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1.
Cell Oncol (Dordr) ; 45(3): 463-477, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35543859

RESUMO

PURPOSE: Reliable biomarkers to predict the outcome and treatment response of estrogen receptor (ER)-negative breast cancer (BC) are urgently needed. Since immune-related signaling plays an important role in the tumorigenesis of ER-negative BC, we asked whether Notch genes, alone or in combination with other immune genes, can be used to predict the clinical outcome and immune checkpoint blockade (ICB) for this type of cancer. METHODS: We analyzed transcriptome data of 6918 BC samples from five independent cohorts, 81 xenograft triple-negative BC tumors that respond differently to ICB treatment and 754 samples of different cancer types from patients treated with ICB agents. RESULTS: We found that among four Notch genes, the expression levels of NOTCH1 and NOTCH4 were positively associated with recurrence of ER-negative BC, and that combined expression of these two genes (named Notch14) further enhanced this association, which was comparable with that of the Notch pathway signature. Analysis of 1182 immune-related genes revealed that the expression levels of most HLA genes, particularly HLA-DMA and -DRA, were reversely associated with recurrence in ER-negative BC with low, but not high Notch14 expression. A combined expression signature of NOTCH1, NOTCH4, HLA-DMA and HLA-DRA was more prognostic for ER-negative and triple-negative BCs than previously reported immune-related signatures. Furthermore, we found that the expression levels of these four genes were also synergistically associated with T cell exclusion score, infiltration of specific T cells and ICB efficacy in ER-negative BC, thereby providing a potential molecular mechanism for the synergistic effect of these genes on BC. CONCLUSIONS: Our data indicate that a gene signature composed of NOTCH1, NOTCH4, HLA-DMA and HLA-DRA may serve as a potential promising biomarker for predicting ICB therapy efficacy and recurrence in ER-negative/triple-negative BCs.


Assuntos
Cadeias alfa de HLA-DR , Receptor Notch1 , Receptor Notch4 , Linfócitos T , Neoplasias de Mama Triplo Negativas , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Feminino , Cadeias alfa de HLA-DR/biossíntese , Cadeias alfa de HLA-DR/genética , Cadeias alfa de HLA-DR/imunologia , Humanos , Receptor Notch1/biossíntese , Receptor Notch1/genética , Receptor Notch1/imunologia , Receptor Notch4/biossíntese , Receptor Notch4/genética , Receptor Notch4/imunologia , Linfócitos T/imunologia , Linfócitos T/patologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
2.
Neurochem Int ; 150: 105156, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34389314

RESUMO

Notch1 not only plays a key role in the development of the nervous system but also modulates synaptic plasticity and memory. However, the role of Notch1 in the brain of diabetes is still unclear. We hypothesize that Notch1 is involved in type I diabetes-induced cognitive dysfunction. In this study, adult male C57BL/6J mice carrying a heterozygous null mutation in the Notch1 gene (Notch1+/-) and wild-type littermate controls were used in this experiment. They were subjected to streptozocin (55 mg/kg, i.p.) for consecutive five days. After 12 weeks, the cognitive function of all mice was detected by novel object recognition (NOR) test and electrophysiological recording. Our results demonstrated that the levels of Notch1 mRNA and Notch1 receptor were increased in the hippocampus of the wild-type diabetic mice at 12 weeks. It suggested that the Notch1 signal pathway was activated. Compared with the wild-type diabetic mice, the discrimination index and the long-term potentiation was further decreased in the Notch1+/- diabetic group, the impairment of neuronal ultrastructure was exacerbated in the hippocampus of the Notch1+/- diabetic mice, and the number of synapses and autophagic vacuoles were significantly reduced in the Notch1+/- diabetic group. Moreover, some postsynaptic associated protein expressions were down-regulated, as well as the Beclin1 expression and the ratio of LC3II/LC3I were reduced in the hippocampus of the Notch1+/- diabetic mice. Interestingly, the phosphorylation of mTOR, Akt, and ERK1/2 were all inhibited in the Notch1+/- diabetic group. Taken together, these results suggest that Notch1 deficiency deteriorates the synaptic plasticity and inhibits the activation of autophagy partly via the mTOR-independent signal pathway in the hippocampus of type I diabetic mice.


Assuntos
Autofagia/fisiologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Hipocampo/metabolismo , Receptor Notch1/biossíntese , Animais , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patologia , Hipocampo/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor Notch1/genética
3.
J Neuroinflammation ; 18(1): 108, 2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-33971906

RESUMO

BACKGROUND: Interleukin 9 (IL-9), produced mainly by T helper 9 (Th9) cells, has been recognized as an important regulator in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Astrocytes respond to IL-9 and reactive astrocytes always associate with blood-brain barrier damage, immune cell infiltration, and spinal injury in MS and EAE. Several long non-coding RNAs (lncRNAs) with aberrant expression have been identified in the pathogenesis of MS. Here, we examined the effects of lncRNA Gm13568 (a co-upregulated lncRNA both in EAE mice and in mouse primary astrocytes activated by IL-9) on the activation of astrocytes and the process of EAE. METHODS: In vitro, shRNA-recombinant lentivirus with glial fibrillary acidic protein (GFAP) promoter were performed to determine the relative gene expression and proinflammatory cytokines production in IL-9 treated-astrocytes using Western blot, real-time PCR, and Cytometric Bead Array, respectively. RIP and ChIP assays were analyzed for the mechanism of lncRNA Gm13568 regulating gene expression. Immunofluorescence assays was performed to measure the protein expression in astrocytes. In vivo, H&E staining and LFB staining were applied to detect the inflammatory cells infiltrations and the medullary sheath damage in spinal cords of EAE mice infected by the recombinant lentivirus. Results were analyzed by one-way ANOVA or Student's t test, as appropriate. RESULTS: Knockdown of the endogenous lncRNA Gm13568 remarkably inhibits the Notch1 expression, astrocytosis, and the phosphorylation of signal transducer and activator of transcription 3 (p-STAT3) as well as the production of inflammatory cytokines and chemokines (IL-6, TNF-α, IP-10) in IL-9-activated astrocytes, in which Gm13568 associates with the transcriptional co-activators CBP/P300 which are enriched in the promoter of Notch1 genes. More importantly, inhibiting Gm13568 with lentiviral vector in astrocytes ameliorates significantly inflammation and demyelination in EAE mice, therefore delaying the EAE process. CONCLUSIONS: These findings uncover that Gm13568 regulates the production of inflammatory cytokines in active astrocytes and affects the pathogenesis of EAE through the Notch1/STAT3 pathway. LncRNA Gm13568 may be a promising target for treating MS and demyelinating diseases.


Assuntos
Astrócitos/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Interleucina-9/metabolismo , RNA Longo não Codificante/imunologia , Receptor Notch1/biossíntese , Fatores de Transcrição de p300-CBP/metabolismo , Animais , Astrócitos/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Regulação da Expressão Gênica/imunologia , Interleucina-9/imunologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Longo não Codificante/metabolismo , Receptor Notch1/imunologia , Fatores de Transcrição de p300-CBP/imunologia
4.
Life Sci ; 271: 119156, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33548289

RESUMO

Arecoline N-oxide (ANO), an oxidative metabolite of the areca nut, is a predictable initiator in carcinogenesis. The mechanisms of arecoline metabolites in human cancer specimens is still limited. This present study aims to estimate the oral squamous cell carcinoma (OSCC) inductive activity between arecoline metabolites in human cancer specimens/OSCC cells. We have collected 22 pairs (tumor and non-tumor part) of patient's specimens and checked for clinical characteristics. The identification of arecoline and its metabolites levels by using LC-MS/MS. The NOD/SCID mice model was used to check the OSCC inductive activity. The tumor part of OSCC samples exhibited higher levels of arecoline and ANO. Besides, ANO treated mice accelerates the NOTCH1, IL-17a and IL-1ß expressions compared to the control mice. ANO exhibited higher cytotoxicity, intracellular ROS levels and decline in antioxidant enzyme levels in OC-3 cells. The protein expression of NOTCH1 and proliferation marker levels are significantly lower in NOM treated cells. Overall, ANO induced initial stage carcinogenesis in the oral cavity via inflammation, ROS and depletion of antioxidant enzymes. Arecoline N-oxide mercapturic acid (NOM) attenuates the initiation of oral carcinogenesis.


Assuntos
Acetilcisteína/uso terapêutico , Arecolina/análogos & derivados , Óxidos N-Cíclicos/toxicidade , Sequestradores de Radicais Livres/uso terapêutico , Neoplasias Bucais/induzido quimicamente , Neoplasias Bucais/prevenção & controle , Adulto , Animais , Arecolina/toxicidade , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Neoplasias Bucais/metabolismo , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/biossíntese , Células Tumorais Cultivadas
5.
Exp Neurol ; 339: 113645, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33600815

RESUMO

Microglia are rapidly activated after acute ischemic stroke, and the polarization of microglial is associated with the prognosis of acute ischemic stroke. Lipoxin A4 (LXA4), an anti-inflammatory agent, has a protective effect against ischemic stroke. However, the role of LXA4 on the polarization of microglial after acute ischemic stroke remains undetermined. We hypothesized that LXA4 may exert the neuroprotective effect though regulating the polarization of microglial. In this study, clinical features of acute ischemic stroke were simulated using a rat model of model of middle cerebral artery occlusion (MCAO) in vivo and the BV2 microglia oxygen-glucose deprivation/reoxygenation model (OGD/R) in vitro. The protective effects of LXA4 on cerebral ischemia-reperfusion injury were determined using TTC staining, HE staining, and TUNEL staining. The expression of targeted genes was assayed using quantitative real-time PCR (qRT-PCR), immunofluorescence, and western blot to investigated the regulation of LXA4 on microglia polarization after acute ischemic stroke. We found that LXA4 exerted protective effects on focal cerebral ischemia-reperfusion injury and reduced the expression of the pro-inflammatory cytokines IL-1ß and TNF-α. Furthermore, LXA4 inhibited the expression of Notch-1, Hes1, iNOS and CD32 all of which are associated with the differentiation into M1 microglia. By contrast, LXA4 upregulated the expression of Hes5, Arg-1 and CD206 all of which are associated with M2 phenotype in microglia. In addition, blocking the Notch signaling pathway with the inhibitor DAPT significantly mitigated the effect of LXA4 on microglia differentiation. These data suggest that LXA4 may regulate the polarization of microglia after cerebral ischemia-reperfusion injury through the Notch signaling pathway.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Polaridade Celular/efeitos dos fármacos , Microglia/efeitos dos fármacos , Receptor Notch1/antagonistas & inibidores , Receptores de Lipoxinas/administração & dosagem , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Isquemia Encefálica/metabolismo , Linhagem Celular , Polaridade Celular/fisiologia , Masculino , Microglia/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Notch1/biossíntese , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
6.
Pharm Biol ; 59(1): 21-30, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33417512

RESUMO

CONTEXT: Berberine (BBR) is used to treat diarrhoea and gastroenteritis in the clinic. It was found to have anticolon cancer effects. OBJECTIVE: To study the anticolon cancer mechanism of BBR by connectivity map (CMAP) analysis. MATERIALS AND METHODS: CMAP based mechanistic prediction was conducted by comparing gene expression profiles of 10 µM BBR treated MCF-7 cells with that of clinical drugs such as helveticoside, ianatoside C, pyrvinium, gossypol and trifluoperazine. The treatment time was 12 h and two biological replications were performed. The DMSO-treated cells were selected as a control. The interaction between 100 µM BBR and target protein was measured by cellular thermal shift assay. The protein expression of 1-9 µM BBR treated SW480 cells were measured by WB assay. Apoptosis, cell cycle arrest, mitochondrial membrane potential (MMP) of 1-9 µM BBR treated SW480 cells were measured by flow cytometry and Hoechst 33342 staining methods. RESULTS: CMAP analysis found 14 Hsp90, HDAC, PI3K or mTOR protein inhibitors have similar functions with BBR. The experiments showed that BBR inhibited SW480 cells proliferation with IC50 of 3.436 µM, induced apoptosis, autophage, MMP depolarization and arrested G1 phase of cell cycle at 1.0 µM. BBR dose-dependently up-regulated PTEN, while inhibited Notch1, PI3K, Akt and mTOR proteins at 1.0-9.0 µM (p < 0.05). BBR also acted synergistically with Hsp90 and HDAC inhibitor (0.01 µM) in SW480 cells at 0.5 and 1.0 µM. DISCUSSION AND CONCLUSIONS: The integrative gene expression-based chemical genomic method using CMAP analysis may be applicable for mechanistic studies of other multi-targets drugs.


Assuntos
Berberina/administração & dosagem , Neoplasias do Colo/metabolismo , PTEN Fosfo-Hidrolase/biossíntese , Fosfatidilinositol 3-Quinases/biossíntese , Proteínas Proto-Oncogênicas c-akt/biossíntese , Receptor Notch1/biossíntese , Serina-Treonina Quinases TOR/biossíntese , Células A549 , Antineoplásicos/administração & dosagem , Benzoquinonas/administração & dosagem , Neoplasias do Colo/tratamento farmacológico , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Células HCT116 , Humanos , Lactamas Macrocíclicas/administração & dosagem , Células MCF-7 , Nylons , Inibidores de Fosfoinositídeo-3 Quinase/administração & dosagem , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Pirróis/administração & dosagem , Receptor Notch1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Células THP-1 , Serina-Treonina Quinases TOR/antagonistas & inibidores
7.
Dig Dis Sci ; 66(1): 131-142, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32144600

RESUMO

BACKGROUND: In liver cirrhosis, intestinal mucus barrier is rarely studied. AIMS: This study aimed to investigate whether mucus barrier in ileum is altered in cirrhotic rats and its underlying mechanisms. METHODS: Thioacetamide was injected to induce liver cirrhosis in rats. Serum from portal vein blood, and ileum and liver tissues were obtained for further analysis. Goblet cell-like Ls174T cells were cultured for in vitro experiments. RESULTS: The ileal mucus was thin, loose, and porous with small bubbles in cirrhotic rats. mRNA expressions of Muc2 and TFF3 were also down-regulated in cirrhotic rats. Bacteria located near to crypts and LPS were increased in the serum from portal vein in cirrhotic rats. Smaller theca area and few goblet cells were found in cirrhotic rats compared with control. Increased proliferation of ileal epithelia was observed in cirrhotic rats. Notch1, Dll1, and Hes1 expressions were enhanced, and KLF4 expression was suppressed in ileum of cirrhotic rats. In Ls174T cells, EDTA and NICD plasmid induced NICD and Hes1 expression and suppressed KLF4 concomitantly, and mucus expression almost vanished in these cells. NICD plasmid induced more proliferation in Ls174T cells. Oppositely, after DBZ treatment, NICD and Hes1 were inhibited along with augmentation of KLF4 and increased mucous expression in Ls174T cells, while proliferation of the cells was suppressed. CONCLUSIONS: In cirrhotic rats, mucus barrier was impaired. This might be attributed to increased proliferation and decreased differentiation of epithelia, which might be mediated by Notch1-Hes1-KLF4 signaling.


Assuntos
Homeostase/fisiologia , Íleo/metabolismo , Mucosa Intestinal/metabolismo , Cirrose Hepática/metabolismo , Receptor Notch1/biossíntese , Animais , Linhagem Celular Tumoral , Homeostase/efeitos dos fármacos , Humanos , Íleo/efeitos dos fármacos , Íleo/patologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Fator 4 Semelhante a Kruppel , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Tioacetamida/toxicidade
8.
Histol Histopathol ; 36(1): 69-76, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33094831

RESUMO

BACKGROUND: The Notch signaling pathway has different roles in many human neoplasms, being either tumor-promoting or anti-proliferative. In addition, Notch signaling in carcinogenesis can be tissue dependent. The aim of the current study is to elucidate the relation between Notch1 protein expression in lung cancer cells and the following Notch related proteins: Hes1, c-Myc, Jagged1 and Jagged2. METHODS: Notch1 and its related proteins were detected in human lung cancer cell lines and in 54 surgically resected different lung carcinoma tissues. Then, we used small interfering RNA (siRNA) technology, to down-regulate the expression of Notch1 in H69AR and SBC3 small cell lung carcinoma (SCLC) cells. Also, we transfected venus Notch1 intracellular domain (v.NICD) plasmid into human SCLC lines; H69. RESULTS: The expression of Hes1, c-Myc and Jagged2 is affected by Notch1 in SCLC. CONCLUSION: There is a strong association between the expression of Notch1 protein and the expression of Hes1, c-Myc and Jagged2 proteins, which could aid in better understanding tumorigenesis in SCLC.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Receptor Notch1/biossíntese , Carcinoma de Pequenas Células do Pulmão/metabolismo , Células A549 , Linhagem Celular Tumoral , Humanos , Proteína Jagged-1/biossíntese , Proteína Jagged-2/biossíntese , Proteínas Proto-Oncogênicas c-myc/biossíntese , RNA Interferente Pequeno/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Fatores de Transcrição HES-1/biossíntese
9.
Neuromolecular Med ; 23(3): 348-362, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33095377

RESUMO

In response to hypoxic-ischemic brain damage (HIBD), microglia activation and its mediated inflammation contribute to neuronal damage. Inhibition of over-activated microglia is deemed to be a potential therapeutic strategy. Our previous studies showed that gastrodin efficiently depressed the neuroinflammation mediated by activated microglia in HIBD neonatal rats. The underlying mechanisms through which gastrodin acts on activated microglia have not been fully elucidated. This study is designed to determine whether gastrodin would regulate the Notch signaling pathway and Sirtuin3 (Sirt3), which are implicated in regulating microglia activation. The present results showed that gastrodin markedly suppressed the expression of members of Notch signaling pathway (Notch-1, NICD, RBP-JK and Hes-1) in activated microglia both in vivo and in vitro. Conversely, Sirt3 expression was enhanced. In BV-2 microglia treated with a γ-secretase inhibitor of Notch pathway- DAPT, the expression of RBP-JK, Hes-1, and NICD was suppressed in activated microglia. Treatment with DAPT and gastrodin further decreased NICD and Hes-1 expression. Sirt3 expression was also decreased after DAPT treatment. However, Sirt3 expression in activated BV-2 microglia given a combined DAPT and gastrodin treatment was not further increased. In addition, combination of DAPT and Gastrodin cumulatively decreased tumor necrosis factor-α (TNF-α) expression. The results suggest that gastrodin regulates microglia activation via the Notch signaling pathway and Sirt3. More importantly, interference of the Notch signaling pathway inhibited Sirt3 expression, indicating that Sirt3 is a downstream gene of the Notch signaling pathway. It is suggested that Notch and Sirt3 synergistically regulate microglia activation such as in TNF-α production.


Assuntos
Álcoois Benzílicos/farmacologia , Glucosídeos/farmacologia , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Microglia/efeitos dos fármacos , Receptor Notch1/fisiologia , Transdução de Sinais/efeitos dos fármacos , Sirtuínas/fisiologia , Animais , Animais Recém-Nascidos , Álcoois Benzílicos/farmacocinética , Artéria Carótida Primitiva , Células Cultivadas , Córtex Cerebral/patologia , Corpo Caloso/patologia , Diaminas/farmacologia , Modelos Animais de Doenças , Sinergismo Farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Glucosídeos/farmacocinética , Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/patologia , Ligadura , Lipopolissacarídeos/farmacologia , Microglia/metabolismo , Doenças Neuroinflamatórias/tratamento farmacológico , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Receptor Notch1/biossíntese , Receptor Notch1/genética , Sirtuínas/biossíntese , Sirtuínas/genética , Tiazóis/farmacologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética
10.
Sci Rep ; 10(1): 12342, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32704077

RESUMO

Gastric cancer (GC) is one of the most prevalent cancers and a major cause of cancer related mortality worldwide. Incidence of GC is affected by various factors, including genetic and environmental factors. Despite extensive research has been done for molecular characterization of GC, it remains largely unknown. Therefore, further studies specially conducted among various ethnicities in different geographic locations, are required to know the precise molecular mechanisms leading to tumorigenesis and progression of GC. The expression patterns of seven candidate genes, including ß-catenin, Notch1, GATA6, CDX2, miR-34a, miR-181a, and miR-93 were determined in 24 paired GC tissues and corresponding non-cancerous tissues by quantitative Real-Time PCR. The association between the expression of these genes and clinicopathologic factors were also investigated. Our results demonstrated that overall mRNA levels of GATA6 were significantly decreased in the tumor samples in comparison with the non-cancerous tissues (median fold change (FC) = 0.3143; P = 0.0003). Overall miR-93 levels were significantly increased in the tumor samples relative to the non-cancerous gastric tissues (FC = 2.441; P = 0.0002). ß-catenin mRNA expression showed a strong positive correlation with miR-34a (r = 0.5784; P = 0.0031), and miR-181a (r = 0.5652; P = 0.004) expression. miR-34a and miR-181a expression showed a significant positive correlation (r = 0.4862; P = 0.016). Moreover, lower expression of Notch1 was related to distant metastasis in GC patients with a borderline statistical significance (p = 0.0549). These data may advance our understanding of the molecular biology that drives GC as well as provide potential targets for defining novel therapeutic strategies for GC treatment.


Assuntos
Fator de Transcrição CDX2/biossíntese , Fator de Transcrição GATA6/biossíntese , Regulação Neoplásica da Expressão Gênica , MicroRNAs/biossíntese , Proteínas de Neoplasias/biossíntese , RNA Neoplásico/biossíntese , Receptor Notch1/biossíntese , Neoplasias Gástricas/metabolismo , beta Catenina/biossíntese , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Gástricas/patologia
11.
Biol Reprod ; 103(3): 548-559, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32507875

RESUMO

Numerous oocytes are retrieved during in vitro fertilization from patients with polycystic ovary syndrome (PCOS). The poor quality of these oocytes leads to lower fertilization and decreases in cleavage and implantation. MiR-155 is one of the microRNA (miRNA) that is increased in serum and granulosa cells of PCOS patients. In this study, we investigate the effects of miR-155 expression and its target genes on oocyte maturation and embryo development. We used the calcium phosphate protocol to transfect vectors that contained miR-155 or miR-off 155 and alone eGFP into cumulus oophorus complex (COCs) of B6D2F1 female mice for in vitro maturation. Cumulus expansion, nuclear, and cytoplasmic maturation, as well as cleavage rates were determined in groups transfected and compared with the control groups. Quantitative real-time polymerase chain reaction was performed to analyze expression levels of miR-155 and the target genes in the cumulus cells, oocytes, and blastocysts. MiR-155 overexpression in COCs suppressed cumulus expansion, oocyte maturation, and inhibition of endogenous miR-155 by miR-off 155 improved cumulus expansion and oocyte maturation by downregulation and expression increase of the Smad2 and Bcl2 genes. On the other hand, overexpression and downregulation of miR-155 in the COCs led to increase and decrease in cleavage rates by changes in expressions of the Mecp2, Jarid2, and Notch1 genes, respectively (P < 0.05). These results suggested that miR-155 overexpression in granulosa cells of PCOS patients can negatively affect nuclear and cytoplasmic maturation, but this miRNA expression has a positive impact on embryo development.


Assuntos
Blastocisto/fisiologia , Células do Cúmulo/fisiologia , MicroRNAs/genética , Oócitos/crescimento & desenvolvimento , Oócitos/fisiologia , Animais , Biologia Computacional , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Glutationa/metabolismo , Técnicas de Maturação in Vitro de Oócitos , Proteína 2 de Ligação a Metil-CpG/biossíntese , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Oogênese , Complexo Repressor Polycomb 2/biossíntese , Complexo Repressor Polycomb 2/genética , Gravidez , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptor Notch1/biossíntese , Receptor Notch1/genética , Proteína Smad2/genética , Proteína Smad2/metabolismo
12.
Protein Expr Purif ; 174: 105681, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32505675

RESUMO

Notch receptors have large extracellular domains containing up to 36 tandem epidermal growth factor-like (EGF) repeats, which facilitate cell signaling by binding ligands on neighboring cells. Notch receptors play major roles in a variety of developmental processes by controlling cell fate decisions. Each EGF repeat consists of about 40 amino acids with 3 conserved disulfide bonds. Many of the EGF repeats are modified by O-linked fucose glycans, and more than half have calcium-binding sites, but the sequences of the EGF repeats vary giving distinct roles to each repeat. EGF repeat 27 (EGF27) from mouse NOTCH1 is modified with O-fucose and is 1 of 7 repeats that is differentially modified by specific Fringe enzymes, which are known to regulate NOTCH1 activation and ligand binding. To better understand the role of EGF27 in NOTCH1 function and regulation, the 3-dimensional structures of EGF27 and its glycoforms are being pursued. E. coli cells were used to produce EGF27 in sufficient quantities for nuclear magnetic resonance analysis. Previous attempts to express the repeat alone and refold the repeat under a steady redox environment were unsuccessful due to low yields and extensive mixed-disulfide bond cross-linking. A new strategy using a cleavable maltose binding protein fusion tag increased the solubility and yield of EGF27. With the fusion tag, EGF27 was refolded to produce the correct disulfide bond arrangement, which was verified enzymatically with the glycosyltransferases, Protein O-fucosyltransferase 1 (POFUT1) and Lunatic Fringe (LFNG).


Assuntos
Fucose , Biossíntese Peptídica , Peptídeos , Receptor Notch1 , Animais , Fucose/química , Fucose/metabolismo , Glicosilação , Camundongos , Peptídeos/química , Peptídeos/genética , Receptor Notch1/biossíntese , Receptor Notch1/química , Receptor Notch1/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sequências Repetitivas de Aminoácidos
13.
Ultrasound Med Biol ; 46(9): 2311-2321, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32522460

RESUMO

The purpose of this study was to investigate the therapeutic effects of focused ultrasound on the expression of notch1, c-fos and transforming growth factor-ß3 (TGF-ß3) in genital skin of SD rats with vulvar lichen simplex chronicus (LSC). Fifty-six female SD rats with LSC were randomly divided into therapy and sham groups. The therapy group was exposed to focused ultrasound. The sham group received the same therapy with an instrument that had no power output. Four wk after a singly focused ultrasound therapy, histologic analyses revealed that recovered SD rats accounted for 75% of SD rats in the therapy group and 10.7% in the sham group. Total collagen fiber density in the superficial layer of dermis in the therapy group was significantly lower than that in the sham group. Notch1 and c-fos protein expression in the therapy group was significantly lower than that in the sham group, with the opposite effect present for TGF-ß3. Focused ultrasound therapy may inhibit superficial collagen fibrosis in the dermis by affecting expression of notch1, c-fos and TGF-ß3 in vulvar skin tissue and consequently reduce the recurrence rate of LSC.


Assuntos
Ablação por Ultrassom Focalizado de Alta Intensidade , Neurodermatite/metabolismo , Neurodermatite/terapia , Proteínas Proto-Oncogênicas c-fos/biossíntese , Receptor Notch1/biossíntese , Pele/metabolismo , Fator de Crescimento Transformador beta3/biossíntese , Vulva/metabolismo , Animais , Feminino , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley
14.
Eur Rev Med Pharmacol Sci ; 24(2): 581-590, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32016959

RESUMO

OBJECTIVE: To explore the effect of long non-coding ribonucleic acid (lncRNA)-maternally expressed gene 3 (MEG3) on the Notch signaling pathway, and its influences on the proliferation and apoptosis of osteosarcoma MG-63 cells. MATERIALS AND METHODS: LncRNA MEG3 was overexpressed in osteosarcoma MG-63 cells, and the cells were divided into Blank group, Len-con group, and Len-MEG3 group. The expression level of MEG3 in each group was detected via quantitative Polymerase Chain Reaction (qPCR), the cell proliferation level in each group was detected via Cell Counting Kit-8 (CCK-8) assay, and the apoptosis in each group was detected via Hoechst 33258 staining. Moreover, the content of the inflammatory factors in each group was determined using the Enzyme-Linked Immunosorbent Assay (ELISA), and the expression levels of apoptosis-related proteins and Notch signaling pathway-related proteins were determined through Western blotting. RESULTS: The expression level of lncRNA MEG3 in Len-MEG3 group was significantly higher than that in the Blank group and Len-con group (p<0.01). The overexpression of lncRNA MEG3 could significantly weaken the proliferation (p<0.01) and enhance the apoptosis of osteosarcoma cells (p<0.01). The overexpression of lncRNA MEG3 could significantly increase the content of the inflammatory factor interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) (p<0.01), and remarkably decrease the content of the anti-inflammatory factor IL-10 in osteosarcoma cells (p<0.01). Besides, the overexpression of lncRNA MEG3 could evidently raise the expression of Caspase3 (p<0.01) and reduce the Bcl-2/Bax expression in osteosarcoma cells (p<0.01). Finally, the overexpression of lncRNA MEG3 could remarkably reduce the protein expressions of Jagged1, Notch1, and NICD1 in osteosarcoma cells (p<0.01). CONCLUSIONS: The overexpression of lncRNA MEG3 can inhibit the proliferation and promote the apoptosis of osteosarcoma MG-63 cells by suppressing the Notch signaling pathway.


Assuntos
Apoptose/fisiologia , Proliferação de Células/fisiologia , Osteossarcoma/metabolismo , RNA Longo não Codificante/biossíntese , Receptor Notch1/biossíntese , Transdução de Sinais/fisiologia , Linhagem Celular Tumoral , Humanos , Osteossarcoma/patologia , Osteossarcoma/prevenção & controle , Receptor Notch1/antagonistas & inibidores
15.
Mol Cell Biochem ; 464(1-2): 181-191, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31758376

RESUMO

Notch is an evolutionarily conserved signaling pathway with an important role in development and cell fate determination. Deregulation of Notch signaling has been associated with several pathological conditions, including cancer. Acting as an oncogene in some types of cancers and as a tumor suppressor in other, Notch effects seem to be highly context-dependent in solid tumors. In the present study, we aimed to investigate gene expression levels of Notch pathway constituents, including ligands, receptors, and target genes, during the early stages of inflammation-associated intestinal carcinogenesis. To achieve so, we used our recently developed mouse model, in which colon cancer arises in the absence of urokinase-type plasminogen activator (uPA) due to colitis induced by dextran sodium sulfate (DSS) treatment. Among the cell surface components, ligands Jag1/Jag2 and receptors Notch1/Notch2 were found to be significantly upregulated in the uPA-deficient protumorigenic inflammatory microenvironment. Moreover, several intracellular Notch modulators, i.e. Hes1, Hey1, and Klf4, were also shown to be deregulated with inflammation, yet irrespective of uPA status. Sox9 transcription factor, however, was significantly downregulated in the uPA-deficient/DSS-treated mice that developed colon adenomas as compared to the wild-type/DSS-treated group with no neoplasia identified. The latter finding supports a tumor suppressive role of Sox9 in intestinal carcinogenesis. Our results point towards an early activation of Notch signaling pathway at the receptor-ligand level in inflammation-associated colon neoplasmatogenesis developed in the absence of uPA. Interestingly, such activation may not be accompanied by deregulation of downstream Notch-target genes, possibly due to the effects of other inter-related signaling pathways.


Assuntos
Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Receptor Notch1/biossíntese , Receptor Notch2/biossíntese , Transdução de Sinais , Ativador de Plasminogênio Tipo Uroquinase/deficiência , Animais , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos Endogâmicos BALB C , Receptor Notch1/genética , Receptor Notch2/genética
16.
Sci Rep ; 9(1): 16059, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31690782

RESUMO

Cardiac Valve Disease is one of the most common heart disorders with an emerging epidemic of cardiac valve degeneration due to aging. Zebrafish can regenerate most of their organs, including their heart. We aimed to explore the regenerative potential of cardiac valves and the underlying molecular mechanisms involved. We used an inducible, tissue-specific system of chemogenetic ablation and showed that zebrafish can also regenerate their cardiac valves. Upon valvular damage at larval stages, the intracardiac flow pattern becomes reminiscent of the early embryonic stages, exhibiting an increase in the retrograde flow fraction through the atrioventricular canal. As a result of the altered hemodynamics, notch1b and klf2a expression are ectopically upregulated, adopting the expression pattern of earlier developmental stages. We find that Notch signaling is re-activated upon valvular damage both at larval and adult stages and that it is required during the initial regeneration phase of cardiac valves. Our results introduce an animal model of cardiac valve specific ablation and regeneration.


Assuntos
Embrião não Mamífero/embriologia , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Doenças das Valvas Cardíacas/embriologia , Valvas Cardíacas/fisiologia , Receptor Notch1/biossíntese , Regeneração , Transdução de Sinais , Proteínas de Peixe-Zebra/biossíntese , Peixe-Zebra/embriologia , Animais
17.
Gene ; 715: 144029, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31376409

RESUMO

Intervertebral disc degeneration (IDD) is a major cause of lower back pain, but the specific molecular mechanisms governing its development are poorly characterized. This study sought to assess to what extent HOTAIR, a long non-coding (Lnc) RNA is expressed in IDD and regulates the apoptotic death of nucleus pulposus (NP) cells. We therefore used real-time qPCR to measure HOTAIR and microRNA(miR)-34a-5p in degenerative NP cells, and then validated their functional relevance via overexpressing them in these NP cells. We further verified the targets of these RNA constructs in 293 T cells through the use of a dual luciferase reporter assay. We further measured NP cell apoptosis via flow cytometry and Notch1 expression via western blotting. Our results indicated that IDD was linked with decreased HOTAIR expression relative to regular NP cells, and overexpressing this lncRNA was linked to reduced apoptotic NP cell death, whereas overexpressing miR-34a-5p had the opposite effect. We found that HOTAIR served as a miR-34a-5p sponge, sequestering this miRNA and thereby down regulating genes linked to apoptosis through the Notch signaling pathway. Even in naturally degenerated NP cells, HOTAIR delayed the onset of apoptosis. Together these results reveal that a HOTAIR/miR-34a-5p/Notch1 signaling pathway may regulate the development of IDD, potentially making HOTAIR a viable target for treatment of this disease.


Assuntos
Apoptose , Regulação para Baixo , Degeneração do Disco Intervertebral/metabolismo , MicroRNAs/biossíntese , RNA Longo não Codificante/biossíntese , Receptor Notch1/biossíntese , Transdução de Sinais , Adulto , Idoso , Feminino , Humanos , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/patologia , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Núcleo Pulposo/patologia , RNA Longo não Codificante/genética , Receptor Notch1/genética
18.
Sci Rep ; 9(1): 12221, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31434993

RESUMO

Recent studies have shown that transforming microglia phenotype from pro-inflammation of M1 phenotype to anti-inflammation and tissue-repairing M2 phenotype may be an effective therapeutic strategy for preventing ischemic stroke brain injury. Isosteviol Sodium (STV-Na) has shown promise as a neuroprotective agent in cerebral ischemia model, although its effect on microglial polarization and chronic recovery after stroke is not clear. Here, we demonstrated that STV-Na treatment significantly reduced cerebral ischemic damage at both acute and chronic time points. STV-Na has a profound regulatory effect on microglia response after stroke. It can promote M2 polarization and inhibit microglia-mediated inflammation (M1) response following stroke in vivo and in vitro. Furthermore, we also found that Growth Arrest-Specific 5 (GAS5) altered OGD/R-induced microglial activation by increasing Notch1 expression via miR-146a-5p, the mRNA level of GAS5 and the protein level of Notch1 in vivo and in vitro, were discovered that both downgraded with STV-Na. Taken together, the present study demonstrated that STV-Na exerted neuroprotective effects by modulating microglia/macrophage polarization in ischemic stroke via the GAS5/miR-146a-5p sponge. These findings provide new evidence that targeting STV-Na could be a treatment for the prevention of stroke-related brain damage.


Assuntos
Isquemia Encefálica , Diterpenos do Tipo Caurano/farmacologia , Macrófagos , MicroRNAs/metabolismo , Microglia , RNA Longo não Codificante/metabolismo , Acidente Vascular Cerebral , Animais , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Isquemia Encefálica/prevenção & controle , Regulação da Expressão Gênica , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Microglia/metabolismo , Microglia/patologia , Receptor Notch1/biossíntese , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/prevenção & controle
19.
Acta Neuropathol Commun ; 7(1): 118, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31345270

RESUMO

Intracellular mislocalization of TAR DNA-binding protein 43 (TDP-43), a nuclear DNA/RNA-binding protein involved in RNA metabolism, is a pathological hallmark of amyotrophic lateral sclerosis (ALS). Although the aggregation-prone, TDP-43 C-terminal domain is widely considered as a key component of TDP-43 pathology in ALS, recent studies including ours suggest that TDP-43 N-terminal fragments (TDP-∆C) may also contribute to the motor dysfunction in ALS. However, the specific pathological functions of TDP-43 N-terminal fragments in mice have not been elucidated. Here, we established TDP-∆C knock-in mice missing a part of exon 6 of murine Tardbp gene, which encodes the C-terminal region of TDP-43. Homozygous TDP-∆C mice showed embryonic lethality, indicating that the N-terminal domain of TDP-43 alone is not sufficient for normal development. In contrast, heterozygous TDP-∆C mice developed normally but exhibited age-dependent mild motor dysfunction with a loss of C-boutons, large cholinergic synaptic terminals on spinal α-motor neurons. TDP-∆C protein broadly perturbed gene expression in the spinal cords of aged heterozygous TDP-∆C mice, including downregulation of Notch1 mRNA. Moreover, the level of Notch1 mRNA was suppressed both by TDP-43 depletion and TDP-∆C expression in Neuro2a cells. Decreased Notch1 mRNA expression in aged TDP-∆C mice was associated with the age-dependent motor dysfunction and loss of Akt surviving signal. Our findings indicate that the N-terminal region of TDP-43 derived from TDP-∆C induces the age-dependent motor dysfunction associated with impaired Notch1-Akt axis in mice.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas Proto-Oncogênicas c-akt/biossíntese , Receptor Notch1/biossíntese , Transdução de Sinais/fisiologia , Fatores Etários , Esclerose Lateral Amiotrófica/genética , Animais , Linhagem Celular Tumoral , Células Cultivadas , Proteínas de Ligação a DNA/genética , Técnicas de Introdução de Genes , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/genética , Receptor Notch1/genética
20.
Sci Rep ; 9(1): 10373, 2019 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-31316119

RESUMO

Notch signaling has been shown to regulate the homeostasis and wound healing of the corneal epithelium. We investigated the effect of Notch inhibition in the human limbal stem/progenitor cells (LSCs) in vitro by using small molecules. Treatment of the LSCs with DAPT and SAHM1 reduced the proliferation rate and maintained the undifferentiated state of the LSCs in a concentration dependent manner. Stratification and differentiation of the corneal epithelium were not reduced after Notch inhibition, indicating that the function of the corneal basal cells is retained. Our findings suggest that Notch signaling plays a role in the proliferation and maintenance of LSCs.


Assuntos
Células-Tronco Adultas/efeitos dos fármacos , Limbo da Córnea/citologia , Receptor Notch1/fisiologia , Receptor Notch2/fisiologia , Adulto , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Idoso , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Diaminas/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Pessoa de Meia-Idade , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/biossíntese , Receptor Notch1/genética , Receptor Notch2/antagonistas & inibidores , Receptor Notch2/biossíntese , Receptor Notch2/genética , Tiazóis/farmacologia , Fatores de Transcrição HES-1/biossíntese , Fatores de Transcrição HES-1/genética , Adulto Jovem
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