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1.
PLoS Genet ; 16(8): e1008976, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866141

RESUMO

Neural circuitry for mating and reproduction resides within the terminal segments of central nervous system (CNS) which express Hox paralogous group 9-13 (in vertebrates) or Abdominal-B (Abd-B) in Drosophila. Terminal neuroblasts (NBs) in A8-A10 segments of Drosophila larval CNS are subdivided into two groups based on expression of transcription factor Doublesex (Dsx). While the sex specific fate of Dsx-positive NBs is well investigated, the fate of Dsx-negative NBs is not known so far. Our studies with Dsx-negative NBs suggests that these cells, like their abdominal counterparts (in A3-A7 segments) use Hox, Grainyhead (Grh) and Notch to undergo cell death during larval development. This cell death also happens by transcriptionally activating RHG family of apoptotic genes through a common apoptotic enhancer in early to mid L3 stages. However, unlike abdominal NBs (in A3-A7 segments) which use increasing levels of resident Hox factor Abdominal-A (Abd-A) as an apoptosis trigger, Dsx-negative NBs (in A8-A10 segments) keep the levels of resident Hox factor Abd-B constant. These cells instead utilize increasing levels of the temporal transcription factor Grh and a rise in Notch activity to gain apoptotic competence. Biochemical and in vivo analysis suggest that Abdominal-A and Grh binding motifs in the common apoptotic enhancer also function as Abdominal-B and Grh binding motifs and maintains the enhancer activity in A8-A10 NBs. Finally, the deletion of this enhancer by the CRISPR-Cas9 method blocks the apoptosis of Dsx-negative NBs. These results highlight the fact that Hox dependent NB apoptosis in abdominal and terminal regions utilizes common molecular players (Hox, Grh and Notch), but seems to have evolved different molecular strategies to pattern CNS.


Assuntos
Apoptose/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Receptores Notch/genética , Fatores de Transcrição/genética , Abdome/crescimento & desenvolvimento , Animais , Sistema Nervoso Central/crescimento & desenvolvimento , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Larva/genética , Larva/crescimento & desenvolvimento , Masculino , Células-Tronco Neurais/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética
2.
Anticancer Res ; 40(10): 5601-5609, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988884

RESUMO

BACKGROUND/AIM: Since pathways involving LRRC17 are related to the survival of patients with various cancers, we analyzed LRRC17 as a prognostic gene in serous ovarian cancer. MATERIALS AND METHODS: Data were collected from Gene Expression Omnibus (GSE9891, GSE13876, and GSE26712) and The Cancer Genome Atlas (TCGA). We performed survival analyses using C statistics, area under the curve, survival plot with optimal cutoff level, and cox proportional regression. Zebrafish embryos were used as an in vivo model. RESULTS: The prognosis of patients with high LRRC17 expression was poorer than that of patients with low LRRC17 expression. Multivariate regression analysis showed that LRRC17 expression, age, and stage were independently related with survival. Knockdown of lrrc17 reduced survival rate and delayed development in zebrafish embryos. We also found that lrrc17 is important for cell viability by protecting from p53-dependent apoptosis. CONCLUSION: LRRC17 could be a prognostic gene in ovarian cancer as it regulates cancer cell viability through the p53 pathway.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Ovarianas/genética , Proteínas/genética , Proteína Supressora de Tumor p53/genética , Idoso , Apoptose/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/patologia , Prognóstico
3.
Anticancer Res ; 40(10): 5631-5639, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988887

RESUMO

BACKGROUND/AIM: DNA damage response (DDR), wherein p21 is a cell fate determinant, is a potential cancer therapeutic target. Molecular expression during DDR was explored in ovarian clear-cell carcinoma (CCC). MATERIALS AND METHODS: CHK1, CHK2, TP53 and p21 expression in DDR was examined using immunostaining in surgical sections of CCC (n=22). Molecular alterations in two types of CCC cell lines, JHOC-5 and JHOC-9, were investigated using western blot analysis. RESULTS: Expression of DDR-associated molecules was noted in most patients. While high p21 expression was found in half of the patients, the remaining patients exhibited low p21 expression. Treatment with UC2288, a p21 inhibitor, attenuated proliferation of both cell lines, more prominently in JHOC-9, resulting in reduced viability and subsequent apoptosis. CONCLUSION: p21 Inhibitor induced cell death in cells with high p21 expression, suggesting that p21 suppression can be a therapeutic strategy to treat patients with CCC.


Assuntos
Quinase 1 do Ponto de Checagem/genética , Quinase do Ponto de Checagem 2/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Neoplasias Ovarianas/genética , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Dano ao DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/patologia
4.
J Toxicol Sci ; 45(9): 549-558, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32879254

RESUMO

Trimethyltin chloride (TMT) is a stabilizer by-product in the process of manufacturing plastic, which is a kind of very strong toxic substance, and has acute, cumulative and chronic toxicity. TMT may cause bradycardia in patients with occupational poisoning, the mechanism of which has not been reported. This study explored the mechanism of TMT resulting in bradycardia of C57BL/6 mice. TMT was administered to mice to measure heart rate, serum succinate dehydrogenase (SDH) level, and myocardial Na+/K+-ATPase activity and expression. The effects of TMT on myocardial apoptosis were observed by changing the expressions of caspase-3, Bax and Bcl-2 in myocardium. It was found that the heart rate and SDH activity in serum of mice gradually decreased with the increase of TMT dose compared with the control group. The activity and the expression of Na+/K+-ATPase in the heart tissue of mice exposed to TMT was measured and gradually decreased with the increase of dose and time. We measured the expression of Bcl-2, Bax, caspase-3 and cleaved caspase-3 in the heart tissues of TMT exposed mice and found that the expressions of Bax, caspase-3 and cleaved caspase-3 increased and the expressions of Bcl-2 decreased in the heart tissues of the TMT-exposed mice at different doses. With the extension of TMT exposure time, the expression of Bax and caspase-3 increased and the expression of Bcl-2 decreased in the heart tissues of TMT exposed mice. Our findings suggest the mechanisms of TMT resulting in bradycardia may be associated with the inhibited activity and decreased content of Na+/K+-ATPase, thus further leading to the changes of Bcl-2, Bax, caspase-3 and cleaved caspase-3 in the mice's ventricular tissues.


Assuntos
Apoptose/efeitos dos fármacos , Bradicardia/etiologia , Miocárdio/metabolismo , Miocárdio/patologia , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Compostos de Trimetilestanho/toxicidade , Animais , Apoptose/genética , Bradicardia/genética , Caspase 3/genética , Caspase 3/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
5.
BMC Bioinformatics ; 21(1): 396, 2020 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-32894041

RESUMO

BACKGROUND: MicroRNAs are a class of important small noncoding RNAs, which have been reported to be involved in the processes of tumorigenesis and development by targeting a few genes. Existing studies show that the imbalance between cell proliferation and apoptosis is closely related to the initiation and development of cancers. However, the impact of miRNAs on this imbalance has not been studied systematically. RESULTS: In this study, we first construct a cell fate miRNA-gene regulatory network. Then, we propose a systematical method for calculating the global impact of miRNAs on cell fate genes based on the shortest path. Results on breast cancer and liver cancer datasets show that most of the cell fate genes are perturbed by the differentially expressed miRNAs. Most of the top-identified miRNAs are verified in the Human MicroRNA Disease Database (HMDD) and are related to breast and liver cancers. Function analysis shows that the top 20 miRNAs regulate multiple cell fate related function modules and interact tightly based on their functional similarity. Furthermore, more than half of them can promote sensitivity or induce resistance to some anti-cancer drugs. Besides, survival analysis demonstrates that the top-ranked miRNAs are significantly related to the overall survival time in the breast and liver cancers group. CONCLUSION: In sum, this study can help to systematically study the important role of miRNAs on proliferation and apoptosis and thereby uncover the key miRNAs during the process of tumorigenesis. Furthermore, the results of this study will contribute to the development of clinical therapy based miRNAs for cancers.


Assuntos
Apoptose/genética , Proliferação de Células/genética , MicroRNAs/metabolismo , Biomarcadores/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Bases de Dados Genéticas , Feminino , Redes Reguladoras de Genes , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , MicroRNAs/genética , RNA Mensageiro/metabolismo , Análise de Sobrevida
6.
Ecotoxicol Environ Saf ; 205: 111283, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32977282

RESUMO

Fine particulate matter (PM2.5) airborne pollution increases the risk of chronic respiratory diseases, such as idiopathic pulmonary fibrosis (IPF), which is characterized by non-specific inflammation of the interstitial lung and extensive deposition of collagen fibers. Type 2 alveolar epithelial cells (AEC2s) are alveolar stem cells in the adult lung that contribute to the lung repair process through complex signaling. Our previous studies demonstrated that OGG1, a kind of DNA repair enzyme, have a critical role in protecting cells from oxidative damage and apoptosis induced by PM2.5, but the contribution of OGG1 in proliferation and self-renewal of AEC2s is not known. Here, we constructed OGG1-/-mice to test the effect and mechanism of OGG1 on PM2.5-induced pulmonary fibrosis and injury in vivo. We detected proliferation and self-renewal of OGG1 overexpression or OGG1 knockout AEC2s after PM2.5 injury by flow cytometry and clone formation. We observed that knockout of OGG1 aggravated pulmonary fibrosis, oxidative stress, and AEC2 cell death in PM2.5-injured mice. In addition, OGG1 is required for the proliferation and renewal of AEC2s after PM2.5 injury. Overexpression of OGG1 promotes the proliferation and self-renewal of AEC2s by inhibiting PM2.5-mediated oxidative stress and NF-κB signaling hyperactivation in vitro. Furthermore, NF-κB inhibitors promoted proliferation and self-renewal of OGG1-deficient AEC2s cells after PM2.5 injury, and attenuated PM2.5-induced pulmonary fibrosis and injury in mice. These data establish OGG1 as a regulator of NF-κB signal that serves to regulate AEC2 cell proliferation and self-renewal, and suggest a mechanism that inhibition of the NF-κB signaling pathway may represent a potential therapeutic strategy for IPF patients with low-expression of OGG1.


Assuntos
Poluentes Atmosféricos/toxicidade , Células Epiteliais Alveolares/efeitos dos fármacos , Autorrenovação Celular/genética , DNA Glicosilases/metabolismo , Material Particulado/toxicidade , Fibrose Pulmonar/induzido quimicamente , Células-Tronco/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , DNA Glicosilases/genética , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Transdução de Sinais , Células-Tronco/metabolismo , Células-Tronco/patologia
7.
Nat Commun ; 11(1): 4527, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913197

RESUMO

Evasion of programmed cell death represents a critical form of oncogene addiction in cancer cells. Understanding the molecular mechanisms underpinning cancer cell survival despite the oncogenic stress could provide a molecular basis for potential therapeutic interventions. Here we explore the role of pro-survival genes in cancer cell integrity during clonal evolution in non-small cell lung cancer (NSCLC). We identify gains of MCL-1 at high frequency in multiple independent NSCLC cohorts, occurring both clonally and subclonally. Clonal loss of functional TP53 is significantly associated with subclonal gains of MCL-1. In mice, tumour progression is delayed upon pharmacologic or genetic inhibition of MCL-1. These findings reveal that MCL-1 gains occur with high frequency in lung adenocarcinoma and can be targeted therapeutically.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico por imagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Evolução Clonal , Variações do Número de Cópias de DNA , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Progressão da Doença , Humanos , Pulmão/diagnóstico por imagem , Pulmão/patologia , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Transgênicos , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Cultura Primária de Células , Estudos Prospectivos , Proteínas Proto-Oncogênicas p21(ras)/genética , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , RNA-Seq , Estudos Retrospectivos , Esferoides Celulares , Tiofenos/farmacologia , Tiofenos/uso terapêutico , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Proteína Supressora de Tumor p53/genética , Microtomografia por Raio-X
8.
Nat Commun ; 11(1): 4534, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913330

RESUMO

Collisions between the DNA replication machinery and co-transcriptional R-loops can impede DNA synthesis and are a major source of genomic instability in cancer cells. How cancer cells deal with R-loops to proliferate is poorly understood. Here we show that the ATP-dependent chromatin remodelling INO80 complex promotes resolution of R-loops to prevent replication-associated DNA damage in cancer cells. Depletion of INO80 in prostate cancer PC3 cells leads to increased R-loops. Overexpression of the RNA:DNA endonuclease RNAse H1 rescues the DNA synthesis defects and suppresses DNA damage caused by INO80 depletion. R-loops co-localize with and promote recruitment of INO80 to chromatin. Artificial tethering of INO80 to a LacO locus enabled turnover of R-loops in cis. Finally, counteracting R-loops by INO80 promotes proliferation and averts DNA damage-induced death in cancer cells. Our work suggests that INO80-dependent resolution of R-loops promotes DNA replication in the presence of transcription, thus enabling unlimited proliferation in cancers.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proliferação de Células/genética , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Neoplasias/genética , Estruturas R-Loop/genética , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Montagem e Desmontagem da Cromatina , Dano ao DNA , Instabilidade Genômica , Humanos , Neoplasias/patologia , Transcrição Genética
9.
Anticancer Res ; 40(9): 5035-5041, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32878791

RESUMO

BACKGROUND/AIM: Based on the cytotoxic agent (-)-zampanolide, N,N'-(arylmethylene)bisamides were designed and synthesized as candidate anti-cancer agents. Among them, N,N'-[(3,4-dimethoxyphenyl)methylene]biscinnamide (DPMBC) was identified as the most potent cytotoxic analog against cancer cells. In this study, we investigated the mechanisms underlying DPMBC-induced cell death in HL-60 human promyelocytic leukemia and PC-3 human prostate cancer cells. MATERIALS AND METHODS: Cell growth was assessed by the WST-8 assay. Induction of apoptosis was assessed by nuclear morphology, DNA ladder formation, and flow cytometry using Annexin V staining. Activation of factors in the apoptotic signaling pathway was assessed by western blot analyses. Knockdown of death receptor 5 (DR5) was performed using siRNA. RESULTS: DPMBC up-regulated expression levels of DR5 protein and induced apoptosis through the extrinsic apoptotic pathway mediated by DR5 and caspases. CONCLUSION: DPMBC is an extrinsic apoptosis inducer, which has potential as a therapeutic agent for cancer therapy.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Macrolídeos/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Antineoplásicos/química , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fragmentação do DNA , Relação Dose-Resposta a Droga , Humanos , Macrolídeos/química , Estrutura Molecular , RNA Interferente Pequeno/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo
10.
Int J Nanomedicine ; 15: 6355-6372, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32922006

RESUMO

Background: Cerium oxide nanoparticles (CeO2NPs) are potent scavengers of cellular reactive oxygen species (ROS). Their antioxidant properties make CeO2NPs promising therapeutic agents for bone diseases and bone tissue engineering. However, the effects of CeO2NPs on intracellular ROS production in osteoclasts (OCs) are still unclear. Numerous studies have reported that intracellular ROS are essential for osteoclastogenesis. The aim of this study was to explore the effects of CeO2NPs on osteoclast differentiation and the potential underlying mechanisms. Methods: The bidirectional modulation of osteoclast differentiation by CeO2NPs was explored by different methods, such as fluorescence microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting. The cytotoxic and proapoptotic effects of CeO2NPs were detected by cell counting kit (CCK-8) assay, TdT-mediated dUTP nick-end labeling (TUNEL) assay, and flow cytometry. Results: The results of this study demonstrated that although CeO2NPs were capable of scavenging ROS in acellular environments, they facilitated the production of ROS in the acidic cellular environment during receptor activator of nuclear factor kappa-Β ligand (RANKL)-dependent osteoclast differentiation of bone marrow-derived macrophages (BMMs). CeO2NPs at lower concentrations (4.0 µg/mL to 8.0 µg/mL) promoted osteoclast formation, as shown by increased expression of Nfatc1 and C-Fos, F-actin ring formation and bone resorption. However, at higher concentrations (greater than 16.0 µg/mL), CeO2NPs inhibited osteoclast differentiation and promoted apoptosis of BMMs by reducing Bcl2 expression and increasing the expression of cleaved caspase-3, which may be due to the overproduction of ROS. Conclusion: This study demonstrates that CeO2NPs facilitate osteoclast formation at lower concentrations while inhibiting osteoclastogenesis in vitro by inducing the apoptosis of BMMs at higher concentrations by modulating cellular ROS levels.


Assuntos
Diferenciação Celular , Cério/química , Osteoclastos/citologia , Espécies Reativas de Oxigênio/metabolismo , Actinas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Reabsorção Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/ultraestrutura , Masculino , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Nanopartículas/ultraestrutura , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ligante RANK/farmacologia , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
11.
Int J Nanomedicine ; 15: 6421-6432, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32922009

RESUMO

Purpose: Expanded research on the biomedical applications of graphene has shown promising results, although interactions between cells and graphene are still unclear. The current study aims to dissect the cellular and molecular effects of graphene nanocomposite in photothermal therapy against cancer, and to evaluate its efficacy. Methods: In this study, a reduced graphene oxide and iron oxide (rGO-Fe3O4) nanocomposite was obtained by chemical synthesis. The nanocomposite was fully characterized by Raman spectroscopy, TEM, VSM and thermal profiling. Cell-nanocomposite interaction was evaluated by confocal microscopy and viability assays on cancer cell line HeLa. The efficacy of the thermal therapy and changes in gene expression of Bcl-2 and Hsp70 was assessed. Results: The resulting rGO-Fe3O4 nanocomposite exhibited superparamagnetic properties and the capacity to increase the surrounding temperature by 18-20°C with respect to the initial temperature. The studies of cell-nanocomposite interaction showed that rGO-Fe3O4 attaches to cell membrane but there is a range of concentration at which the nanomaterial preserves cell viability. Photothermal therapy reduced cell viability to 32.6% and 23.7% with 50 and 100 µg/mL of nanomaterial, respectively. The effect of treatment on the molecular mechanism of cell death demonstrated an overexpression of anti-apoptotic proteins Hsp70 and Bcl-2 as an initial response to the therapy and depending on the aggressiveness of the treatment. Conclusion: The results of this study contribute to understanding the interactions between cell and graphene and support its application in photothermal therapy against cancer due to its promising results.


Assuntos
Compostos Férricos/química , Grafite/química , Hipertermia Induzida , Nanocompostos/química , Neoplasias/terapia , Fototerapia , Apoptose/genética , Comunicação Celular , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Nanocompostos/ultraestrutura , Neoplasias/genética , Neoplasias/patologia , Oxirredução , Análise Espectral Raman , Temperatura , Resultado do Tratamento
12.
J Environ Pathol Toxicol Oncol ; 39(2): 101-111, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32749120

RESUMO

Long noncoding RNAs (lncRNAs) have been reported to be involved in cancer initiation and evolution, including colorectal cancer (CRC). Nuclear-enriched abundant transcript 1 (NEAT1) exerts important functions in multiple cancers; however, the specific modulatory mechanism in CRC demands in-depth exploration. The expression levels of NEAT1, microRNA-195-5p (miR-195-5p), and centrosomal protein 55 (CEP55) were examined using quantitative real-time polymerase chain reaction (qRT-PCR), and protein expression of CEP55 was detected by Western blot assay. Cell proliferation and apoptosis were measured by 3-(4,5-dimethylthiazole-2-y1)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry. Transwell migration and invasion assays were applied to evaluate cell metastasis ability. Dual-luciferase reporter assay was used to analyze the correlation among NEAT1, miR-195-5p and CEP55. The expression of NEAT1 was up-regulated in CRC tissues and cells, and overall survival was lower with high expression of NEAT1. Knockdown of NEAT1 repressed cell proliferation, migration, and invasion, while inducing apoptosis in CRC cells. NEAT1 targeted miR-195-5p and inhibited the expression of miR-195-5p. Silence of NEAT1 inhibited CRC cell proliferation, migration, and invasion, and promoted apoptosis by up-regulating miR-195-5p. MiR-195-5p targeted and suppressed CEP55 expression, and CEP55 reverted the effects induced by miR-195-5p. NEAT1 regulated the expression of CEP55 through miR-195-5p. NEAT1 promotes colorectal cancer cellular processes by regulating CEP55 expression via the sponging of miR-195-5p. Therefore, NEAT1 might play a crucial role in CRC treatments.


Assuntos
Neoplasias Colorretais/genética , RNA Longo não Codificante/genética , Apoptose/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Mucosa Gástrica/citologia , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Estimativa de Kaplan-Meier , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo
13.
Nat Commun ; 11(1): 3822, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732898

RESUMO

Alveolar macrophages (AMs) derived from embryonic precursors seed the lung before birth and self-maintain locally throughout adulthood, but are regenerated by bone marrow (BM) under stress conditions. However, the regulation of AM development and maintenance remains poorly understood. Here, we show that histone deacetylase 3 (HDAC3) is a key epigenetic factor required for AM embryonic development, postnatal homeostasis, maturation, and regeneration from BM. Loss of HDAC3 in early embryonic development affects AM development starting at E14.5, while loss of HDAC3 after birth affects AM homeostasis and maturation. Single-cell RNA sequencing analyses reveal four distinct AM sub-clusters and a dysregulated cluster-specific pathway in the HDAC3-deficient AMs. Moreover, HDAC3-deficient AMs exhibit severe mitochondrial oxidative dysfunction and deteriorative cell death. Mechanistically, HDAC3 directly binds to Pparg enhancers, and HDAC3 deficiency impairs Pparg expression and its signaling pathway. Our findings identify HDAC3 as a key epigenetic regulator of lung AM development and homeostasis.


Assuntos
Histona Desacetilases/genética , Homeostase/genética , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Animais , Apoptose/genética , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Histona Desacetilases/deficiência , Histona Desacetilases/metabolismo , Pulmão/embriologia , Pulmão/crescimento & desenvolvimento , Macrófagos Alveolares/citologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos
14.
PLoS Genet ; 16(8): e1008953, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776944

RESUMO

Apoptosis of cochlear hair cells is a key step towards age-related hearing loss. Although numerous genes have been implicated in the genetic causes of late-onset, progressive hearing loss, few show direct links to the proapoptotic process. By genome-wide linkage analysis and whole exome sequencing, we identified a heterozygous p.L183V variant in THOC1 as the probable cause of the late-onset, progressive, non-syndromic hearing loss in a large family with autosomal dominant inheritance. Thoc1, a member of the conserved multisubunit THO/TREX ribonucleoprotein complex, is highly expressed in mouse and zebrafish hair cells. The thoc1 knockout (thoc1 mutant) zebrafish generated by gRNA-Cas9 system lacks the C-startle response, indicative of the hearing dysfunction. Both Thoc1 mutant and knockdown zebrafish have greatly reduced hair cell numbers, while the latter can be rescued by embryonic microinjection of human wild-type THOC1 mRNA but to significantly lesser degree by the c.547C>G mutant mRNA. The Thoc1 deficiency resulted in marked apoptosis in zebrafish hair cells. Consistently, transcriptome sequencing of the mutants showed significantly increased gene expression in the p53-associated signaling pathway. Depletion of p53 or applying the p53 inhibitor Pifithrin-α significantly rescued the hair cell loss in the Thoc1 knockdown zebrafish. Our results suggested that THOC1 deficiency lead to late-onset, progressive hearing loss through p53-mediated hair cell apoptosis. This is to our knowledge the first human disease associated with THOC1 mutations and may shed light on the molecular mechanism underlying the age-related hearing loss.


Assuntos
Proteínas de Ligação a DNA/genética , Surdez/genética , Células Ciliadas Auditivas Internas/metabolismo , Proteínas de Ligação a RNA/genética , Proteína Supressora de Tumor p53/genética , Animais , Apoptose/genética , Benzotiazóis/farmacologia , Proteína 9 Associada à CRISPR/genética , Proteínas de Ligação a DNA/deficiência , Surdez/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patologia , Células Ciliadas Auditivas Internas/patologia , Humanos , Camundongos , Mutação , RNA Guia/genética , Transdução de Sinais/efeitos dos fármacos , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Sequenciamento Completo do Exoma , Peixe-Zebra/genética
15.
Yonsei Med J ; 61(8): 660-669, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32734729

RESUMO

PURPOSE: Neonatal hypoxic ischemic encephalopathy (HIE) is an essential factor underlying neonatal death and disability. This study sought to explore the role of miR-146b-5p in regulating neonatal HIE. MATERIALS AND METHODS: In vitro and in vivo HIE models were established in PC12 cells and 10-day neonatal Sprague Dawley rats, respectively. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to assess miR-146b-5p expression and inflammatory factors [interleukin (IL)-6 and tumor necrosis factor (TNF)-α] in brain lesions and PC12 cells, while enzyme-linked immunosorbent assay was employed to detect the expression of oxidative stress factors (SOD and GSH-Px). Gain- and loss-assays of miR-146b-5p were conducted to verify its role in modulating the viability and apoptosis of PC12 cells under oxygen-glucose deprivation (OGD) treatment. Expression of TLR4, IRAK1, TRAF6, TAK1, and NF-κB were examined by qRT-PCR and/or Western blot. Dual luciferase activity assay was conducted to identify relationships between miR-146b-5p and IRAK1. RESULTS: In the HIE models, significant oxidative stress and inflammatory responses emerged upon upregulation of TLR4/IRAK1/TRAF6/TAK1/NF-κB signaling. Overexpression of miR-146b-5p greatly inhibited OGD-induced PC12 cell injury, inflammatory responses, and oxidative stress. Inhibiting miR-146b-5p, however, had the opposite effects. IRAK1 was found to be a target of miR-146b-5p, and miR-146b-5p overexpression suppressed the activation of IRAK1/TRAF6/TAK1/NF-κB signaling. CONCLUSION: This study demonstrated that miR-146b-5p overexpression alleviates HIE-induced neuron injury by inhibiting the IRAK1/TRAF6/TAK1/NF-κB pathway.


Assuntos
Hipóxia-Isquemia Encefálica/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , MAP Quinase Quinase Quinases/metabolismo , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/genética , Sequência de Bases , Modelos Animais de Doenças , Regulação para Baixo/genética , Glucose/deficiência , Inflamação/patologia , MicroRNAs/genética , Estresse Oxidativo , Oxigênio , Células PC12 , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismo
16.
Int J Nanomedicine ; 15: 5131-5146, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764941

RESUMO

Background: Gene therapy is considered a novel way to treat osteosarcoma, and microRNAs are potential therapeutic targets for osteosarcoma. miR-214 has been found to promote osteosarcoma aggression and metastasis. Graphene oxide (GO) is widely used for gene delivery for the distinct physiochemical properties and minimal cytotoxicity. Methods: Polyethyleneimine (PEI)-functionalized GO complex was well-prepared and loaded with miR-214 inhibitor at different concentrations. The load efficacy was tested by gel retardation assay and the cy3-labeled fluorescence of cellular uptake. The experiments of wound healing, immunofluorescence staining, Western blot, qRT-PCR and immunohistochemical staining were performed to measure the inhibitory effect of the miR-214 inhibitor systematically released from the complexes against MG63, U2OS cells and xenograft tumors. Results: The systematic mechanistic elucidation of the efficient delivery of the miR-214 inhibitor by GO-PEI indicated that the inhibition of cellular miR-214 caused a decrease in osteosarcoma cell invasion and migration and an increase in apoptosis by targeting phosphatase and tensin homolog (PTEN). The synergistic combination of the GO-PEI-miR-214 inhibitor and CDDP chemotherapy showed significant cell death. In a xenograft mouse model, the GO-PEI-miR-214 inhibitor significantly inhibited tumor volume growth. Conclusion: This study indicates the potential of functionalized GO-PEI as a vehicle for miRNA inhibitor delivery to treat osteosarcoma with low toxicity and miR-214 can be a good target for osteosarcoma therapy.


Assuntos
Grafite/química , MicroRNAs/antagonistas & inibidores , Terapia de Alvo Molecular , Osteossarcoma/tratamento farmacológico , PTEN Fosfo-Hidrolase/metabolismo , Polietilenoimina/química , Polietilenoimina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias Ósseas/patologia , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/genética , Terapia Combinada , Humanos , Camundongos , MicroRNAs/genética , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia
17.
Arch Virol ; 165(10): 2165-2176, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32740830

RESUMO

The PI3K/Akt signalling pathway is a crucial signalling cascade that regulates transcription, protein translation, cell growth, proliferation, cell survival, and metabolism. During viral infection, viruses exploit a variety of cellular pathways, including the well-known PI3K/Akt signalling pathway. Conversely, cells rely on this pathway to stimulate an antiviral response. The PI3K/Akt pathway is manipulated by a number of viruses, including DNA and RNA viruses and retroviruses. The aim of this review is to provide up-to-date information about the role of the PI3K-Akt pathway in infection with members of five different families of negative-sense ssRNA viruses. This pathway is hijacked for viral entry, regulation of endocytosis, suppression of premature apoptosis, viral protein expression, and replication. Although less common, the PI3K/Akt pathway can be downregulated as an immunomodulatory strategy or as a mechanism for inducing autophagy. Moreover, the cell activates this pathway as an antiviral strategy for interferon and cytokine production, among other strategies. Here, we present new data concerning the role of this pathway in infection with the paramyxovirus Newcastle disease virus (NDV). Our data seem to indicate that NDV uses the PI3K/Akt pathway to delay cell death and increase cell survival as a means of improving its replication. The interference of negative-sense ssRNA viruses with this essential pathway might have implications for the development of antiviral therapies.


Assuntos
Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Fosfatidilinositol 3-Quinase/genética , Proteínas Proto-Oncogênicas c-akt/genética , Infecções por Vírus de RNA/genética , Apoptose/genética , Autofagia/genética , Autofagia/imunologia , Citocinas/genética , Citocinas/imunologia , Endocitose/genética , Endocitose/imunologia , Filoviridae/genética , Filoviridae/metabolismo , Filoviridae/patogenicidade , Interações Hospedeiro-Patógeno/imunologia , Interferons/genética , Interferons/imunologia , Orthomyxoviridae/genética , Orthomyxoviridae/metabolismo , Orthomyxoviridae/patogenicidade , Paramyxoviridae/genética , Paramyxoviridae/metabolismo , Paramyxoviridae/patogenicidade , Fosfatidilinositol 3-Quinase/imunologia , Pneumovirinae/genética , Pneumovirinae/metabolismo , Pneumovirinae/patogenicidade , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-akt/imunologia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/virologia , Rhabdoviridae/genética , Rhabdoviridae/metabolismo , Rhabdoviridae/patogenicidade , Transdução de Sinais , Proteínas Virais/genética , Proteínas Virais/imunologia , Internalização do Vírus , Replicação Viral
18.
Nat Commun ; 11(1): 4071, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792491

RESUMO

Arrest of oligodendrocyte (OL) differentiation and remyelination following myelin damage in multiple sclerosis (MS) is associated with neurodegeneration and clinical worsening. We show that Glutathione S-transferase 4α (Gsta4) is highly expressed during adult OL differentiation and that Gsta4 loss impairs differentiation into myelinating OLs in vitro. In addition, we identify Gsta4 as a target of both dimethyl fumarate, an existing MS therapy, and clemastine fumarate, a candidate remyelinating agent in MS. Overexpression of Gsta4 reduces expression of Fas and activity of the mitochondria-associated Casp8-Bid-axis in adult oligodendrocyte precursor cells, leading to improved OL survival during differentiation. The Gsta4 effect on apoptosis during adult OL differentiation was corroborated in vivo in both lysolecithin-induced demyelination and experimental autoimmune encephalomyelitis models, where Casp8 activity was reduced in Gsta4-overexpressing OLs. Our results identify Gsta4 as an intrinsic regulator of OL differentiation, survival and remyelination, as well as a potential target for future reparative MS therapies.


Assuntos
Glutationa Transferase/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Caspase 8/genética , Caspase 8/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Glutationa Transferase/genética , Homeostase/genética , Homeostase/fisiologia , Imuno-Histoquímica , Masculino , Microglia/citologia , Microglia/metabolismo , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Fagocitose/genética , Fagocitose/fisiologia , Processamento de Proteína Pós-Traducional , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Remielinização/genética , Remielinização/fisiologia
19.
Nat Commun ; 11(1): 4147, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811837

RESUMO

Mutated receptor tyrosine kinases (MT-RTKs) such as internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3 ITD) and a point mutation KIT D816V are driver mutations for acute myeloid leukemia (AML). Clathrin assembly lymphoid myeloid leukemia protein (CALM) regulates intracellular transport of RTKs, however, the precise role for MT-RTKs remains elusive. We here show that CALM knock down leads to severely impaired FLT3 ITD- or KIT D814V-dependent cell growth compared to marginal influence on wild-type FLT3- or KIT-mediated cell growth. An antipsychotic drug chlorpromazine (CPZ) suppresses the growth of primary AML samples, and human CD34+CD38- AML cells including AML initiating cells with MT-RTKs in vitro and in vivo. Mechanistically, CPZ reduces CALM protein at post transcriptional level and perturbs the intracellular localization of MT-RTKs, thereby blocking their signaling. Our study presents a therapeutic strategy for AML with MT-RTKs by altering the intracellular localization of MT-RTKs using CPZ.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Clorpromazina/farmacologia , Leucemia Mieloide Aguda/genética , Proteínas Monoméricas de Montagem de Clatrina/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Feminino , Células HL-60 , Humanos , Leucemia Mieloide Aguda/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Proteínas Monoméricas de Montagem de Clatrina/genética , Mutação Puntual , Transdução de Sinais/efeitos dos fármacos , Sequências de Repetição em Tandem/genética , Transplante Heterólogo , Adulto Jovem
20.
PLoS One ; 15(8): e0237669, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32810137

RESUMO

Pancreatic beta cell death is a hallmark of type 1 and 2 diabetes (T1D/T2D), but the underlying molecular mechanisms are incompletely understood. Key proteins of the DNA damage response (DDR), including tumor protein P53 (P53, also known as TP53 or TRP53 in rodents) and Ataxia Telangiectasia Mutated (ATM), a kinase known to act upstream of P53, have been associated with T2D. Here we test and compare the effect of ATM and P53 ablation on beta cell survival in the rat beta cell line Ins1E. We demonstrate that ATM and P53 differentially regulate beta cell apoptosis induced upon fundamentally different types of diabetogenic beta cell stress, including DNA damage, inflammation, lipotoxicity and endoplasmic reticulum (ER) stress. DNA damage induced apoptosis by treatment with the commonly used diabetogenic agent streptozotocin (STZ) is regulated by both ATM and P53. We show that ATM is a key STZ induced activator of P53 and that amelioration of STZ induced cell death by inhibition of ATM mainly depends on P53. While both P53 and ATM control lipotoxic beta cell apoptosis, ATM but not P53 fails to alter inflammatory beta cell death. In contrast, tunicamycin induced (ER stress associated) apoptosis is further increased by ATM knockdown or inhibition, but not by P53 knockdown. Our results reveal differential roles for P53 and ATM in beta cell survival in vitro in the context of four key pathophysiological types of diabetogenic beta cell stress, and indicate that ATM can use P53 independent signaling pathways to modify beta cell survival, dependent on the cellular insult.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sobrevivência Celular/genética , Células Secretoras de Insulina/patologia , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Diabetes Mellitus/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Técnicas de Silenciamento de Genes , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Ratos , Estreptozocina/toxicidade , Tunicamicina/toxicidade
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