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1.
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
2.
Nat Metab ; 2(12): 1443-1458, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33257854

RESUMO

The in vitro differentiation of insulin-producing beta-like cells can model aspects of human pancreatic development. Here, we generate 95,308 single-cell transcriptomes and reconstruct a lineage tree of the entire differentiation process from human embryonic stem cells to beta-like cells to study temporally regulated genes during differentiation. We identify so-called 'switch genes' at the branch point of endocrine/non-endocrine cell fate choice, revealing insights into the mechanisms of differentiation-promoting reagents, such as NOTCH and ROCKII inhibitors, and providing improved differentiation protocols. Over 20% of all detectable genes are activated multiple times during differentiation, even though their enhancer activation is usually unimodal, indicating extensive gene reuse driven by different enhancers. We also identify a stage-specific enhancer at the TCF7L2 locus for diabetes, uncovered by genome-wide association studies, that drives a transient wave of gene expression in pancreatic progenitors. Finally, we develop a web app to visualize gene expression on the lineage tree, providing a comprehensive single-cell data resource for researchers studying islet biology and diabetes.


Assuntos
Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células Secretoras de Insulina/fisiologia , Diferenciação Celular/genética , Linhagem da Célula/genética , Diabetes Mellitus/genética , Células-Tronco Embrionárias , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Silenciamento de Genes , Genes de Troca/genética , Glucose/farmacologia , Humanos , Secreção de Insulina/efeitos dos fármacos , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Fatores de Transcrição HES-1/biossíntese , Fatores de Transcrição HES-1/genética
3.
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
4.
Autoimmunity ; 52(4): 176-184, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31322442

RESUMO

Liver ischemia-reperfusion injury (IRI) represents a risk factor for early graft dysfunction and an obstacle to expanding donor pool in orthotopic liver transplantation (OLT). Kupffer cells (KCs) are the largest antigen-presenting cell (APC) group and the primary modulators of inflammation in liver tissues. The vital role of Notch1/Jagged1 pathway in mouse OLT model has been reported, however, its potential therapeutic mechanism is unknown. Here, we made use of short hairpin RNA-Jagged1 and AAV-Jagged1 to explore the effects of Notch1/Jagged1 pathway in OLT. In vitro, blockade of Notch1/Jagged1 pathway downregulated the expression of Hairy and enhancer of split-1 (Hes1) gene, which in turn increased the proinflammatory effects of KCs. Moreover, the anti-inflammatory effects of Notch1/Jagged1 pathway were induced by inhibiting Hes1/gene of phosphate and tension/protein kinase B/Toll-like receptor 4/nuclear factor kappa B (Hes1/PTEN/AKT/TLR4/NF-κB) axis in KCs. In vivo, we used a well-established mouse model of OLT to mimic clinical transplantation. Mice were stochastically divided into 6 groups: Sham group (n = 15); Normal saline (NS) group (n = 15); Adeno-associated virus-green fluorescent protein (AAV-GFP) group (n = 15); AAV-Jagged1 group (n = 15); Clodronate liposome (CL) group (n = 15); CL+AAV-Jagged1 group (n = 15) . After OLT the liver damage in AAV-Jagged1 group were significantly accentuated compared to the AAV-GFP group. While blockade of Jagged1 aftet clearence of KCs by CL would not lead to further liver injuries. Taken together, our study demonstrated that blockade of Notch1/Jagged1 pathway aggravates inflammation induced by lipopolysaccharide (LPS) via Hes1/PTEN/AKT/TLR4/NF-κB in KCs, and the blockade of Notch1/Jagged1 pathway in donor liver increased neutrophil/macrophage infiltration and hepatocellular apoptosis, which suggested the function of Notch1/Jagged1 pathway in mouse OLT and highlighted the protective function of Notch1/Jagged1 pathway in liver transplantation.


Assuntos
Proteína Jagged-1/antagonistas & inibidores , Células de Kupffer/metabolismo , Transplante de Fígado/efeitos adversos , Receptor Notch1/antagonistas & inibidores , Traumatismo por Reperfusão/patologia , Animais , Apoptose/genética , Células Cultivadas , Ácido Clodrônico/farmacologia , Inflamação/patologia , Proteína Jagged-1/genética , Fígado/citologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/biossíntese , PTEN Fosfo-Hidrolase/biossíntese , Proteínas Proto-Oncogênicas c-akt/biossíntese , Interferência de RNA , RNA Interferente Pequeno/genética , Receptor Notch1/genética , Receptor 4 Toll-Like/biossíntese , Fatores de Transcrição HES-1/biossíntese
5.
Differentiation ; 107: 1-10, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30927641

RESUMO

Androgen signaling is essential for prostate development, morphogenesis, and regeneration. Emerging evidence also indicates a regulatory role of Notch signaling in prostate development, differentiation, and growth. However, the collaborative regulatory mechanisms of androgen and Notch signaling during prostate development, growth, and regeneration are largely unknown. Hairy and Enhancer of Split 1 (Hes1) is a transcriptional regulator of Notch signaling pathways, and its expression is responsive to Notch signaling. Hes1-expressing cells have been shown to possess the regenerative capability to repopulate a variety of adult tissues. In this study, we developed new mouse models to directly assess the role of the androgen receptor in prostatic Hes1-expressing cells. Selective deletion of AR expression in embryonic Hes1-expressing cells impeded early prostate development both in vivo and in tissue xenograft experiments. Prepubescent deletion of AR expression in Hes1-expressing cells resulted in prostate glands containing abnormalities in cell morphology and gland architecture. A population of castration-resistant Hes1-expressing cells was revealed in the adult prostate, with the ability to repopulate prostate epithelium following androgen supplementation. Deletion of AR in Hes1-expressing cells diminishes their regenerative ability. These lines of evidence demonstrate a critical role for the AR in Notch-responsive cells during the course of prostate development, morphogenesis, and regeneration, and implicate a mechanism underlying interaction between the androgen and Notch signaling pathways in the mouse prostate.


Assuntos
Próstata/fisiologia , Receptores Notch/metabolismo , Regeneração , Fatores de Transcrição HES-1 , Androgênios/metabolismo , Animais , Masculino , Camundongos , Modelos Animais , Próstata/embriologia , Receptores Androgênicos/metabolismo , Transdução de Sinais , Fatores de Transcrição HES-1/biossíntese , Fatores de Transcrição HES-1/genética
6.
Eur Rev Med Pharmacol Sci ; 23(3): 923-931, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30779057

RESUMO

OBJECTIVE: The aim of this study was to investigate the influence of micro-ribonucleic acid-34a (miR-34a) on preeclampsia through the Notch signaling pathway. PATIENTS AND METHODS: The expressions of miR-34a, Notch-1, Notch-2, and Notch-3 in the placenta of 39 preeclampsia patients and 42 normal patients were detected by immunohistochemistry and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The correlations between miR-34a expression with the expressions of Notch-1, Notch-2 and Notch-3 were analyzed, respectively. Besides, placental trophoblasts were isolated from preeclampsia patients and cultured in vitro. The expressions of miR-34a, Notch-1, Notch-2 and Notch-3 in placental trophoblasts were analyzed. Furthermore, the influences of miR-34a on the protein expressions of Notch-1, Notch-2, Notch-3, and hairy and enhancer of split-1 (Hes-1) in the Notch signaling pathway were analyzed by Luciferase reporter gene assay and Western blotting. The role of Notch in trophoblast invasion was investigated through the Notch inhibitors. In addition, its influence on the expression of urokinase-type plasminogen activator (uPA) was studied by miR-34a overexpression. RESULTS: The expressions of miR-34a and Notch-1 were correlated with preeclampsia in the placentas of preeclampsia patients and normal patients to a certain degree. The expression of miR-34a in preeclamptic placenta was significantly higher than that of the normal placenta (p<0.05). However, Notch-1 expression was markedly lower in preeclamptic placenta (p<0.05). No significant differences were found in the expressions of Notch-2 and Notch-3 between the two types of placentas (p>0.05). MiR-34a had a remarkable negative correlation with Notch-1 expression in the Notch family (p<0.001, r=-0.5775). RT-PCR results revealed that the mRNA expression of miR-34a in placental trophoblasts of patients with preeclampsia was notably higher than that of normal people (p<0.01). However, Western blotting demonstrated that the protein expressions of Notch-1, Notch-2 and Notch-3 exhibited the opposite results. Additionally, the protein expression of Notch-1, Notch-2, Notch-3 and Hes-1 in trophoblasts transfected with pre-miR-34a was significantly decreased. The treatment with Notch inhibitors markedly reduced the trophoblast invasion. Furthermore, miR-34a overexpression or intracellular domain of Notch (ICN) overexpression regulated uPA expression. CONCLUSIONS: MiR-34a regulates uPA system through the Notch signal transduction, thereby regulating the invasion of placental trophoblasts in patients with preeclampsia.


Assuntos
MicroRNAs/fisiologia , Pré-Eclâmpsia/fisiopatologia , Receptores Notch/fisiologia , Estudos de Casos e Controles , Movimento Celular/fisiologia , Células Cultivadas , Feminino , Humanos , MicroRNAs/biossíntese , Placenta/metabolismo , Gravidez , Receptores Notch/biossíntese , Transdução de Sinais/fisiologia , Fatores de Transcrição HES-1/biossíntese , Trofoblastos/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/biossíntese
7.
J Glob Oncol ; 5: 1-10, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30615540

RESUMO

PURPOSE: Cervical cancer (CC) is the most common cancer affecting women worldwide. Human papillomavirus (HPV) infection is a major contributing factor for the development of CC. The development of CC occurs progressively from precancer stages to cancerous stages (ie, invasive squamous cell carcinoma [ISCC] and adenocarcinoma [ADC]). ADC is a rare form of CC that develops from the mucinous endocervical epithelium. It is believed that the downstream targets of Notch signaling contribute to the etiology of CC. One such target is HES1, whose role in the modulation of ADC is unknown. The purpose of this study is to determine the role of HES1 protein in HPV-associated ADC subtype of CC and also to compare its expression in histologic subtypes of precancer and ISCC. PATIENTS AND METHODS: A total of 148 patients (30 with precancers, 98 with ISCC, and 20 with ADC) and 40 normal control participants were analyzed for the expression of HES1 via immunohistochemistry, with results validated by immunoblotting. RESULTS: The comparison between HPV-16 and HES1 expression was significant in precancer (cervical intraepithelial neoplasia grades 1 to 3; P = .013), ISCC (International Federation of Gynecology and Obstetrics stages I to IV; P = .001), and ADC ( P = .007). An overall significant mean difference was observed between HES1, JAG1, and Notch-3 proteins in precancer ( P = .001), ISCC ( P = .001), and ADC ( P = .001). Pairwise comparisons between HES1 and JAG1 and HES1 and Notch-3 were also found to be significant. CONCLUSION: This study showed that among all HPV-16-positive precancers, the major HES1 positivity signal arises from cervical intraepithelial neoplasia grades 2 and 3 that develops into ISCC. Moreover, HPV-16-positive ADC also showed an association with HES1. The HES1, JAG1, and Notch-3 proteins showed their synergistic role in modulating HPV associated ADC along with histologic subtypes of precancer and ISCC of CC.


Assuntos
Carcinoma de Células Escamosas/virologia , Infecções por Papillomavirus/metabolismo , Fatores de Transcrição HES-1/metabolismo , Displasia do Colo do Útero/virologia , Neoplasias do Colo do Útero/virologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Estudos de Casos e Controles , Feminino , Humanos , Imuno-Histoquímica , Estadiamento de Neoplasias , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Lesões Pré-Cancerosas/virologia , Curva ROC , Receptor Notch3/metabolismo , Fatores de Transcrição HES-1/biossíntese , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Displasia do Colo do Útero/metabolismo , Displasia do Colo do Útero/patologia
8.
Anticancer Drugs ; 29(8): 767-773, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29916899

RESUMO

To investigate effect of long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) on epithelial-to-mesenchymal transition (EMT) of esophageal cancer (EC) and role of enhancer of zeste homolog 2 (Ezh2)-Notch1 signaling pathway in the process. The expression of MALAT1 was determined in four EC cell lines by real-time PCR. TE-1 and EC109 cells were transfected with sh-MALAT1 to inhibit expression of MALAT1 or transfected with pcDNA3.1-Ezh2 to overexpress Ezh2. Invasion and migration assays were conducted to analyze cell metastasis, and expressions of Ezh2-Notch1 signaling-related proteins as well as EMT related proteins were determined using both real-time PCR and western blot. MALAT1 was significantly up-regulated in all EC cell lines compared with the normal cells. Silencing MALAT1 using shRNA could significantly inhibit cell viability (reduced almost 30% of cell viability compared with the control), invasion (reduced almost 30% of cell migration compared with the control), and migration (reduced almost 50% of cell migration compared with the control) of both TE-1 and EC109 cells (P<0.05). Meanwhile, expression of Ezh2, Notch1, Hes1, MMP-9, and Vimentin was significantly decreased and expression of E-cadherin was significantly increased when cells were transfected with sh-MALAT1 compared with the nontransfected cells (P<0.05). However, when cells were cotransfected with both sh-MALAT1 and pcDNA3.1-Ezh2, the protein expression changes induced by sh-MALAT1 were recovered. MALAT1 could affect EMT and metastasis of EC cells through Ezh2-Notch1 signaling pathway. This study can give deeper understandings of the role of MALAT1 in EC and may provide some new directions for treatment of patients with EC.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Neoplasias Esofágicas/genética , RNA Longo não Codificante/genética , Receptor Notch1/metabolismo , Linhagem Celular Tumoral , Proteína Potenciadora do Homólogo 2 de Zeste/biossíntese , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Inativação Gênica , Humanos , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/genética , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/metabolismo , Receptor Notch1/biossíntese , Receptor Notch1/genética , Transdução de Sinais , Fatores de Transcrição HES-1/biossíntese , Fatores de Transcrição HES-1/genética , Transfecção , Vimentina/biossíntese , Vimentina/genética
9.
Proc Natl Acad Sci U S A ; 115(16): E3682-E3691, 2018 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-29610298

RESUMO

Hemodynamic forces and Notch signaling are both known as key regulators of arterial remodeling and homeostasis. However, how these two factors integrate in vascular morphogenesis and homeostasis is unclear. Here, we combined experiments and modeling to evaluate the impact of the integration of mechanics and Notch signaling on vascular homeostasis. Vascular smooth muscle cells (VSMCs) were cyclically stretched on flexible membranes, as quantified via video tracking, demonstrating that the expression of Jagged1, Notch3, and target genes was down-regulated with strain. The data were incorporated in a computational framework of Notch signaling in the vascular wall, where the mechanical load was defined by the vascular geometry and blood pressure. Upon increasing wall thickness, the model predicted a switch-type behavior of the Notch signaling state with a steep transition of synthetic toward contractile VSMCs at a certain transition thickness. These thicknesses varied per investigated arterial location and were in good agreement with human anatomical data, thereby suggesting that the Notch response to hemodynamics plays an important role in the establishment of vascular homeostasis.


Assuntos
Proteína Jagged-1/fisiologia , Mecanotransdução Celular/fisiologia , Contração Muscular/fisiologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/fisiologia , Receptor Notch3/fisiologia , Idoso , Artérias/ultraestrutura , 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 , Simulação por Computador , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Homeostase , Humanos , Proteína Jagged-1/biossíntese , Proteína Jagged-1/genética , Ligantes , Pessoa de Meia-Idade , Modelos Biológicos , Morfogênese/fisiologia , Músculo Liso Vascular/ultraestrutura , Receptor Notch3/biossíntese , Receptor Notch3/genética , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Estresse Mecânico , Fatores de Transcrição HES-1/biossíntese , Fatores de Transcrição HES-1/genética , Gravação em Vídeo
10.
J Cell Physiol ; 233(8): 5747-5755, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29155449

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers. Recent research has demonstrated that chronic pancreatitis (CP) is associated with an increased risk of PDAC, partly due to acinar-to-ductal metaplasia (ADM). Baicalein has been shown to exert anti-inflammatory and anti-tumor effects for CP or PDAC, respectively. The aim of our study was to investigate the effect of baicalein, and the putative underlying mechanism, on inflammatory cytokines-induced ADM of rat pancreatic acinar cell line AR42J. To investigate ADM and baicalein effects in vitro, AR42J were treated with recombinant rat Tumor Necrosis Factor alpha (rTNFα) with or without baicalein for 5 days. Results showed that rTNFα-induced AR42J cells switched their phenotype from dominantly amylase-positive acinar cells to dominantly cytokeratin 19-positive ductal cells. Moreover, expression of the transcripts for TNFα or Hes-1, a Notch target, was up-regulated in these cells. Interestingly, baicalein reduced the population of ADM as well as cytokines gene expression but not Hes-1. Baicalein inhibited NF-κB activation induced by rTNFα in AR42J, but no effect on Notch 1activation. Moreover, baicalein suppressed the secretion of TNFα and Nitric Oxide (NO) in macrophages stimulated with LPS and further inhibited ADM of conditional medium-treated AR42J cells. Baicalein also suppressed the inflammatory response of LPS-activated macrophages, thereby inhibited ADM of AR42J by altering their microenvironment. Taken together, our study indicates that baicalein reduces rTNFα-induced ADM of AR42J cells by inhibiting NF-κB activation. It also sheds new light on Chinese material medica therapy of pancreatitis and thereby prevention of PDAC.


Assuntos
Anti-Inflamatórios/farmacologia , Flavanonas/farmacologia , Metaplasia/patologia , Pancreatite/patologia , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Amilases/metabolismo , Animais , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Queratina-19/metabolismo , Lipopolissacarídeos , Macrófagos/imunologia , Medicina Tradicional Chinesa , Camundongos , Óxido Nítrico/metabolismo , Células RAW 264.7 , Ratos , Receptor Notch1/metabolismo , Fatores de Transcrição HES-1/biossíntese , Fator de Necrose Tumoral alfa/farmacologia
11.
Cancer Res ; 77(20): 5464-5478, 2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28827371

RESUMO

NOTCH signaling exerts essential roles in normal and malignant intestinal physiology and the homeostasis of cancer stem-like cells (CSC), but the basis for this latter role remains obscure. The signaling scaffold protein STRAP is upregulated in several cancers, where it promotes tumorigenicity and metastasis. Here we report a novel oncogenic function for STRAP in maintaining CSC subpopulations in a heterogeneous mixture by antagonizing formation of the chromatin modifier PRC2 and by epigenetically activating NOTCH signals in human colorectal cancer. Silencing STRAP sensitized colorectal cancer cells to chemotherapeutic drugs in vitro and in vivo STRAP depletion also contributed to a reduced stem-like phenotype of colorectal cancer cells, as indicated by reduced expression of the CSC signature and NOTCH signaling regulators in vitro and by diminished tumorigenesis in vivo Genes encoding some upstream activators of NOTCH were highly enriched for H3K27me3, which forms repressive chromatin domains upon STRAP silencing. Mechanistically, STRAP competitively disrupted association of the PRC2 subunits EZH2 and SUZ12, thereby inhibiting PRC2 assembly. Restoring the NOTCH pathway by lentiviral expression of NICD1 or HES1 in STRAP-depleted tumor cells reversed the CSC phenotype. In 90 colorectal cancer clinical specimens, a significant positive correlation was documented between the expression of STRAP and HES1. Overall, our findings illuminated a novel STRAP-NOTCH1-HES1 molecular axis as a CSC regulator in colorectal cancer, with potential implications to improve treatment of this disease. Cancer Res; 77(20); 5464-78. ©2017 AACR.


Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/patologia , Receptor Notch1/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Epigênese Genética , Fluoruracila/farmacologia , Células HCT116 , Células HEK293 , Células HT29 , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Compostos Organoplatínicos/farmacologia , Oxaliplatina , Proteínas de Ligação a RNA , Receptor Notch1/genética , Transdução de Sinais , Fatores de Transcrição HES-1/biossíntese , Fatores de Transcrição HES-1/genética , Fatores de Transcrição HES-1/metabolismo
12.
Virol Sin ; 32(3): 188-198, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28451898

RESUMO

Human cytomegalovirus (HCMV) infection is a leading cause of birth defects, primarily affecting the central nervous system and causing its maldevelopment. As the essential downstream effector of Notch signaling pathway, Hes1, and its dynamic expression, plays an essential role on maintaining neural progenitor /stem cells (NPCs) cell fate and fetal brain development. In the present study, we reported the first observation of Hes1 oscillatory expression in human NPCs, with an approximately 1.5 hour periodicity and a Hes1 protein half-life of about 17 (17.6 ± 0.2) minutes. HCMV infection disrupts the Hes1 rhythm and down-regulates its expression. Furthermore, we discovered that depleting Hes1 protein disturbed NPCs cell fate by suppressing NPCs proliferation and neurosphere formation, and driving NPCs abnormal differentiation. These results suggested a novel mechanism linking disruption of Hes1 rhythm and down-regulation of Hes1 expression to neurodevelopmental disorders caused by congenital HCMV infection.


Assuntos
Citomegalovirus/fisiologia , Interações Hospedeiro-Patógeno , Células-Tronco Neurais/fisiologia , Células-Tronco Neurais/virologia , Fatores de Transcrição HES-1/biossíntese , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Regulação para Baixo , Humanos
13.
Cell Death Dis ; 8(1): e2547, 2017 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-28079885

RESUMO

The deregulation of lineage control programs is often associated with the progression of haematological malignancies. The molecular regulators of lineage choices in the context of tyrosine kinase inhibitor (TKI) resistance remain poorly understood in chronic myeloid leukemia (CML). To find a potential molecular regulator contributing to lineage distribution and TKI resistance, we undertook an RNA-sequencing approach for identifying microRNAs (miRNAs). Following an unbiased screen, elevated miRNA182-5p levels were detected in Bcr-Abl-inhibited K562 cells (CML blast crisis cell line) and in a panel of CML patients. Earlier, miRNA182-5p upregulation was reported in several solid tumours and haematological malignancies. We undertook a strategy involving transient modulation and CRISPR/Cas9 (clustered regularly interspersed short palindromic repeats)-mediated knockout of the MIR182 locus in CML cells. The lineage contribution was assessed by methylcellulose colony formation assay. The transient modulation of miRNA182-5p revealed a biased phenotype. Strikingly, Δ182 cells (homozygous deletion of MIR182 locus) produced a marked shift in lineage distribution. The phenotype was rescued by ectopic expression of miRNA182-5p in Δ182 cells. A bioinformatic analysis and Hes1 modulation data suggested that Hes1 could be a putative target of miRNA182-5p. A reciprocal relationship between miRNA182-5p and Hes1 was seen in the context of TK inhibition. In conclusion, we reveal a key role for miRNA182-5p in restricting the myeloid development of leukemic cells. We propose that the Δ182 cell line will be valuable in designing experiments for next-generation pharmacological interventions.


Assuntos
Proliferação de Células/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , MicroRNAs/genética , Fatores de Transcrição HES-1/genética , Linhagem da Célula/genética , Resistencia a Medicamentos Antineoplásicos/genética , Células Eritroides/metabolismo , Células Eritroides/patologia , Proteínas de Fusão bcr-abl/genética , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , MicroRNAs/metabolismo , Células Mieloides/metabolismo , Células Mieloides/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Fatores de Transcrição HES-1/biossíntese
14.
Drug Res (Stuttg) ; 67(1): 13-19, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27684192

RESUMO

Background: Ovarian cancer is one of the most lethal gynecological malignancies and numerous changes in signaling cascades are involved in the initiation and progression of ovarian cancerous cells. Here, we investigated the role of NF-κB and Notch pathways inhibition on human ovarian cancer OVCAR-3 cells proliferation and IκB-α and Hes-1 expression as 2 key genes in these pathways regulation. Methods: The effects of Bay 11-7085 and DAPT, NF-κB and Notch pathways specific inhibitors, on cell proliferation were evaluated using MTT assay. In addition, the cells were transfected by Notch and IKK-ß siRNAs. mRNA and protein levels of target genes were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot after 48 h incubation with inhibitors and siRNAs. Results: Bay 11-7085 and DAPT significantly decreased the cell proliferation OVCAR-3. IκB-α and Hes-1 mRNA levels decreased to 5 or 3% and 6% or 2% after treatment with Bay 11-7085 or DAPT, respectively (p<0.05). We also found that combination treatment exert a more potent effects on the expression of these gene (p<0.05). Moreover, siRNA transfection caused a significant reduction in IκB-α and Hes-1 mRNA levels (p<0.05). In the protein level, OVCAR-3 cell treatment with both chemichal inhibitors and specific siRNA cause a significant decrease in the expression of target genes (p<0.05) Conclusion: Our findings suggest that inhibition of NF-κB and Notch signaling pathways can effectively reduce OVCAR-3 cells proliferation. Therefore, pharmacological targeting of the NF-κB and Notch signaling pathway could be a promising future treatment of ovarian cancer.


Assuntos
Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Inibidor de NF-kappaB alfa/biossíntese , NF-kappa B/antagonistas & inibidores , Receptores Notch/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição HES-1/biossíntese , Linhagem Celular Tumoral , Dipeptídeos/farmacologia , Sinergismo Farmacológico , Humanos , Quinase I-kappa B/antagonistas & inibidores , Nitrilas/farmacologia , RNA Interferente Pequeno/farmacologia , Sulfonas/farmacologia
15.
FEBS Lett ; 590(12): 1729-38, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27214741

RESUMO

Uveal melanoma (UM) is the most common primary intraocular tumour in adults and has a high incidence. Nearly 50% of patients with UM develop metastases after diagnosis. Long noncoding RNAs (lncRNAs) are involved in both oncogenic and tumour suppression pathways. We show that lncRNA PAUPAR is present at low levels in UM tissues and cell lines and modulates the tumourigenesis of UM in vitro and in vivo. The ectopic expression of PAUPAR in UM cells revealed that PAUPAR acts as a necessary UM suppressor and induces the silencing of HES1 expression, which significantly reduces tumour metastasis. Mechanistically, PAUPAR modulates HES1 expression by inhibiting histone H3K4 methylation. These data support a role of this lncRNA as a novel therapeutic target in cancer prevention and treatment.


Assuntos
Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Histonas/metabolismo , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , RNA Longo não Codificante/biossíntese , RNA Neoplásico/biossíntese , Neoplasias Uveais/metabolismo , Animais , Linhagem Celular Tumoral , Histonas/genética , Humanos , Masculino , Melanoma/genética , Melanoma/patologia , Metilação , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , RNA Longo não Codificante/genética , RNA Neoplásico/genética , Fatores de Transcrição HES-1/biossíntese , Fatores de Transcrição HES-1/genética , Neoplasias Uveais/genética , Neoplasias Uveais/patologia
16.
Tumour Biol ; 37(4): 5413-26, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26563369

RESUMO

Trastuzumab is a humanized monoclonal antibody against the human epidermal growth factor receptor 2 (HER2) that is overexpressed in about 25 % of breast cancer patients. However, primary and/or acquired resistance to trastuzumab develops in most affected persons. In this study, we explored the functional role of miR-182 inhibition with aiming the sensitization of SKBR3 cells to trastuzumab. Cell viability, apoptosis, colony formation, and migration capacities of SKBR3(S) (sensitive) and SKBR3(R) (resistant) cells were assessed to determine the anti-proliferative effects of PNA-antimiR-182. In addition, the expression levels of miR-182, mRNA of FOXO1, and Bim as well as the protein levels of HER2 and Notch1 signaling factors were evaluated by stem-loop RT-qPCR, RT-qPCR, and Western blot, respectively. The results indicated that miR-182 might play a causal role in the mechanism of trastuzumab. In line with that, PNA-antimiR-182 inhibited synergistically the viability of both the sensitive and resistant cell groups. Furthermore, the inhibitory effect of PNA-anitmiR-182 on migration in SKBR3 cells was more than the induction of apoptosis. In addition, PNA-antimiR-182 reduced the levels of NICD, Hes1, HIF-1α, and p-Akt in both cell groups, while it augmented the intracellular content of FOXO1 and Numb suppressor proteins. In other words, PNA-antimiR-182-mediated upregulation of Numb was associated with downregulation of HIF-1α and Hes1. Consequently, downregulation of miR-182 might find therapeutical value for overcoming trastuzumab resistance. Graphical Abstract The crosstalk between HER2 and Notch1 signaling pathway is mediated by miR-182.


Assuntos
Neoplasias da Mama/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteínas de Membrana/genética , MicroRNAs/genética , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição HES-1/genética , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Proteínas de Membrana/biossíntese , MicroRNAs/antagonistas & inibidores , MicroRNAs/biossíntese , Proteínas do Tecido Nervoso/biossíntese , RNA Mensageiro/biossíntese , Receptor ErbB-2/genética , Fatores de Transcrição HES-1/biossíntese , Trastuzumab/administração & dosagem , Trastuzumab/genética
17.
Tumour Biol ; 37(3): 3797-805, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26472724

RESUMO

Delta-like ligand-4 (DLL4)-Notch signaling is known to play a pivotal role in the regulation of tumor angiogenesis. We had previously found that DLL4 was overexpressed, while Notch1 receptor, which binds to DLL4 during angiogenesis, was absent in the majority of human primary glioblastomas. Thus, DLL4-Notch signaling pathway in the regulation of tumor angiogenesis in primary glioblastoma remains unknown. Tumor tissues from 70 patients with primary glioblastoma were analyzed by immunohistochemistry for expression of components of DLL4-Notch signaling, vascular endothelial growth factor (VEGF), and microvessel density (MVD). Immunohistochemistry results showed that the positive staining of DLL4 and Notch4 was primarily distributed in tumor vascular endothelial cells but rarely detected in tumor cells. However, VEGF, hairy/enhancer of split-1 (HES1; a target gene of Notch signaling), and Notch1-3 expression was seen in both tumor vascular endothelial cells and tumor cells. Univariate analysis showed that the expression levels of VEGF and DLL4, HES1, and Notch4 in tumor endothelial cells were significantly associated with MVD in primary glioblastoma (P < 0.001). Binary logistic regression analysis showed that high expression levels of DLL4, HES1, and Notch4 in tumor endothelial cells were associated with a decrease of MVD in primary glioblastoma, while MVD increased with elevated VEGF expression in contrast. In addition, DLL4, Notch4, and HES1 expression were positively correlated in tumor vascular endothelial cells (P < 0.05). We conclude that the vascular DLL4-Notch4 signaling and VEGF signaling complementing each other plays an important role in the progression of tumor angiogenesis in primary glioblastoma. Graphical abstract A, positive staining of DLL4 in human kidney; B, positive staining of VEGF in human breast cancer; C, positive staining of CD34 in human lung cancer; D, positive staining of HES1 in human breast cancer; E-H, positive staining of Notch1-4: E-F in human lung cancer; G-H in human kidney.


Assuntos
Glioblastoma/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Proteínas de Membrana/biossíntese , Neovascularização Patológica/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Receptores Notch/biossíntese , Transdução de Sinais , Adolescente , Adulto , Idoso , Feminino , Glioblastoma/irrigação sanguínea , Glioblastoma/patologia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Receptor Notch1/biossíntese , Receptor Notch2/biossíntese , Receptor Notch3/biossíntese , Receptor Notch4 , Fatores de Transcrição HES-1/biossíntese , Fator A de Crescimento do Endotélio Vascular/biossíntese , Adulto Jovem
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