição SOXC/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Taxa de Sobrevida , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
6.
J Hum Genet ; 64(5): 487-492, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30765867

RESUMO

We herein report two individuals with novel nonsense mutations in STAG2 on Xq25, encoding stromal antigen 2, a component of the cohesion complex. A male fetus (Case 1) clinically presented with holoprosencephaly, cleft palate and lip, blepharophimosis, nasal bone absence, and hypolastic left heart by ultrasonography at 15 gestational weeks. Another female patient (Case 2) showed a distinct phenotype with white matter hypoplasia, cleft palate, developmental delay (DD), and intellectual disability (ID) at 7 years. Whole-exome sequencing identified de novo nonsense mutations in STAG2: c.3097C>T, p.(Arg1033*) in Case 1 and c.2229G>A, p.(Trp743*) in Case 2. X-inactivation was highly skewed in Case 2. To date, only 10 STAG2 pathogenic variants (four nonsense, four missense, and two frameshift) have been reported in patients with multiple congenital anomalies, ID, and DD. Although Case 2 showed similar clinical features to the reported female patients with STAG2 abnormalities, Case 1 showed an extremely severe phenotype, which could be explained by the first detected truncating variant in males.


Assuntos
Anormalidades Múltiplas/genética , Antígenos Nucleares/genética , Códon sem Sentido , Mutação de Sentido Incorreto , Anormalidades Múltiplas/patologia , Criança , Feminino , Humanos , Masculino , Fatores Sexuais
7.
In Vivo ; 33(2): 441-445, 2019 Mar-Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30804123

RESUMO

BACKGROUND/AIM: Cerebral ischemia is a major cause of abnormal brain development. In a cerebral ischemia model, periventricular leukomalacia (PVL), white matter lesion and a decrease in the number of subcortical neurons were observed. The aim of this study was to investigate the effect of hypoxia on neurogenesis and cell survival. MATERIALS AND METHODS: In seven-day postnatal rats, the right carotid artery was ligated. The rats were incubated either in a regular normoxic chamber (control group) or in a hypoxic chamber (PVL group, 8% 02 and 92% N2 at 37°C) for 2 h. Nestin- and NeuN-positive neurons were detected by immunohistochemistry. RESULTS: The densities of nestin-immunoreactivity (IR) cells in the cerebral parietal cortex and subventricular zone were increased with hypoxia. NeuN-IR cells in the cerebral cortex were significantly decreased in the PVL group. CONCLUSION: Perinatal white matter injury induced neurogenesis, while the survival of neurons was decreased in the cerebral cortex.


Assuntos
Córtex Cerebral/metabolismo , Hipóxia-Isquemia Encefálica/genética , Neurogênese/genética , Neurônios/metabolismo , Animais , Animais Recém-Nascidos , Antígenos Nucleares/genética , Artéria Carótida Primitiva/metabolismo , Artéria Carótida Primitiva/patologia , Proliferação de Células/genética , Córtex Cerebral/patologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hipóxia-Isquemia Encefálica/patologia , Imuno-Histoquímica , Ligadura , Proteínas do Tecido Nervoso/genética , Nestina/genética , Neurônios/patologia , Ratos , Substância Branca/lesões , Substância Branca/metabolismo , Substância Branca/patologia
8.
Nucleic Acids Res ; 47(4): e24, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30590765

RESUMO

Chimeric RNAs generated by cis-splicing between adjacent genes (cis-SAGe) are increasingly recognized as a widespread phenomenon. These chimeric messenger RNAs are present in normal human cells, and are also detected in various cancers. The mechanisms for how this group of chimeras is formed are not yet clear, in part due to the lack of a tractable system for their experimental investigation. Here we developed a fast, easy and versatile cell-based reporter system to identify regulators of cis-SAGe. The reporter, consisting of four main cassettes, simultaneously measures the effects of a candidate regulator on cis-SAGe and canonical splicing. Using this cell-based assay, we screened 102 candidate factors involved in RNA pol II cleavage and termination, elongation, splicing, alternative splicing and R-loop formation. We discovered that two factors, SRRM1 and SF3B1, affect not only cis-SAGe chimeras, but also other types of chimeric RNAs in a genome-wide fashion. This system can be used for studying trans-acting factors and cis-acting sequence elements and factors, as well as for screening small molecule inhibitors.


Assuntos
Antígenos Nucleares/genética , Regulação da Expressão Gênica/genética , Proteínas Associadas à Matriz Nuclear/genética , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Processamento de RNA/genética , Proteínas de Ligação a RNA/genética , Processamento Alternativo/genética , Regulação Neoplásica da Expressão Gênica/genética , Fusão Gênica/genética , Genes Reporter/genética , Genoma Humano/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Clivagem do RNA/genética , RNA Polimerase II/genética
9.
Mol Cell ; 73(2): 212-223.e7, 2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30554942

RESUMO

Cohesin subunits are frequently mutated in cancer, but how they function as tumor suppressors is unknown. Cohesin mediates sister chromatid cohesion, but this is not always perturbed in cancer cells. Here, we identify a previously unknown role for cohesin. We find that cohesin is required to repress transcription at DNA double-strand breaks (DSBs). Notably, cohesin represses transcription at DSBs throughout interphase, indicating that this is distinct from its known role in mediating DNA repair through sister chromatid cohesion. We identified a cancer-associated SA2 mutation that supports sister chromatid cohesion but is unable to repress transcription at DSBs. We further show that failure to repress transcription at DSBs leads to large-scale genome rearrangements. Cancer samples lacking SA2 display mutational patterns consistent with loss of this pathway. These findings uncover a new function for cohesin that provides insights into its frequent loss in cancer.


Assuntos
Neoplasias Ósseas/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Quebras de DNA de Cadeia Dupla , Instabilidade Genômica , Interfase , Osteossarcoma/genética , Transcrição Genética , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Reparo do DNA , Regulação para Baixo , Fase G1 , Fase G2 , Regulação Neoplásica da Expressão Gênica , Humanos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
Viruses ; 10(12)2018 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-30572664

RESUMO

Influenza A virus (IAV) infections remain a major human health threat. IAV has enormous genetic plasticity and can rapidly escape virus-targeted anti-viral strategies. Thus, there is increasing interest to identify host proteins and processes the virus requires for replication and maturation. The IAV non-structural protein 1 (NS1) is a critical multifunctional protein that is expressed to high levels in infected cells. Host proteins that interact with NS1 may serve as ideal targets for attenuating IAV replication. We previously developed and characterized broadly cross-reactive anti-NS1 monoclonal antibodies. For the current study, we used these mAbs to co-immunoprecipitate native IAV NS1 and interacting host proteins; 183 proteins were consistently identified in this NS1 interactome study, 124 of which have not been previously reported. RNAi screens identified 11 NS1-interacting host factors as vital for IAV replication. Knocking down one of these, nuclear mitotic apparatus protein 1 (NUMA1), dramatically reduced IAV replication. IAV genomic transcription and translation were not inhibited but transport of viral structural proteins to the cell membrane was hindered during maturation steps in NUMA1 knockdown (KD) cells.


Assuntos
Antígenos Nucleares/metabolismo , Interações Hospedeiro-Patógeno , Vírus da Influenza A/fisiologia , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas não Estruturais Virais/metabolismo , Montagem de Vírus , Células A549 , Antígenos Nucleares/genética , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Imunoprecipitação , Vírus da Influenza A/genética , Proteínas Associadas à Matriz Nuclear/genética , Interferência de RNA , Proteínas não Estruturais Virais/genética , Replicação Viral
11.
Sci Rep ; 8(1): 17891, 2018 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-30559450

RESUMO

Non-homologous end-joining (NHEJ), which can promote genomic instability when dysfunctional, is a major DNA double-strand break (DSB) repair pathway. Although ubiquitylation of the core NHEJ factor, Ku (Ku70-Ku80), which senses broken DNA ends, is important for its removal from sites of damage upon completion of NHEJ, the mechanism regulating Ku ubiquitylation remains elusive. We provide evidence showing that the ubiquitin carboxyl-terminal hydrolase L3 (UCHL3) interacts with and directly deubiquitylates one of the Ku heterodimer subunits, Ku80. Additionally, depleting UCHL3 resulted in reduced Ku80 foci formation, Ku80 binding to chromatin after DSB induction, moderately sensitized cells to ionizing radiation and decreased NHEJ efficiencies. Mechanistically, we show that DNA damage induces UCHL3 phosphorylation, which is dependent on ATM, downstream NHEJ factors and UCHL3 catalytic activity. Furthermore, this phosphorylation destabilizes UCHL3, despite having no effect on its catalytic activity. Collectively, these data suggest that UCHL3 facilitates cellular viability after DSB induction by antagonizing Ku80 ubiquitylation to enhance Ku80 retention at sites of damage.


Assuntos
Cisteína Endopeptidases/genética , Dano ao DNA/genética , Autoantígeno Ku/genética , Ubiquitinação/genética , Antígenos Nucleares/genética , Linhagem Celular , Sobrevivência Celular/genética , Cromatina/genética , DNA/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , Fosforilação/genética , Ligação Proteica/genética
12.
PLoS One ; 13(12): e0208955, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30562372

RESUMO

Inflammatory and microenvironmental factors produced by cancer cells are thought to directly or indirectly promote cancer cell growth. Prostaglandins, including prostaglandin E2, have key roles as a microenvironment factor in influencing the development of tumors, and are produced by the rate limiting enzyme cyclooxygenase 2 (COX-2). In this study, we used canine melanoma cells treated with the proinflammatory cytokine interleukin 1ß (IL-1ß) and investigated the transcriptional factor nuclear factor-κB (NF-κB) signaling in IL-1ß-induced COX-2 expression. IL-1ß induced prostaglandin E2 release and COX-2 mRNA expression in a time- and dose-dependent manner. In the cells treated with the NF-κB inhibitors BAY11-7082 and TPC-1, IL-1ß-mediated prostaglandin E2 release and COX-2 mRNA expression were inhibited. IL-1ß also provoked phosphorylation of p65/RelA and p105/NF-κB1, which are members of the NF-κB families. The IL-1ß-induced phosphorylation of p65 and p105 was attenuated in the presence of both NF-κB inhibitors. In melanoma cells transfected with siRNA of p65 or p105, IL-1ß-mediated COX-2 mRNA expression was inhibited. These findings suggest that canonical activation of NF-κB signaling plays a crucial role for inflammatory states in melanoma cells.


Assuntos
Antígenos Nucleares/genética , Proteínas Cromossômicas não Histona/genética , Ciclo-Oxigenase 2/genética , Melanoma/genética , Fator de Transcrição RelA/genética , Animais , Modelos Animais de Doenças , Cães , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-1beta/genética , Melanoma/patologia , Nitrilos/farmacologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais/genética , Sulfonas/farmacologia
13.
Cell Physiol Biochem ; 46(4): 1365-1380, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29689552

RESUMO

BACKGROUND/AIMS: RBFOX3, an RNA-binding fox protein, plays an important role in the differentiation of neuronal development, but its role in the chemosensitivity of hepatocellular carcinoma (HCC) to 5-FU is unknown. METHODS: In this study, we examined the biological functions of RBFOX3 and its effect on the chemosensitivity of HCC cells to 5-FU in vitro and in a mouse xenograft model. RESULTS: RBFOX3 was found to have elevated expression in HCC cell lines and tissue samples, and its knockdown inhibited HCC cell proliferation. Moreover, knockdown of RBFOX3 improved the inhibitory effect of 5-fluorouracil (5-FU) on cell proliferation, migration and invasion, and enhanced the apoptosis induced by 5-FU. However, overexpression of RBFOX3 reduced the inhibitory effect of 5-fluorouracil (5-FU) on cell proliferation, migration and invasion, and decreased the apoptosis induced by 5-FU. We further elucidated that RBFOX3 knockdown synergized with 5-FU to inhibit the growth and invasion of HCC cells through PI3K/AKT and epithelial-mesenchymal transition (EMT) signaling, and promote apoptosis by activating the cytochrome-c/caspase signaling pathway. Finally, we validated that RBFOX3 regulated 5-FU-mediated cytotoxicity in HCC in mouse xenograft models. CONCLUSIONS: The findings from this study indicate that RBFOX3 regulates the chemosensitivity of HCC to 5-FU in vitro and in vivo. Therefore, targeting RBFOX3 may improve the inhibition of HCC growth and progression by 5-FU, and provide a novel potential therapeutic strategy for HCC.


Assuntos
Antígenos Nucleares/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fluoruracila/toxicidade , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Antígenos Nucleares/genética , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Caspases/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocromos c/metabolismo , Fluoruracila/uso terapêutico , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Metástase Neoplásica , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transplante Heterólogo
14.
Biochem Biophys Res Commun ; 499(1): 30-36, 2018 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-29551686

RESUMO

Alternative splicing is an essential process in eukaryotes, as it increases the complexity of gene expression by generating multiple proteins from a single pre-mRNA. However, information on the regulatory mechanisms for alternative splicing is lacking, because splicing occurs over a short period via the transient interactions of proteins within functional complexes of the spliceosome. Here, we investigated in detail the molecular mechanisms connecting alternative splicing with epigenetic mechanisms. We identified interactions between histone proteins and splicing factors such as Rbfox2, Rbfox3, and splicing factor proline and glutamine rich protein (SFPQ) by in vivo crosslinking and immunoprecipitation. Furthermore, we confirmed that splicing factors were bound to specific modified residues of histone proteins. Additionally, changes in histone methylation due to histone methyltransferase inhibitor treatment notably affected alternative splicing in selected genes. Therefore, we suggested that there may be crosstalk mechanisms connecting histone modifications and RNA-binding proteins that increase the local concentration of RNA-binding proteins in alternative exon loci of nucleosomes by binding specific modified histone proteins, leading to alternative splicing. This crosstalk mechanism may play a major role in epigenetic processes such as histone modification and the regulation of alternative splicing.


Assuntos
Processamento Alternativo , Antígenos Nucleares/genética , Epigênese Genética , Histonas/genética , Proteínas do Tecido Nervoso/genética , Fator de Processamento Associado a PTB/genética , Fatores de Processamento de RNA/genética , Proteínas Repressoras/genética , Antígenos Nucleares/metabolismo , Cromatina/química , Cromatina/metabolismo , Reagentes para Ligações Cruzadas/química , Células HeLa , Histonas/metabolismo , Humanos , Imunoprecipitação , Metilação , Proteínas do Tecido Nervoso/metabolismo , Fator de Processamento Associado a PTB/metabolismo , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Spliceossomos/genética , Spliceossomos/metabolismo , Succinimidas/química
15.
Genes Chromosomes Cancer ; 57(6): 311-319, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29427526

RESUMO

The advent of large scale genomic sequencing technologies significantly improved the molecular classification of acute megakaryoblastic leukaemia (AMKL). AMKL represents a subset (∼10%) of high fatality pediatric acute myeloid leukemia (AML). Recurrent and mutually exclusive chimeric gene fusions associated with pediatric AMKL are found in 60%-70% of cases and include RBM15-MKL1, CBFA2T3-GLIS2, NUP98-KDM5A and MLL rearrangements. In addition, another 4% of AMKL harbor NUP98 rearrangements (NUP98r), with yet undetermined fusion partners. We report a novel NUP98-BPTF fusion in an infant presenting with primary refractory AMKL. In this NUP98r, the C-terminal chromatin recognition modules of BPTF, a core subunit of the NURF (nucleosome remodeling factor) ATP-dependent chromatin-remodeling complex, are fused to the N-terminal moiety of NUP98, creating an in frame NUP98-BPTF fusion, with structural homology to NUP98-KDM5A. The leukemic blasts expressed two NUP98-BPTF splicing variants, containing one or two tandemly spaced PHD chromatin reader domains. Our study also identified an unreported wild type BPTF splicing variant encoding for 2 PHD domains, detected both in normal cord blood CD34+ cells and in leukemic blasts, as with the fly BPTF homolog, Nurf301. Disease course was marked by rapid progression and primary chemoresistance, with ultimately significant tumor burden reduction following treatment with a clofarabine containing regimen. In sum, we report 2 novel NUP98-BPTF fusion isoforms that contribute to refine the NUP98r subgroup of pediatric AMKL. Multicenter clinical trials are critically required to determine the frequency of this fusion in AMKL patients and explore innovative treatment strategies for a disease still plagued with poor outcomes.


Assuntos
Antígenos Nucleares/genética , Leucemia Megacarioblástica Aguda/genética , Proteínas do Tecido Nervoso/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Fatores de Transcrição/genética , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica , Humanos , Lactente , Cariotipagem , Leucemia Megacarioblástica Aguda/tratamento farmacológico , Masculino , Processamento de RNA
16.
PLoS Pathog ; 14(1): e1006865, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29364981

RESUMO

Gammaherpesvirus (GHV) pathogenesis is a complex process that involves productive viral replication, dissemination to tissues that harbor lifelong latent infection, and reactivation from latency back into a productive replication cycle. Traditional loss-of-function mutagenesis approaches in mice using murine gammaherpesvirus 68 (MHV68), a model that allows for examination of GHV pathogenesis in vivo, have been invaluable for defining requirements for specific viral gene products in GHV infection. But these approaches are insufficient to fully reveal how viral gene products contribute when the encoded protein facilitates multiple processes in the infectious cycle and when these functions vary over time and from one host tissue to another. To address this complexity, we developed an MHV68 genetic platform that enables cell-type-specific and inducible viral gene deletion in vivo. We employed this system to re-evaluate functions of the MHV68 latency-associated nuclear antigen (mLANA), a protein with roles in both viral replication and latency. Cre-mediated deletion in mice of loxP-flanked ORF73 demonstrated the necessity of mLANA in B cells for MHV68 latency establishment. Impaired latency during the transition from draining lymph nodes to blood following mLANA deletion also was observed, supporting the hypothesis that B cells are a major conduit for viral dissemination. Ablation of mLANA in infected germinal center (GC) B cells severely impaired viral latency, indicating the importance of viral passage through the GC for latency establishment. Finally, induced ablation of mLANA during latency resulted in complete loss of affected viral genomes, indicating that mLANA is critically important for maintenance of viral genomes during stable latency. Collectively, these experiments provide new insights into LANA homolog functions in GHV colonization of the host and highlight the potential of a new MHV68 genetic platform to foster a more complete understanding of viral gene functions at discrete stages of GHV pathogenesis.


Assuntos
Antígenos Nucleares/genética , Gammaherpesvirinae/genética , Proteínas Virais/genética , Células 3T3 , Animais , Células Cultivadas , Doença Crônica , Embrião de Mamíferos , Feminino , Gammaherpesvirinae/patogenicidade , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutagênese/fisiologia , Células NIH 3T3 , Especificidade de Órgãos , Latência Viral/genética
17.
J Investig Clin Dent ; 9(1)2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28631891

RESUMO

AIM: Saliva can play an important role in human herpesvirus-8 (HHV-8) transmission in endemic regions for Kaposi's sarcoma (KS). Little is known about HHV-8 oral shedding in immunocompetent individuals from non-endemic regions for KS. METHODS: We conducted a prospective study of HHV-8 salivary excretion among 59 healthy, immunocompetent individuals from São Paulo, Brazil, followed up weekly for 4 months, resulting in 16 saliva samples from each participant. Antibodies to HHV-8 latency-associated nuclear antigen (LANA) and lytic-phase antigens were investigated with immunofluorescence assays (IFA). HHV-8 DNA detection was performed using real-time polymerase chain reaction (PCR). RESULTS: All 59 individuals were seronegative to LANA and lytic antibodies. HHV-8 DNA was undetectable in saliva samples in 100% of the participants, totaling 944 samples and being consistently negative during the different periods of sampling, which lasted approximately 120 days. No sequences of HHV-8 DNA were detected in the saliva samples of healthy, immunocompetent adults by using real-time PCR, with the resulting data being consistent with IFA-based serological tests. CONCLUSIONS: Unlike other herpesviruses, HHV-8 is not excreted in the saliva of healthy individuals from non-endemic regions for KS.


Assuntos
Herpesvirus Humano 8/isolamento & purificação , Herpesvirus Humano 8/patogenicidade , Sarcoma de Kaposi/epidemiologia , Sarcoma de Kaposi/virologia , Actinas/metabolismo , Adulto , Anticorpos Antivirais , Antígenos Nucleares/genética , Antígenos Nucleares/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Brasil/epidemiologia , DNA Viral/isolamento & purificação , Feminino , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Projetos Piloto , Estudos Prospectivos , Saliva/virologia , Testes Sorológicos , Proteínas Virais/genética , Proteínas Virais/imunologia , Adulto Jovem
18.
Gut Microbes ; 9(1): 68-75, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-28816579

RESUMO

Factors shaping the human intestinal microbiota range from environmental influences, like smoking and exercise, over dietary patterns and disease to the host's genetic variation. Recently, we could show in a microbiome genome-wide association study (mGWAS) targeting genetic variation influencing the ß diversity of gut microbial communities, that approximately 10% of the overall gut microbiome variation can be explained by host genetics. Here, we report on the application of a new method for genotype-ß-diversity association testing, the distance-based F (DBF) test. With this we identified 4 loci with genome-wide significant associations, harboring the genes CBEP4, SLC9A8, TNFSF4, and SP140, respectively. Our findings highlight the utility of the high-performance DBF test in ß diversity GWAS and emphasize the important role of host genetics and immunity in shaping the human intestinal microbiota.


Assuntos
Bactérias/genética , Biodiversidade , Microbioma Gastrointestinal , Loci Gênicos/genética , Estudo de Associação Genômica Ampla , Modelos Estatísticos , Antígenos Nucleares/genética , Bactérias/classificação , Variação Genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade/genética , Ligante OX40/genética , Proteínas de Ligação a RNA/genética , Reprodutibilidade dos Testes , Trocadores de Sódio-Hidrogênio/genética , Fatores de Transcrição/genética
19.
Kobe J Med Sci ; 64(3): E93-E106, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-30666039

RESUMO

A number of studies have investigated the effects of ischemic injury on functional and cellular characteristics of hippocampus. There is only a limited study on vascular remodeling of it. The present study aimed at examining vascular remodeling in hippocampus and spatial memory disturbances after transient brain ischemia. Male Wistar rats were randomly divided into four groups, i.e. sham operated (SHAM), transient brain ischemia with 1 day reperfusion (IR1), 3 day reperfusion (IR3), and 10 days reperfusion (IR10) groups. Transient brain ischemia was induced by bilateral common carotid artery occlusion (BCCAO). The spatial memory test was performed using the Morris water maze (MWM) in SHAM and IR10 groups. The rats were euthanized at day 1, 3 or 10 after BCCAO depending on the groups. The mRNA expressions of SOD2, Bcl-2, NeuN, eNOS, endothelin-1 (ET-1), CD31, VE-cadherin and vascular remodeling of the hippocampus were examined. There were deteriorations of spatial learning ability in IR10 group. The percentages of SOD2 and Bcl-2, the expression of NeuN, decreased and the vascular remodeling was observed in the ischemic groups. The eNOS and CD31 expressions were less in IR10, the VE-cadherin expression was less in all ischemic groups than in SHAM group, while ET-1 expression in IR1 group was higher than any other groups. The spatial memory deterioration after BCCAO is associated with vascular remodeling in hippocampus, characterized by lumen narrowing and smooth muscle thickening of microvessels.


Assuntos
Hipocampo/irrigação sanguínea , Hipocampo/fisiopatologia , Ataque Isquêmico Transitório/psicologia , Transtornos da Memória/etiologia , Remodelação Vascular/fisiologia , Animais , Antígenos CD/genética , Antígenos Nucleares/genética , Caderinas/genética , Modelos Animais de Doenças , Endotelina-1/genética , Expressão Gênica , Genes bcl-2 , Hipocampo/fisiologia , Ataque Isquêmico Transitório/complicações , Ataque Isquêmico Transitório/patologia , Masculino , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/patologia , Transtornos da Memória/fisiopatologia , Proteínas do Tecido Nervoso/genética , Óxido Nítrico Sintase Tipo III/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Memória Espacial/fisiologia , Superóxido Dismutase/genética , Remodelação Vascular/genética
20.
Cytogenet Genome Res ; 153(2): 96-104, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29186711

RESUMO

The genus Colomesus is the sole representative of the family Tetraodontidae in the Amazon region. Here, Colomesus asellus was analyzed using conventional and molecular cytogenetic protocols. Its diploid chromosome number is 2n = 46 with 12 meta-, 10 submeta-, 16 subtelo-, and 8 acrocentric chromosomes and a fundamental number of FN = 84. An XX/XY sex chromosome system was identified. Mapping of 18S rDNA correlated with the nucleolus organizer regions (Ag-NORs) in the short arms of the 2 X chromosomes in females and in the Y chromosome in males. C-banding revealed heterochromatin in the centromeric regions of all chromosomes, except for pair 3. Prominent sex chromosome-specific heterochromatin amplification was observed, covering the short arms of the Y chromosome almost entirely. FISH with telomeric and tropomyosin (tpm1) sequences, respectively, revealed terminal signals in all chromosomes. The analysis of extended DNA fibers confirmed the colocalization and the interspersed pattern of the telomeric and tpm1 sequences. Thus, this study highlights the remarkable evolutionary dynamism presented by the Amazonian puffer fish regarding the differentiation of a heteromorphic XY sex chromosome system and a particular sex-specific amplification of rDNA sites. This is the first record of such an association in the Tetraodontidae family.


Assuntos
Cromossomos Sexuais/genética , Processos de Determinação Sexual , Tetraodontiformes/genética , Animais , Antígenos Nucleares/genética , Brasil , Bandeamento Cromossômico , DNA Ribossômico/genética , Feminino , Amplificação de Genes , Hibridização in Situ Fluorescente , Masculino , RNA Ribossômico 18S/genética , Sequências Repetitivas de Ácido Nucleico , Telômero/genética , Telômero/ultraestrutura , Tropomiosina/genética
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