Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.605
Filtrar
1.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360789

RESUMO

The erythroid Krüppel-like factor EKLF/KLF1 is a hematopoietic transcription factor binding to the CACCC DNA motif and participating in the regulation of erythroid differentiation. With combined use of microarray-based gene expression profiling and the promoter-based ChIP-chip assay of E14.5 fetal liver cells from wild type (WT) and EKLF-knockout (Eklf-/-) mouse embryos, we identified the pathways and direct target genes activated or repressed by EKLF. This genome-wide study together with the molecular/cellular analysis of the mouse erythroleukemic cells (MEL) indicate that among the downstream direct target genes of EKLF is Tal1/Scl. Tal1/Scl encodes another DNA-binding hematopoietic transcription factor TAL1/SCL, known to be an Eklf activator and essential for definitive erythroid differentiation. Further identification of the authentic Tal gene promoter in combination with the in vivo genomic footprinting approach and DNA reporter assay demonstrate that EKLF activates the Tal gene through binding to a specific CACCC motif located in its promoter. These data establish the existence of a previously unknow positive regulatory feedback loop between two DNA-binding hematopoietic transcription factors, which sustains mammalian erythropoiesis.


Assuntos
Eritropoese , Feto/embriologia , Hematopoese Extramedular , Fatores de Transcrição Kruppel-Like/metabolismo , Fígado/embriologia , Proteína 1 de Leucemia Linfocítica Aguda de Células T/metabolismo , Animais , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Knockout , Elementos de Resposta , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genética
2.
J Immunol ; 207(3): 809-823, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34282003

RESUMO

The transcription factor promyelocytic leukemia zinc finger (PLZF) is encoded by the BTB domain-containing 16 (Zbtb16) gene. Its repressor function regulates specific transcriptional programs. During the development of invariant NKT cells, PLZF is expressed and directs their effector program, but the detailed mechanisms underlying PLZF regulation of multistage NKT cell developmental program are not well understood. This study investigated the role of acetylation-induced PLZF activation on NKT cell development by analyzing mice expressing a mutant form of PLZF mimicking constitutive acetylation (PLZFON) mice. NKT populations in PLZFON mice were reduced in proportion and numbers of cells, and the cells present were blocked at the transition from developmental stage 1 to stage 2. NKT cell subset differentiation was also altered, with T-bet+ NKT1 and RORγt+ NKT17 subsets dramatically reduced and the emergence of a T-bet-RORγt- NKT cell subset with features of cells in early developmental stages rather than mature NKT2 cells. Preliminary analysis of DNA methylation patterns suggested that activated PLZF acts on the DNA methylation signature to regulate NKT cells' entry into the early stages of development while repressing maturation. In wild-type NKT cells, deacetylation of PLZF is possible, allowing subsequent NKT cell differentiation. Interestingly, development of other innate lymphoid and myeloid cells that are dependent on PLZF for their generation is not altered in PLZFON mice, highlighting lineage-specific regulation. Overall, we propose that specific epigenetic control of PLZF through acetylation levels is required to regulate normal NKT cell differentiation.


Assuntos
Fatores de Transcrição Kruppel-Like , Células T Matadoras Naturais , Acetilação , Animais , Diferenciação Celular , Imunidade Inata , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Linfócitos/metabolismo , Camundongos , Células T Matadoras Naturais/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica
3.
Nat Commun ; 12(1): 4362, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272396

RESUMO

Squamous cell carcinomas (SCCs) comprise one of the most common histologic types of human cancer. Transcriptional dysregulation of SCC cells is orchestrated by tumor protein p63 (TP63), a master transcription factor (TF) and a well-researched SCC-specific oncogene. In the present study, both Gene Set Enrichment Analysis (GSEA) of SCC patient samples and in vitro loss-of-function assays establish fatty-acid metabolism as a key pathway downstream of TP63. Further studies identify sterol regulatory element binding transcription factor 1 (SREBF1) as a central mediator linking TP63 with fatty-acid metabolism, which regulates the biosynthesis of fatty-acids, sphingolipids (SL), and glycerophospholipids (GPL), as revealed by liquid chromatography tandem mass spectrometry (LC-MS/MS)-based lipidomics. Moreover, a feedback co-regulatory loop consisting of SREBF1/TP63/Kruppel like factor 5 (KLF5) is identified, which promotes overexpression of all three TFs in SCCs. Downstream of SREBF1, a non-canonical, SCC-specific function is elucidated: SREBF1 cooperates with TP63/KLF5 to regulate hundreds of cis-regulatory elements across the SCC epigenome, which converge on activating cancer-promoting pathways. Indeed, SREBF1 is essential for SCC viability and migration, and its overexpression is associated with poor survival in SCC patients. Taken together, these data shed light on mechanisms of transcriptional dysregulation in cancer, identify specific epigenetic regulators of lipid metabolism, and uncover SREBF1 as a potential therapeutic target and prognostic marker in SCC.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Metabolismo dos Lipídeos/genética , Neoplasias Pulmonares/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Acetilação , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Sequenciamento de Cromatina por Imunoprecipitação , Cromatografia Líquida , Epigenômica , Receptores ErbB/genética , Receptores ErbB/metabolismo , Neoplasias Esofágicas/genética , Ácidos Graxos/biossíntese , Ácidos Graxos/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Histonas/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Neoplasias Pulmonares/genética , Elementos Reguladores de Transcrição , Transdução de Sinais/genética , Esfingolipídeos/biossíntese , Esfingolipídeos/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Espectrometria de Massas em Tandem , Fatores de Transcrição/genética , Transcriptoma/genética , Proteínas Supressoras de Tumor/genética
4.
Front Immunol ; 12: 648815, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34305888

RESUMO

Multiple lines of evidence have demonstrated that cigarette smoke or Chronic Obstructive Pulmonary Disease upregulates angiotensin-converting enzyme 2, the cellular receptor for the entry of the severe acute respiratory syndrome coronavirus 2, which predisposes individuals to develop severe Coronavirus disease 2019. The reason for this observation is unknown. We recently reported that the loss of function of Miz1 in the lung epithelium in mice leads to a spontaneous COPD-like phenotype, associated with upregulation of angiotensin-converting enzyme 2. We also reported that cigarette smoke exposure downregulates Miz1 in lung epithelial cells and in mice, and Miz1 is also downregulated in the lungs of COPD patients. Here, we provide further evidence that Miz1 directly binds to and represses the promoter of angiotensin-converting enzyme 2 in mouse and human lung epithelial cells. Our data provide a potential molecular mechanism for the upregulation of angiotensin-converting enzyme 2 observed in smokers and COPD patients, with implication in severe Coronavirus disease 2019.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Receptores Virais/genética , Transcrição Genética , Células Epiteliais Alveolares/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Domínio BTB-POZ , Linhagem Celular , Fumar Cigarros/efeitos adversos , Fatores de Transcrição Kruppel-Like/química , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Receptores Virais/metabolismo , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Transcrição Genética/efeitos dos fármacos , Fatores de Necrose Tumoral/farmacologia , Internalização do Vírus
5.
Mol Cell Biochem ; 476(10): 3845-3856, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34117589

RESUMO

Endometriosis is an estrogen-dependent disease. Several researches have reported the dysregulated circular RNAs (circRNAs) in endometriosis, whereas the functions of circRNAs are largely unknown. This study aims to explore the role and mechanism of circ_0075503 in migration and invasion of eutopic endometrial stromal cells. 30 paired ectopic and eutopic endometrium tissues were collected from patients with endometriosis. And primary endometrial stromal cells (ESCs) were stimulated with estradiol (E2) to establish the in vitro cellular model of endometriosis. The levels of circ_0075503, miR-15a-5p and Krüppel-like factor 12 (KLF12) were measured by quantitative reverse transcription polymerase chain reaction or western blot assays. Cell viability, migration and invasion were examined via 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide, transwell assay or western blot assays. The target relationship between miR-15a-5p and circ_0075503 or KLF12 was analyzed by dual-luciferase reporter assay and RNA Immunoprecipitation (RIP) assay. Circ_0075503 expression was elevated in ectopic endometrium and ectopic ESCs. Down-regulation of circ_0075503 suppressed E2-induced promotion of cell viability, migration and invasion in eutopic ESCs. Circ_0075503 could act as a sponge for miR-15a-5p, and KLF12 was targeted by miR-15a-5p. Inhibition of miR-15a-5p reversed the effects of circ_0075503 knockdown on E2-treated ESCs migration and invasion. Besides, miR-15a-5p repressed E2-induced promotion effects on cell migration and invasion via targeting KLF12. Circ_0075503 could regulate KLF12 expression by sponging miR-15a-5p. Knockdown of circ_0075503 inhibited E2-induced enhancement of cell migration and invasion in eutopic ESCs by regulating miR-15a-5p/KLF12 axis, indicating a novel target for the treatment of endometriosis.


Assuntos
Movimento Celular , Endometriose/metabolismo , Técnicas de Silenciamento de Genes , Fatores de Transcrição Kruppel-Like/metabolismo , MicroRNAs/metabolismo , RNA Circular/metabolismo , Adulto , Endometriose/genética , Feminino , Humanos , Fatores de Transcrição Kruppel-Like/genética , MicroRNAs/genética , Pessoa de Meia-Idade , RNA Circular/genética , Células Estromais/metabolismo
6.
Life Sci ; 280: 119698, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34111466

RESUMO

AIMS: The purpose of this study was to investigate the effects of miR-431-5p on hepatocyte apoptosis in AIH. MATERIALS AND METHODS: We used intraperitoneal injection of S100 to establish AIH mouse model and injected AAV into tail vein on day 14 of modeling to regulate miR-431-5p expression. The expression of ALT, AST, IgG and apoptosis-related proteins Bax, Bcl-2 and cleaved caspase 3 were measured in each group. Cellular experiments were performed using miR-431-5p mimics or inhibitors to transfect LPS-stimulated AML12 cells, and apoptosis was verified using Western blot and Hoechst 33342/PI Double Staining. The target of miR-431-5p, KLF15, was screened using databases and verified by the luciferase reporter assay. The relationship between KLF15 and p53 was verified by si-KLF15 and PFTß (a p53-specific inhibitor). KEY FINDINGS: Here, we observed that the increase in the level of miR-431-5p was accompanied by a decrease in the expression of Krüppel-like zinc finger transcription factor 15 (KLF15). In addition, the deletion of miR-431-5p significantly reduced hepatocyte apoptosis in AIH mice induced by liver S100 and apoptosis of AML12 cells induced by LPS stimulation, accompanied by decreased expression of Bax and cleaved caspase-3 as well as increased expression of Bcl-2. Moreover, KLF15 was the direct and functional target of miR-431-5p. Furthermore, miR-431-5p negatively regulated the expression of KLF15, and KLF15 deletion partially abolished the inhibitory effect of miR-431-5p deletion on apoptosis by activating p53 signaling. SIGNIFICANCE: In summary, miR-431-5p may be a potential therapeutic target for AIH.


Assuntos
Apoptose , Hepatite Autoimune/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fígado/patologia , MicroRNAs/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Regulação para Baixo , Hepatite Autoimune/etiologia , Hepatite Autoimune/patologia , Fatores de Transcrição Kruppel-Like/genética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/antagonistas & inibidores , Proteínas S100/efeitos adversos , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Regulação para Cima
7.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070901

RESUMO

Glycosaminoglycans (GAGs) and proteoglycans (PGs) are major components of the glycocalyx. The secreted GAG and CD44 ligand hyaluronic acid (HA), and the cell surface PG syndecan-1 (Sdc-1) modulate the expression and activity of cytokines, chemokines, growth factors, and adhesion molecules, acting as critical regulators of tumor cell behavior. Here, we studied the effect of Sdc-1 siRNA depletion and HA treatment on hallmark processes of cancer in breast cancer cell lines of different levels of aggressiveness. We analyzed HA synthesis, and parameters relevant to tumor progression, including the stem cell phenotype, Wnt signaling constituents, cell cycle progression and apoptosis, and angiogenic markers in luminal MCF-7 and triple-negative MDA-MB-231 cells. Sdc-1 knockdown enhanced HAS-2 synthesis and HA binding in MCF-7, but not in MDA-MB-231 cells. Sdc-1-depleted MDA-MB-231 cells showed a reduced CD24-/CD44+ population. Furthermore, Sdc-1 depletion was associated with survival signals in both cell lines, affecting cell cycle progression and apoptosis evasion. These changes were linked to the altered expression of KLF4, MSI2, and miR-10b and differential changes in Erk, Akt, and PTEN signaling. We conclude that Sdc-1 knockdown differentially affects HA metabolism in luminal and triple-negative breast cancer model cell lines and impacts the stem phenotype, cell survival, and angiogenic factors.


Assuntos
Regulação Neoplásica da Expressão Gênica , Glicocálix/metabolismo , Ácido Hialurônico/metabolismo , Sindecana-1/genética , Neoplasias de Mama Triplo Negativas/genética , Via de Sinalização Wnt/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Antígeno CD24/genética , Antígeno CD24/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Bases de Dados Factuais , Feminino , Glicocálix/química , Glicocálix/efeitos dos fármacos , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Hialuronan Sintases/genética , Hialuronan Sintases/metabolismo , Ácido Hialurônico/farmacologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Células MCF-7 , MicroRNAs/genética , MicroRNAs/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Análise de Sobrevida , Sindecana-1/antagonistas & inibidores , Sindecana-1/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/mortalidade , Neoplasias de Mama Triplo Negativas/patologia
8.
Int J Cancer ; 149(6): 1358-1368, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-33997976

RESUMO

In the course of identifying the molecular mechanism that is related to strong cell-cell adhesion in stratified structures of the squamous epithelium, calmodulin-like protein 5 (CALML5) was identified as a spinous structure-associated protein by producing monoclonal antibodies with the use of the crude intercellular portion of squamous tissue as an immunogen and by subsequent morphologic screening. By electrophoretic mobility shift assay (EMSA) and a series of mutagenesis studies, two transcription factors, ZNF750 and KLF4, by binding in line to the CALML5 gene promoter, were found to play a central role in CALML5 transcription. Knockdown of CALML5 by siRNA in the A431 cell line that expresses high levels of CALML5 resulted in the acceleration of wound confluence in a scratch assay, indicating that CALML5 functions as a tumor-suppressor in uterine cervical cancer. Immunohistochemical evaluation of squamous intraepithelial lesions, carcinoma in situ (CIS) and invasive uterine cancer, revealed a reduction in CALML5 expression during the stages of CIS through various molecular pathways including the blockage of the nuclear translocation of KLF4. Conversely, restoration of the nuclear translocation of KLF4 by inhibiting ERK-signaling reactivated CALML5 expression in ME180 cells expressing low levels of CALML5. Thus, alteration of the p63-ZNF750-KLF4 axis may result in critical functional loss of CALM-related genes during cancer progression. Although the morphological association of CALML5 with the spiny-structure in relation to cell motility is not clear, evaluation of CALML5 expression provides a useful diagnostic indicator of differentiating dysplasia, preinvasive and invasive cervical cancers.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Carcinoma de Células Escamosas/patologia , Regulação para Baixo , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Neoplasias do Colo do Útero/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Células HL-60 , Humanos , Estadiamento de Neoplasias , Regiões Promotoras Genéticas , Transporte Proteico , Transcrição Genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo
9.
Cell Prolif ; 54(7): e13072, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34031939

RESUMO

OBJECTIVES: Induction of deactivation and apoptosis of hepatic stellate cells (HSCs) are principal therapeutic strategies for liver fibrosis. Krüppel-like factor 14 (KLF14) regulates various biological processes, however, roles, mechanisms and implications of KLF14 in liver fibrosis are unknown. MATERIALS AND METHODS: KLF14 expression was detected in human, rat and mouse fibrotic models, and its effects on HSCs were assessed. Chromatin immunoprecipitation assays were utilized to investigate the binding of KLF14 to peroxisome proliferator-activated receptor γ (PPARγ) promoter, and the binding of enhancer of zeste homolog 2 (EZH2) to KLF14 promoter. In vivo, KLF14-overexpressing adenovirus was injected via tail vein to thioacetamide (TAA)-treated rats to investigate the role of KLF14 in liver fibrosis progression. EZH2 inhibitor EPZ-6438 was utilized to treat TAA-induced rat liver fibrosis. RESULTS: KLF14 expression was remarkably decreased in human, rat and mouse fibrotic liver tissues. Overexpression of KLF14 increased LD accumulation, inhibited HSCs activation, proliferation, migration and induced G2/M arrest and apoptosis. Mechanistically, KLF14 transactivated PPARγ promoter activity. Inhibition of PPARγ blocked the suppressive role of KLF14 overexpression in HSCs. Downregulation of KLF14 in activated HSCs was mediated by EZH2-regulated histone H3 lysine 27 trimethylation. Adenovirus-mediated KLF14 overexpression ameliorated TAA-induced rat liver fibrosis in PPARγ-dependent manner. Furthermore, EPZ-6438 dramatically alleviated TAA-induced rat liver fibrosis. Importantly, KLF14 expression was decreased in human with liver fibrosis, which was significantly correlated with EZH2 upregulation and PPARγ downregulation. CONCLUSIONS: KLF14 exerts a critical anti-fibrotic role in liver fibrosis, and targeting the EZH2/KLF14/PPARγ axis might be a novel therapeutic strategy for liver fibrosis.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Cirrose Hepática/patologia , PPAR gama/metabolismo , Animais , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Compostos de Bifenilo/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Regulação para Baixo , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/genética , Cirrose Hepática/metabolismo , Camundongos , Morfolinas/farmacologia , PPAR gama/antagonistas & inibidores , PPAR gama/genética , Regiões Promotoras Genéticas , Piridonas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Tioacetamida/farmacologia
10.
Methods Mol Biol ; 2318: 267-279, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019296

RESUMO

Cellular reprogramming is a process by which adult differentiated cells lose their identity and are converted into pluripotent stem cells, known as induced pluripotent stem (iPS) cells. This process can be achieved in vitro and in vivo and is relevant for many fields including regenerative medicine and cancer. Cellular reprogramming is commonly induced by the ectopic expression of a transcription factor cocktail composed by Oct4, Sox2, Klf4, and Myc (abbreviated as OSKM), and its efficiency and kinetics are strongly dependent on the presence of Myc. Here, we describe a versatile method to study reprogramming in vivo based on the use of adeno-associated viral (AAV) vectors, which allows the targeting of specific organs and cell types. This method can be used to test Myc mutations or genes that may replace Myc, or be combined with different Myc regulators. In vivo reprogramming can be scored by the presence of teratomas and the isolation of in vivo iPS, thereby providing a simple surrogate for the function of Myc in dedifferentiation and stemness. Our protocol can be divided into five steps: (1) intravenous inoculation of AAV vectors; (2) monitoring the animals until the appearance of teratomas; (3) analysis of teratomas; (4) histopathological analysis of mouse organs; and (5) isolation of in vivo-generated iPS cells from teratomas, blood, and bone marrow. The information obtained by this in vivo testing platform may provide relevant information on the role of Myc in tissue regeneration, stemness, and cancer.


Assuntos
Técnicas de Reprogramação Celular/métodos , Reprogramação Celular/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Reprogramação Celular/fisiologia , DNA/genética , Dependovirus/genética , Fibroblastos/citologia , Genes myc/genética , Genes myc/fisiologia , Engenharia Genética/métodos , Vetores Genéticos/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/metabolismo , Transdução Genética
12.
Aging (Albany NY) ; 13(10): 13807-13821, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33929970

RESUMO

Pulmonary fibrosis is a common pulmonary interstitial disease of pathogenesis without effective drugs for treatment. Therefore, discovering new and effective drugs is urgently needed. In the present study, we prepared a novel compound named acetyl oxygen benzoate engeletin ester (AOBEE), investigated its effect on experimental pulmonary fibrosis, and proposed a long non-coding RNA (lncRNA)-mediated mechanism of its action. Bleomycin-induced pulmonary fibrosis in mice exhibited that AOBEE improved forced vital capacity (FVC) and alveolar structure and inhibited α-SMA, vimentin, and collagen expression. TGFß1-stimulated fibroblast L929 cells showed that AOBEE reduced these fibrotic proteins expression and inhibited the activated-fibroblast proliferation and migration. Whole transcriptome sequencing was performed to screen out lncRNA-lnc865 and lnc556 with high expression under bleomycin treatment, but AOBEE caused a considerable decrease in lnc865 and lnc556. Mechanistic study elucidated that AOBEE alleviated pulmonary fibrosis through lnc865- and lnc556-mediated mechanism, in which both lnc865 and lnc556 sponged miR-29b-2-5p to target signal transducer and activator of transcription 3 (STAT3). Further signal pathway inhibitors and the Cignal Finder 45-pathway reporter array illustrated that the up- and downstream pathways were TGFß1-smad2/3 and p38MAPK, and Krüppel-like factor 4 (KLF4), respectively. In conclusion, AOBEE promoted KLF4 degradation leading to the attenuation of pulmonary fibrosis by inhibiting TGFß1-smad/p38MAPK-lnc865/lnc556-miR-29b-2-5p-STAT3 signal pathway. We hope this work will provide valuable information to design new drugs and therapeutic targets of lncRNAs for pulmonary fibrosis treatment.


Assuntos
Flavonóis/farmacologia , Glicosídeos/farmacologia , Fatores de Transcrição Kruppel-Like/metabolismo , MicroRNAs/metabolismo , Fibrose Pulmonar/metabolismo , RNA Longo não Codificante/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Flavonóis/química , Glicosídeos/química , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Modelos Biológicos , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo
13.
Genes (Basel) ; 12(4)2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33918002

RESUMO

Inducing apoptosis is an effective treatment for cancer. Conventional cytotoxic anticancer agents induce apoptosis primarily through activation of tumor suppressor p53 by causing DNA damage and the resulting regulation of B-cell leukemia/lymphoma-2 (BCL-2) family proteins. Therefore, the effects of these agents are limited in cancers where p53 loss-of-function mutations are common, such as triple-negative breast cancer (TNBC). Here, we demonstrate that ultraviolet (UV) light-induced p53-independent transcriptional activation of NOXA, a proapoptotic factor in the BCL-2 family, results in apoptosis induction. This UV light-induced NOXA expression was triggered by extracellular signal-regulated kinase (ERK) activity. Moreover, we identified the specific UV light-inducible DNA element of the NOXA promoter and found that this sequence is responsible for transcription factor Krüppel-like factor 4 (KLF4)-mediated induction. In p53-mutated TNBC cells, inhibition of KLF4 by RNA interference reduced NOXA expression. Furthermore, treatment of TNBC cells with a KLF4-inducing small compound, APTO-253, resulted in the induction of NOXA expression and NOXA-mediated apoptosis. Therefore, our results help to clarify the molecular mechanism of DNA damage-induced apoptosis and provide support for a possible treatment method for p53-mutated cancers.


Assuntos
Apoptose , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Imidazóis/farmacologia , Fatores de Transcrição Kruppel-Like/metabolismo , Fenantrolinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Proteína Supressora de Tumor p53/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Feminino , Humanos , Fatores de Transcrição Kruppel-Like/genética , Prognóstico , Proteínas Proto-Oncogênicas c-bcl-2/genética , Taxa de Sobrevida , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética
14.
Aging (Albany NY) ; 13(8): 11942-11953, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33875621

RESUMO

Atherosclerosis is a chronic inflammatory disease known to be mediated by numerous factors, among which endothelial dysfunction plays a critical role. Oscillatory shear stress induces endothelial cells to lose their anti-atherosclerotic properties and downregulates the expression of the innate protective transcription factor, Krüppel-like factor 2 (KLF2), which is typically upregulated in vascular endothelial cells in response to harmful stimuli. Oxidative stress and inflammation impair endothelial function and damage their survival. Oscillatory shear stress also promotes generation of reactive oxygen species and production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), thereby further promoting endothelial dysfunction and formation of atherosclerotic plaque. A major event in the development of atherosclerotic plaque is rolling and adhesion of monocytes to endothelial cells, which is mediated by adhesion molecules including vascular cellular adhesion molecule 1 and endothelial-selectin. Expression of these molecules is also upregulated by oscillatory shear stress. Estrogen has long been recognized as a protective agent against atherosclerosis, but the mechanisms through which estrogen receptors prevent atherogenesis remain unclear. In the present study, we investigated the role of the G-coupled protein estrogen receptor (GPR30) in oscillatory shear stress- induced endothelial dysfunction. We show that agonism of GPR30 by its specific agonist G1 prevented oscillatory shear stress -induced oxidative stress markers and production of inflammatory cytokines and adhesion molecules. As a result, GPR30 activation suppresses monocytes adhesion to endothelial cells. Furthermore, we demonstrate that GPR30 prevents oscillatory shear stress- induced downregulation of KLF2 via ERK5 pathway. These findings suggest that endothelial GPR30 is potential target to suppress oscillatory shear stress mediated atherogenesis.


Assuntos
Aterosclerose/tratamento farmacológico , Adesão Celular/efeitos dos fármacos , Fatores de Transcrição Kruppel-Like/metabolismo , Quinolinas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Aterosclerose/patologia , Adesão Celular/genética , Selectina E/metabolismo , Células Endoteliais , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Quinolinas/uso terapêutico , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Estresse Mecânico , Células THP-1 , Molécula 1 de Adesão de Célula Vascular/metabolismo
15.
Nature ; 594(7862): 271-276, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33910229

RESUMO

Vascular malformations are thought to be monogenic disorders that result in dysregulated growth of blood vessels. In the brain, cerebral cavernous malformations (CCMs) arise owing to inactivation of the endothelial CCM protein complex, which is required to dampen the activity of the kinase MEKK31-4. Environmental factors can explain differences in the natural history of CCMs between individuals5, but why single CCMs often exhibit sudden, rapid growth, culminating in strokes or seizures, is unknown. Here we show that growth of CCMs requires increased signalling through the phosphatidylinositol-3-kinase (PI3K)-mTOR pathway as well as loss of function of the CCM complex. We identify somatic gain-of-function mutations in PIK3CA and loss-of-function mutations in the CCM complex in the same cells in a majority of human CCMs. Using mouse models, we show that growth of CCMs requires both PI3K gain of function and CCM loss of function in endothelial cells, and that both CCM loss of function and increased expression of the transcription factor KLF4 (a downstream effector of MEKK3) augment mTOR signalling in endothelial cells. Consistent with these findings, the mTORC1 inhibitor rapamycin effectively blocks the formation of CCMs in mouse models. We establish a three-hit mechanism analogous to cancer, in which aggressive vascular malformations arise through the loss of vascular 'suppressor genes' that constrain vessel growth and gain of a vascular 'oncogene' that stimulates excess vessel growth. These findings suggest that aggressive CCMs could be treated using clinically approved mTORC1 inhibitors.


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Mutação , Neoplasias/genética , Animais , Animais Recém-Nascidos , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Mutação com Ganho de Função , Hemangioma Cavernoso do Sistema Nervoso Central/irrigação sanguínea , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Mutação com Perda de Função , MAP Quinase Quinase Quinase 3/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo
16.
Biochem Biophys Res Commun ; 557: 334-341, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-33915432

RESUMO

Atherosclerosis is a chronic lipid disfunction and inflammatory disease, which is characterized with enriched foam cells and necrotic core underneath the vascular endothelium. Therefore, the inhibition of foam cell formation is a critical step for atherosclerosis treatment. Metformin, a first-line treatment for Type 2 diabetes, is reported to be beneficial to cardiovascular disease. However, the mechanism underlying the antiatherogenic effect of metformin remains unclear. Macrophage autophagy is reported to be a highly anti-atherogenic process that promotes the catabolism of cytosolic lipid to maintain cellular lipid homeostasis. Notably, dysfunctional autophagy in macrophages plays a detrimental role during atherogenesis. Krueppel-like factor 2 (KLF2) is an important transcription factor that functions as a key regulator of the autophagy-lysosome pathway. While the role of KLF2 in foam cell formation during the atherogenesis remains elusive. In this study, we first investigated whether metformin could protect against atherogenesis via enhancing autophagy in high fat diet (HFD)-induced apoE-/- mice. Subsequently, we further determined the molecular mechanism that whether metformin could inhibit foam cell formation by activating KLF2-mediated autophagy. We show that metformin protected against HFD-induced atherosclerosis and enhanced plaque stability in apoE-/- mice. Metformin inhibits foam cell formation and cellular apoptosis partially through enhancing autophagy. Mechanistically, metformin promotes autophagy via modulating KLF2 expression. Taken together, our study demonstrates a novel antiatherogenic mechanism of metformin by upregulating KLF2-mediated autophagy.


Assuntos
Apolipoproteínas E/genética , Aterosclerose/tratamento farmacológico , Autofagia/efeitos dos fármacos , Células Espumosas/efeitos dos fármacos , Fatores de Transcrição Kruppel-Like/metabolismo , Metformina/farmacologia , Placa Aterosclerótica/tratamento farmacológico , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/patologia , Apoptose/efeitos dos fármacos , Aterosclerose/genética , Aterosclerose/metabolismo , Colesterol/metabolismo , Dieta Hiperlipídica , Células Espumosas/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Metformina/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Placa Aterosclerótica/genética , Placa Aterosclerótica/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
17.
Int J Mol Sci ; 22(7)2021 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-33801599

RESUMO

MYC is a proto-oncogene regulating a large number of genes involved in a plethora of cellular functions. Its deregulation results in activation of MYC gene expression and/or an increase in MYC protein stability. MYC overexpression is a hallmark of malignant growth, inducing self-renewal of stem cells and blocking senescence and cell differentiation. This review summarizes the latest advances in our understanding of MYC-mediated molecular mechanisms responsible for its oncogenic activity. Several recent findings indicate that MYC is a regulator of cancer genome and epigenome: MYC modulates expression of target genes in a site-specific manner, by recruiting chromatin remodeling co-factors at promoter regions, and at genome-wide level, by regulating the expression of several epigenetic modifiers that alter the entire chromatin structure. We also discuss novel emerging therapeutic strategies based on both direct modulation of MYC and its epigenetic cofactors.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ativação Transcricional , Animais , Apoptose , Carcinogênese , Diferenciação Celular , Proliferação de Células , Cromatina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Epigênese Genética , Epigenoma , Genoma Humano , Células-Tronco Hematopoéticas/citologia , Homeostase , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Leucemia/metabolismo , Linfoma/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo
18.
BMC Cancer ; 21(1): 456, 2021 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-33892667

RESUMO

BACKGROUND: Endometrial cancer (UCEC) is one of the most common gynecological malignancies. We previously found that overexpression of G protein α subunit 14 (GNA14) promoted UCEC growth. Krüppel-like factor 7 (KLF7) acts as an oncogene in various cancer types, whereas the connection between GNA14 and KLF7 in UCEC is unclear. We herein explored the involvement of GNA14/KLF7 in UCEC development. METHODS: Clinical relevance of GNA14, KLF7 and HAS2 in UCEC was analyzed from TCGA and by immunohistochemical staining. Knockdown and overexpression of indicated genes were conducted by transfecting the cells with siRNAs and lentivirus, respectively. mRNA and protein expression was detected by qRT-PCR and Western blot. CCK8, colony formation, cell cycle, apoptosis, transwell and wound healing were performed to check cell biology function in vitro. Tumor growth in nude mice was conducted to check in vivo function. RNA sequencing was used to determine dys-regulated genes. RESULTS: We demonstrated that GNA14 stimulated the expression of KLF7 in UCEC cells. There was a positive correlation between GNA14 and KLF7 in normal and UCEC tissues. In vitro, KLF7 promoted cell proliferation, colony formation, cell cycle progression, and migration of UCEC cells. Apoptosis was inhibited by KLF7. Xenografted tumorigenesis of UCEC cells was suppressed by KLF7 knockdown. Furthermore, RNA sequencing results showed that KLF7 regulated the expression of a large amount of genes, among which hyaluronan synthase 2 (HAS2) was downregulated in KLF7 knockdown cells. Based on TCGA database and immunoblotting assays, KLF7 positively regulated HAS2 in UCEC cells and tissues. Lastly, knockdown of HAS2 reversed the oncogenic role of KLF7 on UCEC cell proliferation, migration, and xenografted tumor development. CONCLUSION: Taken together, we reveal that GNA14/KLF7/HAS2 signaling cascade exerts tumor promoting function during UCEC development.


Assuntos
Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Hialuronan Sintases/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Animais , Sequência de Bases , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação para Baixo , Neoplasias do Endométrio/genética , Feminino , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Xenoenxertos , Humanos , Hialuronan Sintases/genética , Fatores de Transcrição Kruppel-Like/genética , Lentivirus , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Transfecção/métodos , Regulação para Cima
19.
BMC Cancer ; 21(1): 377, 2021 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-33827480

RESUMO

BACKGROUND: Epiplakin1 (Eppk1) is part of epidermal growth factor (EGF) signal and takes part in reorganization of cytoskeleton and cell proliferation. However, the role of Eppk1 in cervical cancer (CC) remains unknown. METHODS: To express Eppk1 and KLF5 and their correlation, we used RNA-sequence, RT-qPCR, TCGA database and immunofluorescence staining in vitro and in different pathological cervical tissues. In CC cell lines, we tested adenovirus-mediated over expression or knockdown of KLF5 and siRNA-mediated knockdown of Eppk1 and a suiting assessment of cell proliferation and cell signaling by western blot and CCK8 tests. We studied the mechanism by which KLF5 regulates Eppk1 expression by reporter gene test and chromatin immunoprecipitation test. RESULTS: Eppk1 expression promoted in CC tissues and cell lines compared with increased KLF5 expression. The results of immunofluorescence staining further showed the increased co-expression of Eppk1 and KLF5 correlated substantially with tumorigenesis in cervical tissues. Overexpression of KLF5 significantly increased Eppk1 expression at transcription and translation levels. Conversely, the knockdown of KLF5 by siRNA against KLF5 decreased Eppk1 expression. Mechanically, KLF5 activated Eppk1 transcription by direct binding to the Eppk1 promoter. Gain- and loss-of-function experiments reported that KLF5 promoted cell proliferation in Hela partly dependent on Eppk1 upregulation. Besides, KLF5-mediated activation of p38 signaling significantly decreased after Eppk1 knockdown compared with decline of proliferation, suggesting that Eppk1 lies upstream of p38 signaling affecting cell proliferation. Finally, Eppk1 expression is positively correlated with tumor size in clinicopathological features of CC. CONCLUSIONS: Eppk1 may be an effective therapeutic target for affecting p38 signaling pathway and cell proliferation in cervical cancer.


Assuntos
Autoantígenos/genética , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Kruppel-Like/metabolismo , Sistema de Sinalização das MAP Quinases , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Adulto , Idoso , Autoantígenos/metabolismo , Biópsia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Imunofluorescência , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Regiões Promotoras Genéticas , Ligação Proteica , Neoplasias do Colo do Útero/patologia
20.
Int J Mol Sci ; 22(7)2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810223

RESUMO

DNA can adopt various structures besides the B-form. Among them, cruciform structures are formed on inverted repeat (IR) sequences. While cruciform formable IRs (CFIRs) are sometimes found in regulatory regions of transcription, their function in transcription remains elusive, especially in eukaryotes. We found a cluster of CFIRs within the mouse Pou5f1 enhancer. Here, we demonstrate that this cluster or some member(s) plays an active role in the transcriptional regulation of not only Pou5f1, but also Sox2, Nanog, Klf4 and Esrrb. To clarify in vivo function of the cluster, we performed genome editing using mouse ES cells, in which each of the CFIRs was altered to the corresponding mirror repeat sequence. The alterations reduced the level of the Pou5f1 transcript in the genome-edited cell lines, and elevated those of Sox2, Nanog, Klf4 and Esrrb. Furthermore, transcription of non-coding RNAs (ncRNAs) within the enhancer was also upregulated in the genome-edited cell lines, in a similar manner to Sox2, Nanog, Klf4 and Esrrb. These ncRNAs are hypothesized to control the expression of these four pluripotency genes. The CFIRs present in the Pou5f1 enhancer seem to be important to maintain the integrity of ES cells.


Assuntos
Elementos Facilitadores Genéticos , Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Animais , Linhagem Celular , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Conformação de Ácido Nucleico , Fator 3 de Transcrição de Octâmero/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Ativação Transcricional , Regulação para Cima
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...