RESUMO
ISL1 is expressed in cardiac progenitor cells and plays critical roles in cardiac lineage differentiation and heart development. Cardiac progenitor cells hold great potential for clinical and translational applications. However, the mechanisms underlying ISL1 function in cardiac progenitor cells have not been fully elucidated. Here we uncover a hierarchical role of ISL1 in cardiac progenitor cells, showing that ISL1 directly regulates hundreds of potential downstream target genes that are implicated in cardiac differentiation, through an epigenetic mechanism. Specifically, ISL1 promotes the demethylation of tri-methylation of histone H3K27 (H3K27me3) at the enhancers of key downstream target genes, including Myocd and Mef2c, which are core cardiac transcription factors. ISL1 physically interacts with JMJD3, a H3K27me3 demethylase, and conditional depletion of JMJD3 leads to impaired cardiac progenitor cell differentiation, phenocopying that of ISL1 depletion. Interestingly, ISL1 is not only responsible for the recruitment of JMJD3 to specific target loci during cardiac progenitor differentiation, but also modulates its demethylase activity. In conclusion, ISL1 and JMJD3 partner to alter the cardiac epigenome, instructing gene expression changes that drive cardiac differentiation.
Assuntos
Diferenciação Celular , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Miocárdio/citologia , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/genética , Metilação de DNA/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Células HEK293 , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Proteínas com Homeodomínio LIM/genética , Lisina/metabolismo , Camundongos , Células NIH 3T3 , Especificidade de Órgãos , Ligação Proteica , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Sepsis-induced cardiac apoptosis is one of the major pathogenic factors in myocardial dysfunction. As it enhances numerous proinflammatory factors, lipopolysaccharide (LPS) is considered the principal mediator in this pathological process. However, the detailed mechanisms involved are unclear. In this study, we attempted to explore the mechanisms involved in LPS-induced cardiomyocyte apoptosis. We found that LPS stimulation inhibited microRNA (miR)-499 expression and thereby upregulated the expression of SOX6 and PDCD4 in neonatal rat cardiomyocytes. We demonstrate that SOX6 and PDCD4 are target genes of miR-499, and they enhance LPS-induced cardiomyocyte apoptosis by activating the BCL-2 family pathway. The apoptosis process enhanced by overexpression of SOX6 or PDCD4, was rescued by the cardiac-abundant miR-499. Overexpression of miR-499 protected the cardiomyocytes against LPS-induced apoptosis. In brief, our results demonstrate the existence of a miR-499-SOX6/PDCD4-BCL-2 family pathway in cardiomyocytes in response to LPS stimulation.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Apoptose/imunologia , Lipopolissacarídeos/farmacologia , MicroRNAs/metabolismo , Miócitos Cardíacos/fisiologia , Fatores de Transcrição SOXD/fisiologia , Animais , Células Cultivadas , Células HeLa , Ventrículos do Coração/citologia , Humanos , MicroRNAs/genética , Interferência de RNA/imunologia , Ratos Sprague-DawleyRESUMO
The regulation of cardiac differentiation is critical for maintaining normal cardiac development and function. The precise mechanisms whereby cardiac differentiation is regulated remain uncertain. Here, we have identified a GATA-4 target, EGF, which is essential for cardiogenesis and regulates cardiac differentiation in a dose- and time-dependent manner. Moreover, EGF demonstrates functional interaction with GATA-4 in inducing the cardiac differentiation of P19CL6 cells in a time- and dose-dependent manner. Biochemically, GATA-4 forms a complex with STAT3 to bind to the EGF promoter in response to EGF stimulation and cooperatively activate the EGF promoter. Functionally, the cooperation during EGF activation results in the subsequent activation of cyclin D1 expression, which partly accounts for the lack of additional induction of cardiac differentiation by the GATA-4/STAT3 complex. Thus, we propose a model in which the regulatory cascade of cardiac differentiation involves GATA-4, EGF, and cyclin D1.
Assuntos
Diferenciação Celular/fisiologia , Fator de Crescimento Epidérmico/metabolismo , Fator de Transcrição GATA4/metabolismo , Coração/embriologia , Modelos Biológicos , Miocárdio/citologia , Transdução de Sinais/fisiologia , Animais , Western Blotting , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Técnicas Histológicas , Imunoprecipitação , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de TempoRESUMO
BACKGROUND: Insulin enhancer binding protein-1 (ISL-1), a LIM-homeodomain transcription factor, is essential for the heart, motor neuron and pancreas development. Recently, ISL-1 has been found in some types of human cancers. However, how ISL-1 exerts the role in tumor development is not clear. METHODS AND RESULTS: The expression of ISL-1 was assessed in 211 human lymphoma samples and 23 normal lymph node samples. Immunohistochemistry results demonstrated a markedly higher expression of ISL-1 in 75% of non-Hodgkin lymphoma (NHL) samples compared with that in normal lymph nodes or Hodgkin lymphoma (HL) samples. CCK-8 analysis, cell cycle assay and xenograft model were performed to characterize the association between ISL-1 expression level and biological functions in NHL. The results showed that ISL-1 overexpression obviously promoted NHL cells proliferation, changed the cell cycle distribution in vitro and significantly enhanced xenografted lymphoma development in vivo. Real-time PCR, Western blot, luciferase assay and ChIP assay were used to explore the potential regulatory targets of ISL-1 and the results demonstrated that ISL-1 activated the c-Myc expression in NHL by direct binding to a conserved binding site on the c-Myc enhancer. Further results revealed that ISL-1 could be positively regulated by the c-Jun N-terminal kinase (JNK) and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways. Both the JNK and JAK/STAT signaling inhibitors could significantly suppressed the growth of NHL cells through the down-regulation of ISL-1 as demonstrated by CCK-8 and Western blot assays. Bioinformatic analysis and luciferase assay exhibited that ISL-1 was a novel target of p-STAT3 and p-c-jun. ChIP, Co-IP and ChIP-re-IP analysis revealed that ISL-1 could participate with p-STAT3 and p-c-Jun to form a p-STAT3/p-c-Jun/ISL-1 transcriptional complex that binds directly on the ISL-1 promoter, demonstrating a positive feedback regulatory mechanism for ISL-1 expression in NHL. CONCLUSIONS: Our results provide the first evidence that ISL-1 is tightly linked to NHL proliferation and development by promoting c-Myc transcription, and its aberrant expression was regulated by p-STAT3/p-c-Jun/ISL-1 complex activation.
Assuntos
Proteínas com Homeodomínio LIM/metabolismo , Linfoma não Hodgkin/metabolismo , Linfoma não Hodgkin/patologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição STAT3/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Janus Quinases/metabolismo , Proteínas com Homeodomínio LIM/genética , Dados de Sequência Molecular , Fosforilação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Insulin enhancer binding protein-1 (ISL-1), a LIM-homeodomain transcription factor, has been reported to play essential roles in promoting adult pancreatic ß-cells proliferation. Recent studies indicate that ISL-1 may also involve in the occurrence of a variety of tumors. However, whether ISL-1 has any functional effect on tumorigenesis, and what are the differences on ISL-1 function in distinct conditions, are completely unknown. In this study, we found that ISL-1 was highly expressed in human pancreatic ß-cells, as well as in diffuse large B cell lymphoma (DLBCL), but to a much less extent in other normal tissues or tumor specimens. Further study revealed that ISL-1 promoted the proliferation of pancreatic ß-cells and DLBCL cells, and also accelerated the tumorigenesis of DLBCL in vivo. We also found that ISL-1 could activate c-Myc transcription not only in pancreatic ß-cells but also in DLBCL cells. However, a cell-specific feedback regulation was detectable only in DLBCL cells. This auto-regulatory loop was established by the interaction of ISL-1 and c-Myc to form an ISL-1/c-Myc transcriptional complex, and synergistically to promote ISL-1 transcription through binding on the ISL-1 promoter. Taken together, our results demonstrate a positive feedback regulation of ISL-1 in DLBCL but not in pancreatic ß-cells, which might result in the functional diversities of ISL-1 in different physiological and pathological processes.
Assuntos
Retroalimentação Fisiológica , Células Secretoras de Insulina/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Fatores de Transcrição/metabolismo , Animais , Carcinogênese , Cricetinae , Humanos , Proteínas com Homeodomínio LIM/genética , Linfoma Difuso de Grandes Células B/genética , Camundongos , Camundongos Endogâmicos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Islet 1 (ISL1), a marker of second heart field progenitors, plays a crucial role in cardiomyocyte differentiation and proliferation. However, little is known about transcriptional regulating mechanisms on Isl1 gene expression. Recent studies have demonstrated that Wnt/ß-catenin signaling regulates Isl1 expression during heart development. However, the detailed mechanisms still remain unclear. In the present study performed during differentiation of P19CL6 into cardiomyocytes, we explored the underlying regulating mechanisms on Wnt/ß-catenin-mediated Isl1 expression after we first confirmed that Wnt/ß-catenin signaling promoted cardiomyocyte differentiation partly through Isl1 activation. We found a novel TCF/LEF1 binding site that was located 2300 bp upstream of the Isl1 ATG. Furthermore, Wnt/ß-catenin signaling upregulated histone H3K9 acetylation on TCF/LEF1 binding sites on the Isl1 promoter, resulting in upregulation of Isl1 expression. This Wnt-mediated H3K9 acetylation on the Isl1 promoter was modulated by the acetyltransferase CREB-binding protein (CBP), instead of p300, through interaction with ß-catenin. Collectively, these results suggest that in early stages of cardiomyocyte differentiation Wnt/ß-catenin signaling promotes Isl1 expression via two ways: a novel TCF/LEF1 binding site and H3K9 acetylation conducted by CBP on the Isl1 promoter. To our knowledge, this is the first study reporting Wnt/ß-catenin-regulated H3K9 acetylation on promoters of its target genes. And this study gives new insights into transcriptional regulating mechanisms of Wnt-mediated Isl1 expression during cardiomyocyte differentiation.
Assuntos
Diferenciação Celular , Histonas/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , Acetilação , Animais , Sequência de Bases , Sítios de Ligação , Proteína de Ligação a CREB/metabolismo , Linhagem Celular Tumoral , Proteínas com Homeodomínio LIM/genética , Lisina/metabolismo , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Ligação Proteica , Fatores de Transcrição/genética , beta Catenina/metabolismoRESUMO
Background microRNAs (miRNAs) are a class of small, non-coding endogenous RNAs that post-transcriptionally regulate some protein-coding genes. miRNAs play an important role in many cardiac pathophysiological processes, including myocardial infarction, cardiac hypertrophy, and heart failure. miR-499, specifically expressed in skeletal muscle and cardiac cells, is differentially regulated and functions in heart development. However, the function of miR-499 in mature heart is poorly understood. Results We report that cardiac-abundant miR-499 could protect neonatal rat cardiomyocytes against H 2O 2-induced apoptosis. Increased miR-499 level favored survival, while decreased miR-499 level favored apoptosis. We identified three proapoptotic protein-coding genes-Pdcd4, Pacs2, and Dyrk2-as targets of miR-499. miR-499 inhibited cardiomyocyte apoptosis through its suppressive effect on Pdcd4 and Pacs2 expression, thereby blocking Bid expression and BID mitochondrial translocation. We also found that H 2O 2-induced phosphorylation of c-Jun transcriptionally upregulated miR-499 expression via binding of phosphorylated c-Jun to the Myh7b promoter. Conclusions Our results revealed that miR-499 played an inhibiting role in the mitochondrial apoptosis pathway, and had protective effects against H 2O 2-induced injury in cardiomyocytes.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Apoptose/genética , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , Proteínas de Transporte Vesicular/genética , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Peróxido de Hidrogênio/farmacologia , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Interferência de RNA , RNA Mensageiro , Ratos , Espécies Reativas de Oxigênio , Quinases DyrkRESUMO
As embryonic stem cells (ESCs) represent an attractive candidate cell source for obtaining cardiomyocytes to be used in cell replacement therapy, it is thus of considerable importance to understand the mechanism by which cardiac differentiation is regulated. In previous studies, we have shown that angiotensin type 1 receptor (AT1R) expressed in cardiomyocytes derived from mouse embryonic stem cells. However, little is known about the role of AT1R in cardiac differentiation, which plays a key role in cardiac physiology and pharmacology. In the present study, we demonstrated that AT1R agonist significantly enhanced cardiac differentiation as determined by increased percentage of beating embryoid bodies and a higher expression level of cardiac markers. On the contrary, AT1R agonist stimulated differentiation was reversed in the presence of AT1R antagonist. In addition, by administering selective inhibitors we found that the effect of AT1R was driven via extracellular-signal regulated kinase, c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase pathways. These findings suggest that AT1R signaling plays a key role in cardiac differentiation of ESCs.
Assuntos
Angiotensina II/farmacologia , Diferenciação Celular , Corpos Embrioides/citologia , Mioblastos Cardíacos/citologia , Miócitos Cardíacos/citologia , Receptor Tipo 1 de Angiotensina/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Ácido Ascórbico/farmacologia , Corpos Embrioides/efeitos dos fármacos , Corpos Embrioides/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Losartan/farmacologia , Camundongos , Mioblastos Cardíacos/efeitos dos fármacos , Mioblastos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Receptor Tipo 1 de Angiotensina/agonistas , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Insulin is a secreted peptide hormone identified in human pancreas to promote glucose utilization. Insulin has been observed to induce cell proliferation and myogenesis in C2C12 cells. The precise mechanisms underlying the proliferation of C2C12 cells induced by insulin remain unclear. In this study, we observed for the first time that 10 nM insulin treatment promotes C2C12 cell proliferation. Additionally, 50 and 100 nM insulin treatment induces C2C12 cell apoptosis. By utilizing real-time PCR and Western blotting analysis, we found that the mRNA levels of cyclinD1 and BAD are induced upon 10 and 50 nM/100 nM insulin treatment, respectively. The similar results were observed in C2C12 cells expressing GATA-6 or PPARα. Our results identify for the first time the downstream targets of insulin, cyclin D1, and BAD, elucidate a new molecular mechanism of insulin in promoting cell proliferation and apoptosis.
Assuntos
Proliferação de Células , Ciclina D1/genética , Insulina/genética , Proteína de Morte Celular Associada a bcl/genética , Apoptose/genética , Linhagem Celular , Linhagem Celular Tumoral , Citometria de Fluxo , Fator de Transcrição GATA6/genética , Fator de Transcrição GATA6/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/genética , Neoplasias/patologia , PPAR alfa/genética , PPAR alfa/metabolismo , Transdução de Sinais , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
BACKGROUND: Early prediction of treatment response is crucial for the optimal treatment of advanced breast cancer. We aimed to explore whether monitoring early changes in plasma human epidermal growth factor receptor 2 (HER2) levels using digital PCR (dPCR) could predict the treatment response in advanced breast cancer. METHODS: This was a multicenter, prospective, noninterventional clinical study of patients with advanced breast cancer. All enrolled patients underwent blood testing to measure the HER2 levels by digital PCR before treatment initiation and once every 3 weeks during the study. The primary endpoints werea the diagnostic value of dPCR for detecting HER2 status in the blood andb the relevance of potential changes in the plasma HER2 level at 3 weeks from baseline for predicting treatment response. RESULTS: Overall, 85 patients were enrolled between October 9, 2018, and January 23, 2020. dPCR had a specificity of 91.67% (95% CI: 80.61% to 97.43%) for detecting HER2 amplification, and the area under the receiver operating characteristic (ROC) curve was 0.84 (p < 0.01). A clinically relevant specificity threshold of approximately 90%, which was equivalent to a ≥15% decrease in the plasma HER2 ratio at 3 weeks from baseline, showed a positive predictive value of 97.37% (95% CI: 77.11% to 98.65%) in terms of predicting clinical benefit. Patients whose plasma HER2 ratio was reduced by ≥15% had a longer median progression-free survival (PFS) than those whose ratio was reduced by <15% (9.20 months vs. 4.50 months, p < 0.01). CONCLUSIONS: Early changes in the plasma HER2 ratio may predict the treatment response in patients with advanced breast cancer and could facilitate optimal treatment selection.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/terapia , Neoplasias da Mama/tratamento farmacológico , Biomarcadores Tumorais/metabolismo , Estudos Prospectivos , Valor Preditivo dos Testes , Curva ROCRESUMO
Notch and Wnt/ß-catenin signaling both play essential roles and interact closely in cardiomyocyte differentiation but the mechanism of interaction is largely unknown. Here we show that activation of Notch signaling in undifferentiated P19CL6 cells promoted cardiac differentiation, indicated by upregulated expression of early cardiac markers and activated the canonical Wnt pathway, suggested by augmented nuclear translocation of ß-catenin. Further activation of the Notch pathway in early differentiating cells (at day 3) inhibited expression of a specific cardiac progenitor marker Islet1 but had no influence on ß-catenin translocation. Notch signaling thus played biphasic roles in the early stage of cardiomyocyte differentiation and Wnt/ß-catenin signaling. Unlike Notch signaling, Wnt signaling promoted cardiomyocyte differentiation and activated the Notch pathway in either undifferentiated or early differentiating cells. Additionally, ß-catenin, recombination signal sequence binding protein-Jkappa (RBP-Jκ), and Notch1 intracellular domain (NICD-1) formed a transcriptional complex which was recruited to the Hes1 promoter region, indicating direct transcriptional regulation of Hes1. We thus document a specific reciprocal interaction between these two signaling pathways during early stage cardiac differentiation of P19CL6 cells.
Assuntos
Diferenciação Celular , Miócitos Cardíacos/fisiologia , Receptor Notch1/metabolismo , Via de Sinalização Wnt , Proteína Wnt3A/fisiologia , beta Catenina/metabolismo , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Cloreto de Lítio/farmacologia , Luciferases de Renilla/biossíntese , Luciferases de Renilla/genética , Camundongos , Complexos Multiproteicos/metabolismo , Miócitos Cardíacos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Receptor Cross-Talk , Receptor Notch1/química , Receptor Notch1/genética , Fatores de Transcrição HES-1 , Transcrição Gênica , Proteína Wnt3A/farmacologiaRESUMO
Islet 1 (ISL1), a marker of cardiac progenitors, plays a crucial role in cardiogenesis. However, the precise mechanism underlying the activation of its expression is not fully understood. Using the cardiac differentiation model of P19CL6 cells, we show that POU homeodomain protein, OCT1, modulates Isl1 expression in the process of cardiac differentiation. Oct1 knock-down resulted in reduction of Isl1 expression and downregulated mesodermal, cardiac-specific, and signal pathway gene expression. Additionally, the octamer motif located in the proximal region of Isl1 promoter is essential to Isl1 transcriptional activation. Mutation of this motif remarkably decreased Isl1 transcription. Although both OCT1 and OCT4 bound to this motif, it was OCT1 rather than OCT4 that modulated Isl1 expression. Furthermore, the correlation of OCT1 in regulation of Isl1 was revealed by in situ hybridization in early embryos. Collectively, our data highlight a novel role of OCT1 in the regulation of Isl1 expression.
Assuntos
Diferenciação Celular , Proteínas de Homeodomínio/metabolismo , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Transportador 1 de Cátions Orgânicos/metabolismo , Sequência de Bases , Western Blotting , Células Cultivadas , Imunofluorescência , Marcadores Genéticos , Proteínas de Homeodomínio/genética , Proteínas com Homeodomínio LIM , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TranscriçãoRESUMO
Background: The Ilizarov technique of distraction osteogenesis is an effective treatment for tibia defect. However, repeated attempts to reduce due to the complexity of the bone defect during the operation will increase the operation time and iatrogenic injury, and excessive radiation exposure. Three-dimensional (3D)-printed patient-specific instrument (PSI) for preoperative 3D planning and intraoperative navigation have the advantages of accuracy and visualization. The purpose of this study is to investigate whether 3D-printed PSI is helpful to correct tibial bone defects accurately and effectively. Method: From May 2019 to September 2022, 19 patients with tibial bone defects were treated, including 9 males and 10 females, aged 37 to 64 years. There were 4 cases in proximal tibia, 9 in midshaft tibia and 6 in distal tibia. All were treated with Ilizarov technique of distraction osteogenesis. 3D-printed PSI was used in 9 cases, while traditional surgery was used in 10 cases. All patients underwent computed tomography before surgery. Computer software was used to analyze the measurement results, design and print PSI. During the operation, PSI was used to assist in reduction of tibia. Operation times were recorded in all cases, the number of fluoroscopy during the operation, and the varus/valgus, anteversion/reversion angle after the operation were measured. All measurement data were expressed by means ± SD, and Student's t test was used to examine differences between groups. The chi square test or Fisher's precise test was used to compare the counting data of the two groups. Result: All PSI matched well with the corresponding tibia bone defect, and were consistent with the preoperative plan and intraoperative operation. The affected limb had a good reduction effect. The operation time from the beginning of PSI installation to the completion of Ilizarov ring fixator installation was 31.33 ± 3.20â min, while that in the traditional operation group was 64.10 ± 6.14â min (p < 0.001). The times of fluoroscopy in the PSI group during operation was 10.11 ± 1.83, and that in the traditional operation group was 27.60 ± 5.82. The reduction effect of tibia in PSI group was better than that in traditional operation group, with the average angle of PSI group is 1.21 ± 0.24°, and that of traditional operation group is 2.36 ± 0.33° (p < 0.001). Conclusion: The PSI simplifies procedures, reduces the difficulty of the operation, improves the accuracy of the operation, and provides a good initial position when used in distraction osteogenesis to treat the tibial defects.
RESUMO
Cardiac hypertrophy in response to multiple stimuli has important physiological and pathological significances. GATA4 serves as a nuclear integrator of several signalling pathways during cardiac hypertrophy. Sp1 and Sp3 are also reported to be involved in this process. However, the mechanism by which GATA4 acts as a mediator, integrating these ubiquitously expressed transcriptional factors, is poorly understood. We found that the expression of GATA4 and Sp1 was up-regulated in the myocardium of a pressure overload hypertrophy rat model, as well in phenylephrine-induced (PE-induced) hypertrophic growth of neonatal cardiomyocytes. GST pull-down assays demonstrated that GATA4 could interact with Sp1 in vitro. Therefore, we proposed that GATA4 cooperates with Sp1 in regulating ANF expression, as its reactivation is closely linked with hypertrophy. Further studies demonstrated that GATA4 could activate the ANF promoter synergistically with Sp1 through direct interaction. In contrast, Sp3 exhibited antagonistic function, and overexpression of Sp3 repressed the transcriptional synergy between Sp1 and GATA4. We also found that Sp1 alone could activate the ANF promoter in cardiomyocytes, whereas Sp3 exerted negative effects on ANF expression. Bioinformatics analysis revealed novel Sp-binding sites on the ANF promoter. The recruitment of GATA4 and Sp1 on the ANF promoter was enhanced during phenylephrine-mediated hypertrophy, whereas the recruitment of Sp3 was reduced. The phosphorylation of GATA4 by ERK1/2 kinase could enhance the affinity between GATA4 and Sp1. Thus, our findings revealed the critical interaction of GATA4 and Sp1 in modulating ANF expression, indicating their involvement in cardiac hypertrophy.
Assuntos
Fator Natriurético Atrial/genética , Cardiomegalia/genética , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica , Fator de Transcrição Sp1/metabolismo , Animais , Fator Natriurético Atrial/metabolismo , Sequência de Bases , Sítios de Ligação , Cardiomegalia/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Dados de Sequência Molecular , Fenilefrina/farmacologia , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Fator de Transcrição Sp3/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
The inhibitor of DNA binding (Id) family of genes encodes negative regulators of basic helix-loop-helix transcription factors and has been implicated in such diverse cellular processes as differentiation, proliferation, apoptosis and migration. Id knockout mouse embryos display multiple cardiac defects but the specific role of Id1 in cardiac differentiation is unclear. In the present study, we investigated the function of Id1 in DMSO-induced P19CL6 cells, a widely-accepted cell model of cardiac differentiation. We found that Id1 was upregulated during the cardiac differentiation of P19CL6 cells. The expression of cardiac specific marker genes, Gata4, α-MHC and ISL1, was upregulated in P19CL6 cells stably transfected with Id1 (P19CL6-Id1) during cardiac differentiation. The overexpression of Id1 reduced the number of cells in G1 phase and increased the cell population in G2, M and S phases, while knockdown of Id1 increased the number of cells in G1 phase from 48.6 ± 2.51% to 62.2 ± 1.52% at day 0 of cardiac induction, and from 52.5 ± 3.41% to 63.7 ± 1.02% at day 3 after cardiac induction, indicating that Id1 promoted proliferation of P19CL6 cells. Luciferase assays showed that the activity of TOP flash was higher in P19CL6-Id1 cells than wildtype P19CL6 cells, while Id1 expression was also upregulated in P19CL6 cells treated with Wnt3a or LiCl. This indicates that there may be positive feedback between Id1 and Wnt signaling which plays an important role in cardiac differentiation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Proteína 1 Inibidora de Diferenciação/metabolismo , Mioblastos Cardíacos/citologia , Mioblastos Cardíacos/metabolismo , Organogênese/genética , Animais , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Fator de Transcrição GATA4/genética , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Proteína 1 Inibidora de Diferenciação/genética , Proteínas com Homeodomínio LIM , Cloreto de Lítio/farmacologia , Camundongos , Organogênese/efeitos dos fármacos , Fatores de Transcrição , Proteínas Wnt/metabolismo , Proteínas Wnt/farmacologia , Proteína Wnt3 , Proteína Wnt3ARESUMO
As embryonic stem cell-derived cardiomyocytes (ESC-CMs) have the potential to be used in cell replacement therapy, an understanding of the signaling mechanisms that regulate their terminal differentiation is imperative. In previous studies, we discovered the presence of adrenergic and muscarinic receptors in mouse embryonic stem cells (ESCs). However, little is known about the role of these receptors in cardiac differentiation and development, which is critically important in cardiac physiology and pharmacology. Here, we demonstrated that a ß-adrenergic receptor (ß-AR) agonist significantly enhanced cardiac differentiation as indicated by a higher percentage of beating embryoid bodies and a higher expression level of cardiac markers. Application of ß1-AR and ß2-AR antagonists partly abolished the effect of the ß-AR agonist. In addition, by administering selective inhibitors we found that the effect of ß-AR was driven via p38 mitogen-activated protein kinase and extracellular-signal regulated kinase pathway. These findings suggest that ESCs are also a target for ß-adrenergic regulation and ß-adrenergic signaling plays a role in ESC cardiac differentiation.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Diferenciação Celular , Células-Tronco Embrionárias/fisiologia , Sistema de Sinalização das MAP Quinases , Miócitos Cardíacos/fisiologia , Receptores Adrenérgicos beta 1/metabolismo , Antagonistas Adrenérgicos beta/farmacologia , Animais , Biomarcadores/metabolismo , Western Blotting , Relação Dose-Resposta a Droga , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Imunofluorescência , Isoproterenol/farmacologia , Camundongos , Microscopia Confocal , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fosforilação , Propranolol/farmacologia , Receptores Adrenérgicos beta 2/metabolismo , Troponina T/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVE: Anlotinib is a novel tyrosine kinase inhibitor blocking angiogenesis. This study was performed to assess the efficacy and safety of anlotinib in patients with metastatic breast cancer. METHODS: Patients with HER2-negative breast cancer, who were pre-treated with anthracycline or taxanes in a neoadjuvant, adjuvant, or metastatic setting, and had treatment failure after at least one prior chemotherapy regimen in the metastatic setting were enrolled. Anlotinib was administered at 12 mg daily for 14 days in a 21-day cycle until disease progression or unacceptable toxicity occurred. Simultaneously, 5-10 mL of venous blood was collected to perform circulating tumor DNA (ctDNA) testing every 2 treatment cycles. The primary endpoint was the objective response rate (ORR). Secondary endpoints included the disease control rate (DCR), progression-free survival (PFS), overall survival, safety, and biomarkers. RESULTS: Twenty-six eligible patients were enrolled, with a median age of 56 (30-75) years. The median follow-up time was 10.5 months. The ORR was 15.4%, the DCR was 80.8%, and the median PFS was 5.22 months (95% confidence interval 2.86-6.24). Fourteen (53.8%) patients survived for more than 10 months. The changes in the detectable ctDNA variant allele frequency were consistent with the tumor response. The most common treatment-related adverse events were hypertension (57.7%), thyroidstimulating hormone elevation (34.6%), and hand-foot syndrome (23.1%). CONCLUSIONS: Anlotinib showed objective efficacy with tolerable toxicity in heavily pre-treated, metastatic HER2-negative breast cancer. The dynamic changes in the ctDNA variant allele fraction may be predictive of the tumor response.
RESUMO
The cardiac transcription factor NKX2.5 plays a crucial role in cardiomyogenesis, but its mechanism of regulation is still unclear. Recently, epigenetic regulation has become increasingly recognized as important in differentiation and development. In this study, we used P19CL6 cells to investigate the regulation of Nkx2.5 expression by methylation and acetylation during cardiomyocyte differentiation. During the early stage of differentiation, Nkx2.5 expression was upregulated, but the methylation status of the Nkx2.5 promoter did not undergo significant change; while the acetylation levels of histones H3 and H4 were increased, accompanied by a significant reduction in Hdac1 expression. Suppression of Hdac1 activity stimulated cardiac differentiation accompanied by increased expression of cardiac-specific genes and cell cycle arrest. Overexpression of Hdac1 inhibited cardiomyocyte formation and downregulated the expressions of Gata4 and Nkx2.5. Mimicking induction of the WNT pathway inhibited Hdac1 expression with upregulated Nkx2.5 expression. WNT3a and WNT3 downregulated the expression of Hdac1, contrary to the effect of SFRP2 and GSK3beta. Cotransfection of beta-catenin and Lef1 significantly downregulated the expression of Hdac1. Our data suggest that WNT signaling pathway plays important roles in the regulation of Hdac1 during the early stage of cardiomyocyte differentiation and that the downregulation of Hdac1 promotes cardiac differentiation.
Assuntos
Diferenciação Celular , Regulação para Baixo/genética , Histona Desacetilases/metabolismo , Proteínas de Homeodomínio/genética , Miócitos Cardíacos/citologia , Transdução de Sinais , Fatores de Transcrição/genética , Proteínas Wnt/metabolismo , Acetilação , Animais , Linhagem Celular , Proliferação de Células , Ilhas de CpG/genética , Metilação de DNA , Regulação da Expressão Gênica no Desenvolvimento , Histona Desacetilase 1 , Histonas , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Camundongos , Miócitos Cardíacos/enzimologia , Fenótipo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Regulação para Cima/genética , beta Catenina/metabolismoRESUMO
beta-Catenin/TCF/LEF1 signaling is implicated in cardiac hypertrophy. We demonstrate that knockdown of beta-catenin attenuates phenylephrine (PE)-induced cardiomyocyte hypertrophy and the up-regulation of the fetal gene Anf. We explore the mechanism through which beta-catenin regulates Anf expression and find a consensus binding sequence on the Anf promoter for TCF/LEF1 family members. LEF1 binds directly to the Anf promoter via this sequence, which shows functional significance, and PE stimulation enhances recruitment of beta-catenin onto the Anf promoter. Thus, we document a direct positive role of beta-catenin on PE-induced cardiomyocyte hypertrophy and identify a new target gene for beta-catenin/TCF/LEF1.
Assuntos
Fator Natriurético Atrial/genética , Cardiomegalia/metabolismo , Regulação da Expressão Gênica , Miócitos Cardíacos/metabolismo , beta Catenina/metabolismo , Animais , Cardiomegalia/induzido quimicamente , Cardiomegalia/genética , Técnicas de Silenciamento de Genes , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Fenilefrina/farmacologia , Regiões Promotoras Genéticas , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição TCF/metabolismo , Transcrição Gênica , Regulação para Cima , beta Catenina/genéticaRESUMO
A new methodology for the one-pot enantioselective construction of 2-pyrrolidinone derivatives bearing a trifluoromethylated all-carbon quaternary stereocenter at the 4-position has been described. This strategy combines an organocatalytic conjugate addition of nitroalkanes to isatin-derived α-trifluoromethyl acrylates and a reduction/lactamization process, affording the corresponding products in moderate to high yields (50-95%) with generally excellent stereoselectivities (up to 96% ee and >20:1 dr).