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1.
Stem Cell Res Ther ; 10(1): 40, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30670081

RESUMO

BACKGROUND: Sertoli cells are the most important somatic cells contributing to the microenvironment (named niche) for spermatogonial stem cells (SSCs). They produce amounts of crucial growth factors and structure proteins that play essential roles in the complex processes of male SSCs survival, proliferation, and differentiation. It has been suggested that Sertoli cell abnormalities could result in spermatogenesis failure, eventually causing azoospermia in humans. However, to the end, the gene expression characteristics and protein functions of human Sertoli cells remained unknown. In this study, we aimed to evaluate the effect of fibroblast growth factor-5 (FGF5), a novel growth factor downregulated in Sertoli cells from Sertoli cell-only syndrome (SCOS) patients compared to Sertoli cells from obstructive azoospermia (OA) patients, on SSCs. METHODS: We compared the transcriptome between Sertoli cell from SCOS and OA patients. Then, we evaluated the expression of FGF5, a growth factor which is downregulated in SCOS Sertoli cells, in human primary cultured Sertoli cells and testicular tissue. Also, the proliferation effect of FGF5 in mice SSCs was detected using EDU assay and CCK-8 assay. To investigate the mechanism of FGF5, Phospho Explorer Array was performed. And the results were verified using Western blot assay. RESULTS: Using RNA-Seq, we found 308 differentially expressed genes (DEGs) between Sertoli cells from SCOS and OA patients. We noted and verified that the expression of fibroblast growth factor-5 (FGF5) was higher in Sertoli cells of OA patients than that of SCOS patients at both transcriptional and translational levels. Proliferation assays showed that rFGF5 enhanced the proliferation of mouse SSCs line C18-4 in a time- and dose-dependent manner. Moreover, we demonstrated that ERK and AKT were activated and the expression of Cyclin A2 and Cyclin E1 was enhanced by rFGF5. CONCLUSION: The distinct RNA profiles between Sertoli cells from SCOS and OA patients were identified using RNA-Seq. Also, FGF5, a growth factor that downregulated in SCOS Sertoli cells, could promote SSCs proliferation via ERK and AKT activation.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator 5 de Crescimento de Fibroblastos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células de Sertoli/fisiologia , Espermatogônias/metabolismo , Adulto , Animais , Azoospermia/genética , Azoospermia/metabolismo , Azoospermia/patologia , Proliferação de Células/fisiologia , Ativação Enzimática , Fator 5 de Crescimento de Fibroblastos/biossíntese , Fator 5 de Crescimento de Fibroblastos/genética , Humanos , Masculino , Camundongos , Proteínas Recombinantes/farmacologia , Síndrome de Células de Sertoli/genética , Síndrome de Células de Sertoli/metabolismo , Síndrome de Células de Sertoli/patologia , Células de Sertoli/metabolismo , Células de Sertoli/patologia , Espermatogônias/citologia , Transcriptoma
2.
J Hepatol ; 63(4): 874-85, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25998163

RESUMO

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. However, the detailed molecular mechanisms underlying HCC progression are still not completely clear. Given the crucial role of microRNAs (miRNAs) in cancer metastasis, we aimed to analyze the expression and function of a metastasis-associated miRNA named miR-188-5p in HCC. METHODS: miRNA array analysis was performed to search for metastasis-associated miRNAs in HCC. miR-188-5p expressions in tumor tissues and adjacent non-tumorous liver tissues of HCC patients and cell lines were evaluated by real-time PCR. The protein expression levels were analyzed by Western blot and immunohistochemistry. Luciferase reporter assays was used to validate the target of miR-188-5p. The effect of miR-188-5p on HCC progression was studied in vitro and in vivo. RESULTS: miR-188-5p was significantly decreased in HCC and its expression levels were highly correlated with multiple nodules, microvascular invasion, overall and disease-free survival of HCC. Ectopic expression of miR-188-5p suppressed HCC cell proliferation and metastasis in vitro and in vivo. Fibroblast growth factor 5 (FGF5) was identified as a major target of miR-188-5p. Enforced expression of miR-188-5p inhibited the expression of FGF5 significantly and the restoration of FGF5 expression reversed the inhibitory effects of miR-188-5p on HCC cell proliferation and metastasis. CONCLUSIONS: These findings collectively demonstrate a tumor suppressor role of miR-188-5p in HCC progression via targeting FGF5, suggesting that miR-188-5p could serve as a potential prognostic biomarker and therapeutic target for HCC.


Assuntos
Carcinoma Hepatocelular/genética , Fator 5 de Crescimento de Fibroblastos/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , MicroRNAs/genética , RNA Neoplásico/genética , Western Blotting , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/secundário , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Feminino , Fator 5 de Crescimento de Fibroblastos/biossíntese , Seguimentos , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , MicroRNAs/biossíntese , Pessoa de Meia-Idade , Metástase Neoplásica , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos
3.
FASEB J ; 25(4): 1208-17, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21183594

RESUMO

Shear stress is a ubiquitous environmental cue experienced by stem cells when they are being differentiated or expanded in perfusion cultures. However, its role in modulating self-renewing stem cell phenotypes is unclear, since shear is usually only studied in the context of cardiovascular differentiation. We used a multiplex microfluidic array, which overcomes the limitations of macroperfusion systems in shear application throughput and precision, to initiate a comprehensive, quantitative study of shear effects on self-renewing mouse embryonic stem cells (mESCs), where shear stresses varying by >1000 times (0.016-16 dyn/cm(2)) are applied simultaneously. When compared with static controls in the presence or absence of a saturated soluble environment (i.e., mESC-conditioned medium), we ascertained that flow-induced shear stress specifically up-regulates the epiblast marker Fgf5. Epiblast-state transition in mESCs involves heparan sulfate proteoglycans (HSPGs), which have also been shown to transduce shear stress in endothelial cells. By disrupting (with sulfation inhibitors and heparinase) and partially reconstituting (with heparin) HSPG function, we show that mESCs also mechanically sense shear stress via HSPGs to modulate Fgf5 expression. This study demonstrates that self-renewing mESCs possess the molecular machinery to sense shear stress and provides quantitative shear application benchmarks for future scalable stem cell culture systems.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Proteoglicanas de Heparan Sulfato/metabolismo , Estresse Mecânico , Animais , Células Cultivadas , Células-Tronco Embrionárias/fisiologia , Fator 5 de Crescimento de Fibroblastos/biossíntese , Camundongos , Microfluídica/instrumentação , Regulação para Cima
4.
J Immunol ; 184(6): 3016-24, 2010 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-20154207

RESUMO

Peptide splicing is a newly described mode of production of antigenic peptides presented by MHC class I molecules, whereby two noncontiguous fragments of the parental protein are joined together after excision of the intervening segment. Three spliced peptides have been described. In two cases, splicing involved the excision of a short intervening segment of 4 or 6 aa and was shown to occur in the proteasome by transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by the N terminus of the other peptide fragment. For the third peptide, which is derived from fibroblast growth factor-5 (FGF-5), the splicing mechanism remains unknown. In this case, the intervening segment is 40 aa long. This much greater length made the transpeptidation model more difficult to envision. Therefore, we evaluated the role of the proteasome in the splicing of this peptide. We observed that the spliced FGF-5 peptide was produced in vitro after incubation of proteasomes with a 49-aa-long precursor peptide. We evaluated the catalytic mechanism by incubating proteasomes with various precursor peptides. The results confirmed the transpeptidation model of splicing. By transfecting a series of mutant FGF-5 constructs, we observed that reducing the length of the intervening segment increased the production of the spliced peptide, as predicted by the transpeptidation model. Finally, we observed that trans-splicing (i.e., splicing of fragments from two distinct proteins) can occur in the cell, but with a much lower efficacy than splicing of fragments from the same protein.


Assuntos
Apresentação de Antígeno/imunologia , Antígenos/metabolismo , Fator 5 de Crescimento de Fibroblastos/metabolismo , Fragmentos de Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/fisiologia , Processamento de Proteína Pós-Traducional/imunologia , Processamento de Proteína/imunologia , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/genética , Antígenos/biossíntese , Antígenos/genética , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Testes Imunológicos de Citotoxicidade , Fator 5 de Crescimento de Fibroblastos/biossíntese , Fator 5 de Crescimento de Fibroblastos/genética , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/química , Inibidores de Proteassoma , Precursores de Proteínas/biossíntese , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína/genética , Transfecção
5.
Arthritis Res Ther ; 7(1): R118-26, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15642131

RESUMO

Fibroblast-like synoviocytes (FLSs) play a major role in the pathogenesis of rheumatoid arthritis (RA) by secreting effector molecules that promote inflammation and joint destruction. How these cells become and remain activated is still elusive. Both genetic and environmental factors probably play a role in transforming FLSs into inflammatory matrix-degrading cells. As bacterial products have been detected in the joint and shown to trigger joint inflammation, this study was undertaken to investigate whether a bacterial ligand of integrin alpha5beta1, protein I/II, could contribute to the aggressive behavior of RA FLSs. Protein I/II is a pathogen-associated molecular pattern (PAMP) isolated from oral streptococci that have been identified in the joints of RA patients. The response of RA and osteoarthritis FLSs to protein I/II was analyzed using human cancer cDNA expression arrays. RT-PCR and pro-MMP-3 (pro-matrix metalloproteinase) assays were then performed to confirm the up-regulation of gene expression. Protein I/II modulated about 6% of all profiled genes. Three of these, those encoding IL-6, leukemia inhibitory factor, and MMP-3, showed a high expression level in all RA FLSs tested, whereas the expression of genes encoding other members of the cytokine or MMP-family was not affected. Furthermore, the up-regulation of MMP-3 gene expression was followed by an increase of pro-MMP-3 release. The expression of interferon regulatory factor 1 and fibroblast growth factor-5 was also up-regulated, although the expression levels were lower. Only one gene, that for insulin-like growth factor binding protein-4, was down-regulated in all RA FLSs. In contrast, in osteoarthritis FLSs only one gene, that for IL-6, was modulated. These results suggest that a bacterial ligand of integrin alpha5beta1 may contribute to the aggressive behavior of RA FLSs by inducing the release of pro-inflammatory cytokines and a cartilage-degrading enzyme, such as IL-6 and MMP-3, respectively.


Assuntos
Artrite Reumatoide/enzimologia , Proteínas de Bactérias/farmacologia , Fibroblastos/enzimologia , Integrina alfa5beta1/efeitos dos fármacos , Metaloproteinase 3 da Matriz/biossíntese , Streptococcus mutans/química , Membrana Sinovial/patologia , Artrite Reumatoide/patologia , Proteínas de Bactérias/genética , Parede Celular/química , Indução Enzimática/efeitos dos fármacos , Precursores Enzimáticos/biossíntese , Precursores Enzimáticos/genética , Fator 5 de Crescimento de Fibroblastos/biossíntese , Fator 5 de Crescimento de Fibroblastos/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Integrina alfa5beta1/fisiologia , Fator Regulador 1 de Interferon/biossíntese , Fator Regulador 1 de Interferon/genética , Interleucina-6/biossíntese , Interleucina-6/genética , Fator Inibidor de Leucemia , Ligantes , Metaloproteinase 3 da Matriz/metabolismo , Osteoartrite/enzimologia , Osteoartrite/patologia , Proteínas Recombinantes de Fusão/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/metabolismo
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