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1.
Immunity ; 51(6): 1043-1058.e4, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31810882

RESUMO

T cell dysfunction is a characteristic feature of chronic viral infection and cancer. Recent studies in chronic lymphocytic choriomeningitis virus (LCMV) infection have defined a PD-1+ Tcf-1+ CD8+ T cell subset capable of self-renewal and differentiation into more terminally differentiated cells that downregulate Tcf-1 and express additional inhibitory molecules such as Tim3. Here, we demonstrated that expression of the glycoprotein CD101 divides this terminally differentiated population into two subsets. Stem-like Tcf-1+ CD8+ T cells initially differentiated into a transitory population of CD101-Tim3+ cells that later converted into CD101+ Tim3+ cells. Recently generated CD101-Tim3+ cells proliferated in vivo, contributed to viral control, and were marked by an effector-like transcriptional signature including expression of the chemokine receptor CX3CR1, pro-inflammatory cytokines, and granzyme B. PD-1 pathway blockade increased the numbers of CD101-Tim3+ CD8+ T cells, suggesting that these newly generated transitional cells play a critical role in PD-1-based immunotherapy.


Assuntos
Antígenos CD/metabolismo , Linfócitos T CD8-Positivos/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Animais , Biomarcadores/metabolismo , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Feminino , Granzimas/genética , Granzimas/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/biossíntese , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Coriomeningite Linfocítica/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/genética
2.
Environ Toxicol ; 34(12): 1313-1319, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31423742

RESUMO

Syzygium cumini (Myrtaceae) is commonly called as Jamun or Jambolan. It has antidiabetic, anti-inflammatory, antipyretic, and antioxidant activities. Hepatocellular carcinoma is the most frequent and deadliest cancers worldwide. We investigated the cytotoxic potentials of S. cumini methanolic seed kernel extract against human hepatoma HepG2 cells. HepG2 cells were treated with 10, 20, and 40 µg/mL of seed kernel extract for 24 hours and cytotoxic analysis was performed by MTT assay. S. cumini induced apoptosis related morphological changes in HepG2 cells were analyzed by annexin V and propidium iodide double staining. Nuclear fragmentation and chromatin condensation were analyzed by Hoechst nuclear staining. Mitochondrial membrane potential (MMP) was investigated by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) staining. Protein expressions of hepatocyte nuclear factor-1α (HFN-1α) was performed using western blotting. S. cumini treatments caused a significant and a concentration-dependent increase in the cytotoxicity of HepG2 cells. S. cumini treatments increased the percentage of cells in an early and late apoptosis stage. This treatment also caused chromatin condensation and nuclear fragmentation. Further, S. cumini treatments decreased MMP and also caused a significant downregulation of HFN-1α protein expression. The present study demonstrated that S. cumini seed extract induced apoptosis in HepG2 cells through decrease in MMP and downregulation of HFN-1α.


Assuntos
Apoptose/efeitos dos fármacos , Myrtaceae/química , Extratos Vegetais/farmacologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Regulação para Baixo/efeitos dos fármacos , Células Hep G2 , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Metanol/química , Myrtaceae/metabolismo , Extratos Vegetais/análise , Extratos Vegetais/química , Sementes/química , Sementes/metabolismo
3.
Med Sci Monit ; 25: 5327-5335, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31317882

RESUMO

BACKGROUND Previous studies of human and animal models indicate that inflammation alters lipid metabolism. The pro-protein convertase subtilisin kexin type 9 (PCSK9) plays an important role in lipid metabolism. MATERIAL AND METHODS We examined the effect of inflammation on PCSK9 expression and lipid deposition in the kidneys of mice with Adriamycin-induced nephropathy. RESULTS The results indicated an increased expression of inflammatory cytokines and lipid deposition over 12 weeks. During this time, the expression of PCSK9 and its transcriptional activator (hepatocyte nuclear factor 1alpha, HNF1alpha) decreased, and the expression of the low-density lipoprotein receptor (LDLR) and its transcriptional activator (sterol regulatory element binding protein-2, SREBP-2) increased. Exogenous inflammation appeared to further aggravate this process. CONCLUSIONS Our mouse model of nephropathy suggests that a key step in the inflammation-induced deposition of lipids in the kidneys is the downregulation renal PCSK9 expression.


Assuntos
Doxorrubicina/toxicidade , Metabolismo dos Lipídeos/efeitos dos fármacos , Síndrome Nefrótica/induzido quimicamente , Síndrome Nefrótica/metabolismo , Pró-Proteína Convertase 9/metabolismo , Animais , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Doxorrubicina/farmacologia , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Inflamação/metabolismo , Rim/metabolismo , Nefropatias , Lipídeos/fisiologia , Lipoproteínas LDL/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nefrite/induzido quimicamente , Nefrite/metabolismo , Pró-Proteína Convertase 9/biossíntese , Pró-Proteína Convertases/metabolismo , Pró-Proteína Convertases/farmacologia , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo
4.
Nature ; 571(7764): 265-269, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31207605

RESUMO

Cytotoxic T cells are essential mediators of protective immunity to viral infection and malignant tumours and are a key target of immunotherapy approaches. However, prolonged exposure to cognate antigens often attenuates the effector capacity of T cells and limits their therapeutic potential1-4. This process, known as T cell exhaustion or dysfunction1, is manifested by epigenetically enforced changes in gene regulation that reduce the expression of cytokines and effector molecules and upregulate the expression of inhibitory receptors such as programmed cell-death 1 (PD-1)5-8. The underlying molecular mechanisms that induce and stabilize the phenotypic and functional features of exhausted T cells remain poorly understood9-12. Here we report that the development and maintenance of populations of exhausted T cells in mice requires the thymocyte selection-associated high mobility group box (TOX) protein13-15. TOX is induced by high antigen stimulation of the T cell receptor and correlates with the presence of an exhausted phenotype during chronic infections with lymphocytic choriomeningitis virus in mice and hepatitis C virus in humans. Removal of its DNA-binding domain reduces the expression of PD-1 at the mRNA and protein level, augments the production of cytokines and results in a more polyfunctional T cell phenotype. T cells with this deletion initially mediate increased effector function and cause more severe immunopathology, but ultimately undergo a massive decline in their quantity, notably among the subset of TCF-1+ self-renewing T cells. Altogether, we show that TOX is a critical factor for the normal progression of T cell dysfunction and the maintenance of exhausted T cells during chronic infection, and provide a link between the suppression of effector function intrinsic to CD8 T cells and protection against immunopathology.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Hepatite C Crônica/imunologia , Hepatite C Crônica/virologia , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Homeodomínio/metabolismo , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Animais , Proliferação de Células , Doença Crônica , Citocinas/imunologia , Citocinas/metabolismo , Epigênese Genética , Feminino , Regulação da Expressão Gênica/imunologia , Hepacivirus/imunologia , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Memória Imunológica , Vírus da Coriomeningite Linfocítica/imunologia , Masculino , Camundongos , Fenótipo , Timócitos/citologia , Timócitos/imunologia , Transcrição Genética
5.
J Diabetes Res ; 2019: 5483946, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31223625

RESUMO

This study is aimed at evaluating the effects, functions, and mechanism of HNF1α on hepatic glycolipid metabolism. In this study, free fatty acid- (FFA-) induced steatosis of hepatocyte liver cell LO2 was used as an in vitro model. The methods of Oil Red O staining, RT-qPCR, western blot, and immunofluorescence staining were used to detect LO2-regulated HNF1α expression and its effects on FFA-induced LO2 cell steatosis, the insulin signaling and SOCS-3-STAT3 signaling pathways, the expression of lipid metabolism-related regulators, and phosphorylation. With increased FFA induction time, the expression of HNF1α in the LO2 fatty degeneration hepatic cells gradually decreased. Downregulation of HNF1α expression aggravated FFA-induced steatosis of LO2 hepatocytes. HNF1α promotes activation of the insulin pathway and oxidative breakdown of fat and inhibits lipid anabolism. Inhibitors of STAT3 can reverse the regulation of decreased HNF1α expression on the insulin signaling pathway and fat metabolism. We also confirmed this pathway using HNF1α-/- mice combining treatment with STAT3 inhibitor NSC 74859 in vivo. HNF1α regulates hepatic lipid metabolism by promoting the expression of SOCS-3 and negatively regulating the STAT3 signaling pathway.


Assuntos
Fator 1-alfa Nuclear de Hepatócito/metabolismo , Resistência à Insulina , Metabolismo dos Lipídeos , Fígado/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Animais , Regulação da Expressão Gênica , Glicolipídeos/metabolismo , Hepatócitos/citologia , Humanos , Insulina/metabolismo , Lipídeos/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Fosforilação , Transdução de Sinais
6.
PLoS One ; 14(5): e0217110, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31145732

RESUMO

Maturity-onset diabetes of the young (MODY) is a heterozygous monogenic diabetes; more than 14 disease genes have been identified. However, the pathogenesis of MODY is not fully understood because the patients' pancreatic beta cells are inaccessible. To elucidate the pathology of MODY, we established MODY3 patient-derived iPS (MODY3-iPS) cells using non-integrating Sendai virus (SeV) vector and examined the mutant mRNA and protein of HNF1A (Hepatocyte Nuclear factor 1A) after pancreatic lineage differentiation. Our patient had a cytosine insertion in the HNF1A gene (P291fsinsC) causing frameshift and making a premature termination codon (PTC). We confirmed these MODY3-iPS cells possessed the characteristics of pluripotent stem cells. After we differentiated them into pancreatic beta cells, transcripts of HNF1A gene were cloned and sequenced. We found that P291fsinsC mutant transcripts were much less frequent than wild ones, but they increased after adding cycloheximide (CHX) to the medium. These results suggested that mutant mRNA was destroyed by nonsense-mediated mRNA decay (NMD). Moreover, we were not able to detect any band of mutant proteins in pancreatic lineage cells which were differentiated from MODY3-iPSCs by western blot (WB) analysis. A scarcity of the truncated form of mutant protein may indicate that MODY3 might be caused by a haplo-insufficiency effect rather than a dominant negative manner.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Células Secretoras de Insulina/patologia , Mutação , RNA Mensageiro/genética , Células Cultivadas , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Feminino , Haploinsuficiência , Fator 1-alfa Nuclear de Hepatócito/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Secretoras de Insulina/metabolismo , RNA Mensageiro/metabolismo
7.
Cell Mol Life Sci ; 76(19): 3891-3898, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31053884

RESUMO

Statins are potent lipid-lowering drugs. Large prospective clinical trials have shown the anti-thrombotic effect of statins, e.g., preventing deep vein thrombosis. However, the mechanism underlying the beneficial effect of statins in reducing thrombus formation remains to be established. We, thus, conduct this study to investigate the potential molecular mechanisms. The cultured human hepatoma cells (HepG2) were used as the in vitro model. The human protein C gene promoter was cloned into the luciferase reporter to study the transcriptional regulation of human protein C gene. Wistar rats fed with simvastatin (5 mg/kg day) were used as the in vivo model. We found that simvastatin increased the expression of protein C in hepatocytes (361 ± 64% and 313 ± 59% after 2 h and 6 h of stimulation, respectively, both p < 0.01). In the animal study, the serum protein C levels were increased in the simvastatin-treated group (7 ± 2.2 unit/ml vs 23.4 ± 19.3 unit/ml and 23.4 ± 18.2 unit/ml and 1 and 2 weeks of treatment, respectively, both p < 0.05). Regarding the possible molecular mechanism, we found that the level of hepatocyte nuclear factor 1α (HNF1α) was also increased in both the in vivo and in vitro models. We found that the protein C promoter activity was increased by simvastatin, and this effect was inhibited by HNF1α knockdown and constitutively active Rac1. Therefore, stains may modulate protein C expression through small GTPase Rac 1 and HNF1α.


Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Proteína C/genética , Animais , Células Hep G2 , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteína C/metabolismo , Ratos Wistar , Sinvastatina/farmacologia , Transcrição Genética/efeitos dos fármacos , Proteínas rac1 de Ligação ao GTP/genética
8.
EBioMedicine ; 44: 403-418, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31103629

RESUMO

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with poor prognosis, and gemcitabine-based chemotherapy remains an effective option for the majority of PDAC patients. Hepatocyte nuclear factor 1α (HNF1A) is a tumor-suppressor in PDAC, but its role in gemcitabine chemoresistance of PDAC has not been clarified. METHODS: The function of HNF1A in gemcitabine was detected by overexpression and knockdown of HNF1A in vitro and in vitro. The regulatory network between HNF1A and ABCB1 was further demonstrated by luciferase assays, deletion/mutation reporter construct assays and CHIP assays. FINDINGS: Here, we found that HNF1A expression is significantly associated with gemcitabine sensitivity in PDAC cell lines. Moreover, we identified that HNF1A overexpression enhanced gemcitabine sensitivity of PDAC both in vitro and in vitro, while inhibition of HNF1A had the opposite effect. Furthermore, by inhibiting and overexpressing HNF1A, we revealed that HNF1A regulates the expression of MDR genes (ABCB1 and ABCC1) in PDAC cells. Mechanistically, we demonstrated that HNF1A regulates ABCB1 expression through binding to its specific promoter region and suppressing its transcription levels. Finally, the survival analyses revealed the clinical value of HNF1A in stratification of gemcitabine sensitive pancreatic cancer patients. INTERPRETATION: Our study paved the road for finding novel treatment combinations using conventional cytotoxic agents with functional restoration of the HNF1A protein, individualized treatment through HNF1A staining and improvement of the prognosis of PDAC patients. FUND: National Natural Science Foundations of China and National Natural Science Foundation of Guangdong Province.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Fator 1-alfa Nuclear de Hepatócito/antagonistas & inibidores , Neoplasias Pancreáticas/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Adulto , Idoso , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Desoxicitidina/farmacologia , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Expressão Gênica , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Prognóstico , Regiões Promotoras Genéticas , Ligação Proteica
9.
Acta Diabetol ; 56(8): 883-888, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30963309

RESUMO

AIMS: HNF1A is a gene coding for the transcription factor HNF1-α, mutated in some forms of MODY and type 2 diabetes mellitus characterized by a strong genetic component. The penetrance of HNF1A variants differs considerably; thus, to assess the genetic risk of diabetes in carrier subjects of a HNF1A mutant allele, a functional characterization of mutant forms is of paramount importance. METHODS: The HNF1A gene was sequenced in two patients with partly discordant diabetic phenotype, carrying the p.Pro409His variant. To evaluate the pathogenicity of the variant, we measured the transactivation power of the corresponding P408H HNF1-α mutant mouse form on HNF1-α target promoters. RESULTS: We found a lower but detectable activity of transactivation of the mutant form compared with the wild-type form and we excluded mechanisms of protein degradation or nuclear mislocalization. CONCLUSIONS: The HNF1A mutation p.Pro409His can be considered a mild variant that confers a moderate risk of type 2 diabetes mellitus in heterozygous carriers.


Assuntos
Diabetes Mellitus Tipo 2/genética , Fator 1-alfa Nuclear de Hepatócito/genética , Células Secretoras de Insulina/metabolismo , Mutação de Sentido Incorreto , Adulto , Animais , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Células HeLa , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Heterozigoto , Humanos , Camundongos , Fenótipo
10.
Immunity ; 50(1): 181-194.e6, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30635236

RESUMO

An improved understanding of the anti-tumor CD8+ T cell response after checkpoint blockade would enable more informed and effective therapeutic strategies. Here we examined the dynamics of the effector response of CD8+ tumor-infiltrating lymphocytes (TILs) after checkpoint blockade therapy. Bulk and single-cell RNA profiles of CD8+ TILs after combined Tim-3+PD-1 blockade in preclinical models revealed significant changes in the transcriptional profile of PD-1- TILs. These cells could be divided into subsets bearing characterstics of naive-, effector-, and memory-precursor-like cells. Effector- and memory-precursor-like TILs contained tumor-antigen-specific cells, exhibited proliferative and effector capacity, and expanded in response to different checkpoint blockade therapies across different tumor models. The memory-precursor-like subset shared features with CD8+ T cells associated with response to checkpoint blockade in patients and was compromised in the absence of Tcf7. Expression of Tcf7/Tcf1 was requisite for the efficacy of diverse immunotherapies, highlighting the importance of this transcriptional regulator in the development of effective CD8+ T cell responses upon immunotherapy.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Experimentais/terapia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Subpopulações de Linfócitos T/imunologia , Animais , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Proliferação de Células , Receptor Celular 2 do Vírus da Hepatite A/antagonistas & inibidores , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Memória Imunológica/genética , Imunoterapia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/imunologia , Transcriptoma
11.
Immunity ; 50(1): 195-211.e10, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30635237

RESUMO

Checkpoint blockade mediates a proliferative response of tumor-infiltrating CD8+ T lymphocytes (TILs). The origin of this response has remained elusive because chronic activation promotes terminal differentiation or exhaustion of tumor-specific T cells. Here we identified a subset of tumor-reactive TILs bearing hallmarks of exhausted cells and central memory cells, including expression of the checkpoint protein PD-1 and the transcription factor Tcf1. Tcf1+PD-1+ TILs mediated the proliferative response to immunotherapy, generating both Tcf1+PD-1+ and differentiated Tcf1-PD-1+ cells. Ablation of Tcf1+PD-1+ TILs restricted responses to immunotherapy. Tcf1 was not required for the generation of Tcf1+PD-1+ TILs but was essential for the stem-like functions of these cells. Human TCF1+PD-1+ cells were detected among tumor-reactive CD8+ T cells in the blood of melanoma patients and among TILs of primary melanomas. Thus, immune checkpoint blockade relies not on reversal of T cell exhaustion programs, but on the proliferation of a stem-like TIL subset.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Melanoma/terapia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Células-Tronco/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Diferenciação Celular , Proliferação de Células , Receptor Celular 2 do Vírus da Hepatite A/antagonistas & inibidores , Fator 1-alfa Nuclear de Hepatócito/genética , Humanos , Imunoterapia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Melanoma/imunologia , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL
12.
Methods Mol Biol ; 1905: 93-101, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30536093

RESUMO

Primary hepatocytes are widely used in regenerative medicine, drug metabolism analysis, and in vitro drug screens. To overcome the shortage of liver donors, several strategies, such as differentiation of pluripotent stem cells and transdifferentiation from somatic cells, were developed to generate hepatocytes from alternative sources. Here, we describe in detail lenti-virus-based procedure for direct conversion of human fibroblasts to hepatocytes (hiHep cells) in vitro. A detailed protocol for preparation of human fibroblasts from scar tissues is also provided. Based on this protocol, FOXA3, HNF1A, and HNF4A are introduced into SV40-large-T-antigen-expressing human scar fibroblasts by lenti-virus. It usually takes about 5-7 days to get epithelial hiHep colonies. SV40-large-T-antigen-expressing hiHep (hiHepLT) cells are proliferative and can be expanded to a large number for potential uses.


Assuntos
Técnicas de Cultura de Células/métodos , Cicatriz/patologia , Fibroblastos/citologia , Hepatócitos/citologia , Lentivirus/genética , Antígenos Transformantes de Poliomavirus/genética , Linhagem da Célula , Proliferação de Células , Transdiferenciação Celular , Reprogramação Celular , Cicatriz/genética , Cicatriz/metabolismo , Fibroblastos/metabolismo , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Fator 3-gama Nuclear de Hepatócito/genética , Fator 3-gama Nuclear de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/metabolismo , Humanos , Vírus 40 dos Símios/genética
13.
J Cell Biochem ; 120(1): 519-532, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30191603

RESUMO

Hepatocyte nuclear factor 4α (HNF4α) is a master regulator of development and function of digestive tissues. The HNF4A gene uses two separate promoters P1 and P2, with P1 products predominant in adult liver, whereas P2 products prevalent in fetal liver, pancreas, and liver/colon cancer. To date, the mechanisms for the regulation of HNF4A and the dynamic switch of P1-HNF4α and P2-HNF4α during ontogenesis and carcinogenesis are still obscure. Our study validated the previously reported self-stimulation of P1-HNF4α but invalidated the reported synergism between HNF4α and HNF1α. HNF4A-AS1, a long noncoding RNA, is localized between the P2 and P1 promoters of HNF4A. We identified critical roles of P1-HNF4α in regulating the expression of HNF4A-AS1 and its mouse ortholog Hnf4a-os. Paired box 6 (PAX6), a master regulator of pancreas development overexpressed in colon cancer, cooperated with HNF1α to induce P2-HNF4α but antagonized HNF4α in HNF4A-AS1 expression. Thus, PAX6 may be important in determining ontogenic and carcinogenic changes of P2-HNF4α and HNF4A-AS1 in the pancreas and intestine. We also interrogated transactivation activities on multiple gene targets by multiple known and novel HNF4α mutants identified in patients with maturity onset diabetes of the young 1 (MODY1) and liver cancer. Particularly, HNF4α-D78A and HNF4α-G79S, two mutants found in liver cancer with mutations in DNA-binding domain, displayed highly gene-specific transactivation activities. Interestingly, HNF4α-Q277X, a MODY1 truncation mutant, antagonized the transactivation activities of HNF1α and farnesoid X receptor, key regulators of insulin secretion. Taken together, our study provides novel mechanistic insights regarding the transcriptional regulation and transactivation activity of HNF4α in digestive tissues.


Assuntos
Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Transcrição Genética , Ativação Transcricional/genética , Regiões 5' não Traduzidas/genética , Diabetes Mellitus Tipo 2/genética , Éxons/genética , Células HEK293 , Células Hep G2 , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Íntrons/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Mutação , Fator de Transcrição PAX6/genética , Fator de Transcrição PAX6/metabolismo , Regiões Promotoras Genéticas , RNA Longo não Codificante/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Transfecção
14.
Oncol Res ; 27(7): 739-750, 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-30180922

RESUMO

The activated form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], regulates numerous cellular processes, including inhibition of cancer progression. IL-1ß has been reported to facilitate cancer development, especially by inducing an epithelial-to-mesenchymal transition (EMT) in several malignant tumors. However, the underlying mechanism of 1,25(OH)2D3 and IL-1ß in colorectal cancer (CRC) still remains largely unknown. To fill in this knowledge gap, we measured cell proliferation and invasion by CCK-8 and Transwell assays after stimulation with 1,25(OH)2D3 and IL-1ß. E-cadherin and vimentin were chosen as markers of EMT measured by immunofluorescence, quantitative real-time PCR (qRT-PCR), and Western blot. The expression and function of the vitamin D receptor (VDR) was evaluated by Western blot and luciferase reporter assay. qRT-PCR and RNA-FISH were performed to detect the expression and location of lncTCF7 in vitro. The binding sites of VDR in the lncTCF7 promoter were confirmed by a chromatin immunoprecipitation assay. Based on the above experiments, we found that 1,25(OH)2D3 attenuates IL-1ß-induced increased proliferation and invasion in colorectal cancer through enhancing VDR, which inhibits the expression of lncTCF7 by directly binding to its promoter region.


Assuntos
Fator 1-alfa Nuclear de Hepatócito/metabolismo , Interleucina-1beta/metabolismo , Vitamina D/análogos & derivados , Animais , Proliferação de Células , Transição Epitelial-Mesenquimal , Humanos , Masculino , Camundongos , Camundongos Nus , Vitamina D/farmacologia , Vitamina D/uso terapêutico
15.
Nat Commun ; 9(1): 5452, 2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30575739

RESUMO

Ezh2 is an histone methyltransferase (HMT) that catalyzes H3K27me3 and functions in TH1, TH2, and Treg cells primarily via HMT activity. Here we show that Ezh2 ablation impairs T follicular helper (TFH) cell differentiation and activation of the TFH transcription program. In TFH cells, most Ezh2-occupied genomic sites, including the Bcl6 promoter, are associated with H3K27ac rather than H3K27me3. Mechanistically, Ezh2 is recruited by Tcf1 to directly activate Bcl6 transcription, with this function requiring Ezh2 phosphorylation at Ser21. Meanwhile, Ezh2 deploys H3K27me3 to repress Cdkn2a expression in TFH cells, where aberrantly upregulated p19Arf, a Cdkn2a protein product, triggers TFH cell apoptosis and antagonizes Bcl6 function via protein-protein interaction. Either forced expression of Bcl6 or genetic ablation of p19Arf in Ezh2-deficient cells improves TFH cell differentiation and helper function. Thus, Ezh2 orchestrates TFH-lineage specification and function maturation by integrating phosphorylation-dependent transcriptional activation and HMT-dependent gene repression.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/fisiologia , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Linfócitos T Auxiliares-Indutores/fisiologia , Animais , Diferenciação Celular , Sobrevivência Celular , Epigênese Genética , Regulação da Expressão Gênica , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação , Viroses/imunologia
16.
Int J Mol Sci ; 19(12)2018 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-30477246

RESUMO

Wingless-type mouse mammary tumor virus (MMTV) integration site (Wnt) signaling is one of the most critical pathways in developing and adult tissues. In the brain, Wnt signaling contributes to different neurodevelopmental aspects ranging from differentiation to axonal extension, synapse formation, neurogenesis, and neuroprotection. Canonical Wnt signaling is mediated mainly by the multifunctional ß-catenin protein which is a potent co-activator of transcription factors such as lymphoid enhancer factor (LEF) and T-cell factor (TCF). Accumulating evidence points to dysregulation of Wnt/ß-catenin signaling in major neurodegenerative disorders. This review highlights a Wnt/ß-catenin/glial connection in Parkinson's disease (PD), the most common movement disorder characterized by the selective death of midbrain dopaminergic (mDAergic) neuronal cell bodies in the subtantia nigra pars compacta (SNpc) and gliosis. Major findings of the last decade document that Wnt/ß-catenin signaling in partnership with glial cells is critically involved in each step and at every level in the regulation of nigrostriatal DAergic neuronal health, protection, and regeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD, focusing on Wnt/ß-catenin signaling to boost a full neurorestorative program in PD.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Neurogênese/genética , Transtornos Parkinsonianos/genética , Regeneração/genética , Transdução de Sinais/genética , Proteínas Wnt/genética , beta Catenina/genética , Animais , Sobrevivência Celular , Terapia Baseada em Transplante de Células e Tecidos/métodos , Modelos Animais de Doenças , Neurônios Dopaminérgicos/patologia , Regulação da Expressão Gênica , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Camundongos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neuroglia/metabolismo , Neuroglia/patologia , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologia , Transtornos Parkinsonianos/terapia , Parte Compacta da Substância Negra/metabolismo , Parte Compacta da Substância Negra/patologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
17.
Cell Physiol Biochem ; 50(1): 261-276, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30282072

RESUMO

BACKGROUND/AIMS: Treatment options for metastatic castrate-resistant prostate cancer (mCRPC) are limited and typically centered on paclitaxel-based chemotherapy. In this study, we aimed to evaluate whether miR-34a attenuates chemoresistance to paclitaxel by regulating target genes associated with drug resistance. METHODS: We used data from The Cancer Genome Atlas to compare miR-34a expression levels in prostate cancer (PC) tissues with normal prostate tissues. The effects of miR-34a inhibition and overexpression on PC proliferation were evaluated in vitro via Cell Counting Kit-8 (CCK-8) proliferation, colony formation, apoptosis, and cell-cycle assays. A luciferase reporter assay was employed to identify the interactions between miR-34a and specific target genes. To determine the effects of up-regulation of miR-34a on tumor growth and chemo-resistance in vivo, we injected PC cells overexpressing miR-34a into nude mice subcutaneously and evaluated the rate of tumor growth during paclitaxel treatment. We examined changes in the expression levels of miR-34a target genes JAG1 and Notch1 and their downstream genes via miR-34a transfection by quantitative reverse transcription PCR (qRT-PCR) and western blot assay. RESULTS: miR-34a served as an independent predictor of reduced patient survival. MiR-34a was down-regulated in PC-3PR cells compared with PC-3 cells. The CCK-8 assay showed that miR-34a overexpression resulted in increased sensitivity to paclitaxel while miR-34a down-regulation resulted in chemoresistance to paclitaxel in vitro. A study of gain and loss in a series of functional assays revealed that PC cells expressing miR-34a were chemosensitive. Furthermore, the overexpression of miR-34a increased the sensitivity of PC-3PR cells to chemotherapy in vivo. The luciferase reporter assay confirmed that JAG1 and Notch1 were directly targeted by miR-34a. Interestingly, western blot analysis and qRT-PCR confirmed that miR-34a inhibited the Notch1 signaling pathway. We found that miR-34a increased the chemosensitivity of PC-3PR cells by directly repressing the TCF1/ LEF1 axis. CONCLUSION: Our results showed that miR-34a is involved in the development of chemosensitivity to paclitaxel. By regulating the JAG1/Notch1 axis, miR-34a or its target genes JAG1 or Notch1 might serve as potential predictive biomarkers of response to paclitaxel-based chemotherapy and/or therapeutic targets that will help to overcome chemoresistance at the mCRPC stage.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Proteína Jagged-1/metabolismo , MicroRNAs/metabolismo , Paclitaxel/farmacologia , Neoplasias da Próstata/patologia , Receptor Notch1/metabolismo , Regiões 3' não Traduzidas , Animais , Antagomirs/metabolismo , Antagomirs/uso terapêutico , Antineoplásicos Fitogênicos/uso terapêutico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Proteína Jagged-1/genética , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Paclitaxel/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/mortalidade , Receptor Notch1/genética , Transdução de Sinais/efeitos dos fármacos
18.
Diabetologia ; 61(12): 2520-2527, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30229274

RESUMO

AIMS/HYPOTHESIS: Treatment change following a genetic diagnosis of MODY is frequently indicated, but little is known about the factors predicting future treatment success. We therefore conducted the first prospective study to determine the impact of a genetic diagnosis on individuals with GCK-, HNF1A- or HNF4A-MODY in the UK, and to identify clinical characteristics predicting treatment success (i.e. HbA1c ≤58 mmol/mol [≤7.5%]) with the recommended treatment at 2 years. METHODS: This was an observational, prospective, non-selective study of individuals referred to the Exeter Molecular Genetic Laboratory for genetic testing from December 2010 to December 2012. Individuals from the UK with GCK- or HNF1A/HNF4A-MODY who were not on recommended treatment at the time of genetic diagnosis, and who were diagnosed below the age of 30 years and were currently aged less than 50 years, were eligible to participate. RESULTS: A total of 44 of 58 individuals (75.9%) changed treatment following their genetic diagnosis. Eight individuals diagnosed with GCK-MODY stopped all diabetes medication without experiencing any change in HbA1c (49.5 mmol/mol [6.6%] both before the genetic diagnosis and at a median of 1.25 years' follow-up without treatment, p = 0.88). A total of 36 of 49 individuals (73.5%) diagnosed with HNF1A/HNF4A-MODY changed treatment; however, of the 21 of these individuals who were being managed with diet or sulfonylurea alone at 2 years, only 13 (36.1% of the population that changed treatment) had an HbA1c ≤58 mmol/mol (≤7.5%). These individuals had a shorter diabetes duration (median 4.6 vs 18.1 years), lower HbA1c (58 vs 73 mmol/mol [7.5% vs 8.8%]) and lower BMI (median 24.2 vs 26.0 kg/m2) at the time of genetic diagnosis, compared with individuals (n = 23/36) with an HbA1c >58 mmol/mol (>7.5%) (or <58 mmol/mol [<7.5%] on additional treatment) at the 2 year follow-up. Overall, 64% (7/11) individuals with a diabetes duration of ≤11 years and an HbA1c of ≤69 mmol/mol (≤8.5%) at time of the genetic test achieved good glycaemic control (HbA1c ≤58 mmol/mol [≤7.5%]) with diet or sulfonylurea alone at 2 years, compared with no participants with a diabetes duration of >11 years and an HbA1c of >69 mmol/mol (>8.5%) at the time of genetic diagnosis. CONCLUSIONS/INTERPRETATION: In participants with GCK-MODY, treatment cessation was universally successful, with no change in HbA1c at follow-up. In those with HNF1A/HNF4A-MODY, a shorter diabetes duration, lower HbA1c and lower BMI at genetic diagnosis predicted successful treatment with sulfonylurea/diet alone, supporting the need for early genetic diagnosis and treatment change. Our study suggests that, in individuals with HNF1A/HNF4A-MODY with a longer duration of diabetes (>11 years) at time of genetic test, rather than ceasing current treatment, a sulfonylurea should be added to existing therapy, particularly in those who are overweight or obese and have a high HbA1c.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Insulina/uso terapêutico , Metformina/uso terapêutico , Adolescente , Adulto , Glicemia/efeitos dos fármacos , Diabetes Mellitus Tipo 2/sangue , Feminino , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Hipoglicemiantes/uso terapêutico , Masculino , Estudos Prospectivos , Adulto Jovem
19.
Sci Rep ; 8(1): 12780, 2018 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-30143652

RESUMO

The transcription factor hepatocyte nuclear factor-1α (HNF-1A) is involved in normal pancreas development and function. Rare variants in the HNF1A gene can cause monogenic diabetes, while common variants confer type 2 diabetes risk. The precise mechanisms for regulation of HNF-1A, including the role and function of post-translational modifications, are still largely unknown. Here, we present the first evidence for HNF-1A being a substrate of SUMOylation in cellulo and identify two lysine (K) residues (K205 and K273) as SUMOylation sites. Overexpression of protein inhibitor of activated STAT (PIASγ) represses the transcriptional activity of HNF-1A and is dependent on simultaneous HNF-1A SUMOylation at K205 and K273. Moreover, PIASγ is a novel HNF-1A interaction partner whose expression leads to translocation of HNF-1A to the nuclear periphery. Thus, our findings support that the E3 SUMO ligase PIASγ regulates HNF-1A SUMOylation with functional implications, representing new targets for drug development and precision medicine in diabetes.


Assuntos
Diabetes Mellitus/metabolismo , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citosol/metabolismo , DNA/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Fator 1-alfa Nuclear de Hepatócito/química , Fator 1-alfa Nuclear de Hepatócito/genética , Humanos , Lisina/metabolismo , Camundongos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Ratos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Ativação Transcricional/genética
20.
Cell Physiol Biochem ; 48(1): 87-98, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30001529

RESUMO

BACKGROUND/AIMS: Chemoresistance is largely responsible for relapses of bladder cancer during clinical therapy. However, the molecular mechanisms involved in the chemoresistance of bladder cancer are unclear. Growing evidence supports the theory that microRNAs (miRNAs) play an important role in chemotherapeutic drug resistance because they are downregulated in many malignancies that have been implicated in the regulation of diverse processes in cancer cells. More specifically, the extent and precise mechanism of the involvement of miR-34as in chemoresistance to epirubicin (EPI) in the treatment of bladder cancer remains unclear. METHODS: In this study, real-time quantitative polymerase chain reaction (PCR) was used to analyze the expression of miR-34a in bladder cancer cell line BIU87 and its EPI chemoresistant cell line BIU87/ADR. The miR-34a profiles in bladder cancer tissues were obtained from The Cancer Genome Atlas database. The effect of miR-34a on chemosensitivity was evaluated by cell viability assays, colony formation assays, and in vivo experimentation. Apoptosis and the cell cycle were examined by flow cytometry. A luciferase reporter assay was used to assess the target genes of miR-34a. Western blot and qPCR were used to analyze the expression of target proteins and downstream molecules. RESULTS: The downregulation of miR-34a in bladder cancer serves as an independent predictor of reduced patient survival. The CCK-8 assay showed that miR-34a overexpression resulted in increased sensitivity to EPI, while miR-34a downregulation resulted in chemoresistance to EPI in vitro. Moreover, it was found that miR-34a increased the sensitivity of BIU87/ADR cells to chemotherapy in vivo. The luciferase reporter assay ascertained that TCF1 and LEF1 are direct target genes of miR-34a. It was found that miR-34a increased chemosensitivity in BIU87/ADR cells by inhibiting the TCF1/LEF1 axis. CONCLUSIONS: The results of this study indicate that miR-34a contributes to the chemosensitivity of BIU87/ADR by inhibiting the TCF1/LEF1 axis. Consequently, miR-34a is a determinant of BIU87 chemosensitivity and may therefore serve as a potential therapeutic target in bladder cancer treatment.


Assuntos
Fator 1-alfa Nuclear de Hepatócito/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , MicroRNAs/metabolismo , Neoplasias da Bexiga Urinária/patologia , Regiões 3' não Traduzidas , Animais , Antagomirs/metabolismo , Apoptose , Linhagem Celular Tumoral , Bases de Dados Genéticas , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Epirubicina/farmacologia , Pontos de Checagem da Fase G1 do Ciclo Celular , Fator 1-alfa Nuclear de Hepatócito/química , Fator 1-alfa Nuclear de Hepatócito/genética , Humanos , Estimativa de Kaplan-Meier , Fator 1 de Ligação ao Facilitador Linfoide/química , Fator 1 de Ligação ao Facilitador Linfoide/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Metástase Neoplásica , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/mortalidade
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