RESUMO
Adipose-derived stem cells (ADSCs) show nearly unlimited potential in medical and animal science. Currently, understanding of the biological mechanisms regulating ADSC growth in vitro remains very limited. Histone acetylation, an epigenetic modification, plays a key role in maintaining stem cell properties. To further study its effect on ADSC growth characteristics in vitro, we treated goat ADSCs with the histone deacetylase inhibitors trichostatin A (TSA) and vorinostat (SAHA). This inhibited SIRT1 expression and increased histone H3K9 acetylation, leading to decreased cell viability, cell cycle arrest, and apoptosis. Quantitative real-time polymerase chain reaction revealed that H3K9 hyperacetylation stimulated transcription of NANOG, OCT4, SOX2, and TERT, but inhibited that of PCNA, P53, and BAX. Western blotting indicated that TSA and SAHA increased protein expression of NANOG, reduced that of SOX2, TERT, PCNA, P53, and BAX, and did not change that of OCT4. These findings provide new experimental evidence contributing to our understanding of the mechanisms underlying ADSC growth characteristics in vitro.
Assuntos
Tecido Adiposo/citologia , Histonas/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Acetilação , Animais , Apoptose/genética , Western Blotting , Ciclo Celular/genética , Proliferação de Células , Sobrevivência Celular/genética , Citometria de Fluxo , Regulação da Expressão Gênica , Cabras , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Transgene silencing, which is common in transgenic plants and animals, limits the generation and application of genetically modified organisms, and is associated with the exogenous gene copy number, the methylation status of its promoters, and histone modification abnormalities. Here, we analyzed the expression of the exogenous gene DsRed and the methylation status of its cytomegalovirus (CMV) promoter in six healthy transgenic cashmere goats and transgenic nuclear donor cells. The CMV promoter exhibited high methylation levels (74.4-88.2%) in four of the goats, a moderate methylation level (58.7%) in one, and a low methylation level (21.2%) in one, while the methylation level of the transgenic nuclear donor cells was comparatively low (14.3%). DsRed expression was negatively correlated with promoter methylation status. Transgenic cashmere goats carried one to three copies of the CMV promoter fragment and one to six copies of the DsRed fragment, but copy number showed no obvious correlation with DsRed expression. After treatment with the methylation inhibitor 5-azacytidine, DsRed expression in transgenic goat cells significantly increased and CMV promoter methylation significantly decreased; this indicated an inverse correlation between promoter methylation status and DsRed expression. After treatment with the histone deacetylase inhibitor trichostatin A, DsRed expression increased, indicating that an abnormal histone modification in transgenic goats is also involved in exogenous gene silencing. These findings indicate the potential of trichostatin A and 5-azacytidine to rescue the biological activity of silenced exogenous transgenes in adult-derived transgenic cells under culture conditions.
Assuntos
Azacitidina/farmacologia , Cabras/genética , Histonas/genética , Ácidos Hidroxâmicos/farmacologia , Proteínas Luminescentes/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Animais , Animais Geneticamente Modificados , Citomegalovirus/genética , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos dos fármacos , Feminino , Dosagem de Genes , Expressão Gênica , Inativação Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Proteínas Luminescentes/agonistas , Proteínas Luminescentes/antagonistas & inibidores , Proteínas Luminescentes/metabolismo , Masculino , TransgenesRESUMO
Pinelliae rhizoma is the dried tuber of Pinellia ternata (Thunb.) Breit., and has been used for thousands of years as a traditional Chinese medicine. However, its genomic background is little known. With the development of high-throughput genomic sequencing, it is now easy and cheap to obtain genomic information. In this study, 193,032,910 high-quality clean reads were generated using the Illumina Hiseq 2000 platform. A total of 53,544 unigenes were identified from the contigs assembled. Functional annotation analysis annotated 37,318, 27,697, 23,043, 22,869, 23,328, and 27,415 unigenes. KEGG analysis revealed that five pathways (169 genes) were associated with alkaloid synthesis, 201 unigenes were related to fatty acid biosynthesis (ko00061), and 133 unigenes were involved in the biosynthesis of unsaturated fatty acids (ko01040). In addition, 6703 simple sequence repeats were designed based on the unigene sequences for screening germplasm resources in the future. These data are a valuable resource for genomic studies on Pinellia plants.
Assuntos
Pinellia/genética , Perfilação da Expressão Gênica , Genes de Plantas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA , TranscriptomaRESUMO
White adipose tissue and brown adipose tissue play critical roles in controlling energy homeostasis and the development of obesity and diabetes. We isolated mouse white adipocytes from inguinal white fat tissues and brown adipocytes from interscapular brown fat tissues, and employed a variety of approaches, including immunofluorescent staining, quantitative real-time PCR, western blotting analysis, and differentiation assay, to characterize those adipocytes. Both white and brown adipocytes stained positively for CD44 and CD29, and lipid droplets were observed after the induction of adipogenesis. The Asc1 expression level in the white adipocytes was 2.5-fold higher than that in the brown adipocytes (P < 0.05), and the expression of Ucp1 in the white adipocytes was approximately 50% of that in the brown adipocytes (P < 0.05). The expression of α-tubulin in the brown adipocytes was approximately 70% of that in the white adipocytes. The brown adipocytes had a higher Cidea mRNA level (P < 0.05) and a lower Pparγ mRNA level (P < 0.05) than the white adipocytes. The results demonstrate that white and brown adipocytes have different gene expression signatures, and may represent two useful cell models to study the mechanisms involved in obesity.
Assuntos
Adipócitos Marrons/citologia , Adipócitos Marrons/metabolismo , Adipócitos Brancos/citologia , Adipócitos Brancos/metabolismo , Expressão Gênica , Adipogenia , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/citologia , Tecido Adiposo Branco/metabolismo , Animais , Proliferação de Células , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Masculino , CamundongosRESUMO
Propofol is one of the extensively and commonly used intravenous anesthetic agents. The current study aimed to evaluate the effects of propofol on the behavior of human gastric cancer cells and the molecular mechanisms of this activity. The effects of propofol on SGC7901 and AGS cell proliferation, apoptosis, and invasion were detected by MTT assay, flow cytometric analysis, and matrigel invasion assay. Real-time polymerase chain reaction (PCR) was used to assess microRNA (miR)-221 expression. miR-221 mimics were transfected into SGC7901 and AGS cells to assess the role of miR- 221 in propofol-induced anti-tumor activity. Propofol significantly inhibited cell proliferation and invasion and promoted apoptosis of SGC7901 and AGS cells. Propofol also efficiently reduced miR-221 expression. Moreover, transfection of miR-221 mimics reversed the effects of propofol on the biological behavior of gastric cancer cells. Propofol can effectively inhibit proliferation and invasion and induce apoptosis of gastric cancer cells through, at least partly, downregulation of miR-221 expression.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , MicroRNAs/genética , Propofol/farmacologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/genética , Humanos , MicroRNAs/metabolismo , Invasividade NeoplásicaRESUMO
Propofol is a commonly used intravenous anesthetic. We evaluated its effects on the behavior of human pancreatic cancer cells and the underlying molecular mechanisms. The effects of propofol on Panc-1 cell proliferation, apoptosis, and invasion were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, caspase-3 activity measurement, and Matrigel invasion assay. Quantitative polymerase chain reaction (qPCR) was used to assess microRNA-133a (miR-133a) expression. Anti-miR-133a was transfected into Panc-1 cells to assess the role of miR-133a in propofol-induced antitumor activity. Propofol significantly inhibited Panc-1 cell proliferation and invasion, and promoted apoptosis. Propofol also efficiently elevated miR-133a expression. Moreover, transfection of anti-miR-133a reversed the effects of propofol on the biological behavior of Panc-1 cells. Propofol can effectively inhibit proliferation and invasion, and induce apoptosis of pancreatic cancer cells, at least partly through the upregulation of miR-133a expression.
Assuntos
MicroRNAs/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Propofol/farmacologia , Regulação para Cima/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/metabolismo , Invasividade Neoplásica , Propofol/químicaRESUMO
The insulin-like growth factor-binding protein-5 (IGFBP-5) is one of the 6 members of the IGFBP family and is involved in the regulation of cell growth, apoptosis, and other IGF-stimulated signaling pathways. To determine the significance of IGFBP-5 in the Inner Mongolia Cashmere goat (Capra hircus), a hair follicle-specific expression vector of IGFBP-5, pCDsRed2-K-IGFBP5 (6.7 kb), was constructed by cloning IGFBP-5 downstream of the keratin-association protein (KAP)6-1 promoter and inserting this fragment into pCDsRed2, which contains a red fluorescent protein (DsRed) expression unit. Inner Mongolia Cashmere goat fetal fibroblast (GFb) cells were transfected with the expression vector by using Lipofectamine(TM) 2000. Cell clones that stably expressed red fluorescence were obtained after selection with Geneticin (G418). The transgene in the cell clones was examined by polymerase chain reaction to verify that exogenous DNA (pKAP6-1 and IGFBP-5) had integrated stably into GFb cells. These data suggest that this method can be used for the construction of a hair follicle-specific expression vector for functional genetic analyses and for obtaining stable transfection donor cells for nuclear transfer.