RESUMO
OBJECTIVE: Tie1 (tyrosine kinase containing immunoglobulin and epidermal growth factor homology 1), an endothelial and hematopoietic cell-specific receptor tyrosine kinase, is an important regulator of angiogenesis and critical for maintaining vascular integrity. The post-transcriptional regulation of tie1 mRNA expression is not understood, but it might partly explain Tie1's differential expression pattern in endothelium. Following up on our previous work that identified natural antisense transcripts from the tie1 locus-tie1 antisense (tie1AS), which regulates tie1 mRNA levels in zebrafish-we attempted to identify the mechanism of this regulation. APPROACH AND RESULTS: Through in vitro and in vivo ribonucleoprotein binding studies, we demonstrated that tie1AS long noncoding RNA interacts with an RNA binding protein-embryonic lethal and abnormal vision Drosophila-like 1 (Elavl1)-that regulates tie1 mRNA levels. When we disrupted the interaction between tie1AS and Elavl1 by using constitutively active antisense morpholino oligonucleotides or photoactivatable morpholino oligonucleotides, tie1 mRNA levels increased between 26 and 31 hours post-fertilization, particularly in the head. This increase correlated with dilation of primordial midbrain channels, smaller eyes, and reduced ventricular space. We also observed these phenotypes when we used CRISPR (clustered regularly interspaced short palindromic repeats)-mediated CRISPRi (CRISPR-mediated interference) to knock down tie1AS. Treatment of the morpholino oligonucleotide-injected embryos with a small molecule that decreased tie1 mRNA levels rescued all 3 abnormal phenotypes. CONCLUSIONS: We identified a novel mode of temporal and spatial post-transcriptional regulation of tie1 mRNA. It involves long noncoding RNA, tie1AS, and Elavl1 (an interactor of tie1AS).
Assuntos
Vasos Sanguíneos/enzimologia , Encéfalo/irrigação sanguínea , Neovascularização Fisiológica/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Vasos Sanguíneos/embriologia , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Receptor de TIE-1/genética , Receptor de TIE-1/metabolismo , Fatores de Tempo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
The mechanisms underlying many of the human disease phenotypes associated with ciliary dysfunction and abnormal centrosome amplification have yet to be fully elucidated. Here, we present for the first time that SIRT2, a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, regulates ciliogenesis and centrosome amplification. Overexpression of SIRT2 in renal epithelial cells appeared to disrupt cilia formation, causing decreased numbers of cells with cilia and decreased cilia length, while inhibition of SIRT2 activity by nicotinamide treatment or knockdown of SIRT2 with siRNA was shown to block cilia disassembly during the cell cycle. Overexpression of SIRT2 in zebrafish decreased cilia numbers in Kupffer's vesicle, while morpholino knock down of SIRT2 increased cilia length. Aberrant centrosome amplification and polyploidy were seen with overexpression of SIRT2 in mouse inner medullary collecting duct 3 cells, similar to that observed following Pkd1 knockdown. SIRT2 was up-regulated in both Pkd1 mutant and knockdown cells. Depletion of SIRT2 prevented the abnormal centrosome amplification and polyploidy associated with loss of polycystin-1 (PC1) alone. Thus, we conclude that the aberrant centrosome amplification and polyploidy in Pkd1 mutant or depleted cells was mediated through overexpression of SIRT2. Our results suggest a novel function of SIRT2 in cilia dynamics and centrosome function, and in ciliopathy-associated disease progression.
Assuntos
Centrossomo/patologia , Cílios/metabolismo , Rim/metabolismo , Sirtuína 2/metabolismo , Canais de Cátion TRPP/metabolismo , Animais , Linhagem Celular , Cílios/efeitos dos fármacos , Amplificação de Genes , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Rim/efeitos dos fármacos , Camundongos , Niacinamida/farmacologia , Sirtuína 2/genética , Canais de Cátion TRPP/genética , Peixe-ZebraRESUMO
Murine sickle cell disease (SCD) results in damage to multiple organs, likely mediated first by vasculopathy. While the mechanisms inducing vascular damage remain to be determined, nitric oxide bioavailability and sterile inflammation are both considered to play major roles in vasculopathy. Here, we investigate the effects of high mobility group box-1 (HMGB1), a pro-inflammatory damage-associated molecular pattern (DAMP) molecule on endothelial-dependent vasodilation and lung morphometrics, a structural index of damage in sickle (SS) mice. SS mice were treated with either phosphate-buffered saline (PBS), hE-HMGB1-BP, an hE dual-domain peptide that binds and removes HMGB1 from the circulation via the liver, 1-[4-(aminocarbonyl)-2-methylphenyl]-5-[4-(1H-imidazol-1-yl)phenyl]-1H-pyrrole-2-propanoic acid (N6022) or N-acetyl-lysyltyrosylcysteine amide (KYC) for three weeks. Human umbilical vein endothelial cells (HUVEC) were treated with recombinant HMGB1 (r-HMGB1), which increases S-nitrosoglutathione reductase (GSNOR) expression by â¼80%, demonstrating a direct effect of HMGB1 to increase GSNOR. Treatment of SS mice with hE-HMGB1-BP reduced plasma HMGB1 in SS mice to control levels and reduced GSNOR expression in facialis arteries isolated from SS mice by â¼20%. These changes were associated with improved endothelial-dependent vasodilation. Treatment of SS mice with N6022 also improved vasodilation in SS mice suggesting that targeting GSNOR also improves vasodilation. SCD decreased protein nitrosothiols (SNOs) and radial alveolar counts (RAC) and increased GSNOR expression and mean linear intercepts (MLI) in lungs from SS mice. The marked changes in pulmonary morphometrics and GSNOR expression throughout the lung parenchyma in SS mice were improved by treating with either hE-HMGB1-BP or KYC. These data demonstrate that murine SCD induces vasculopathy and chronic lung disease by an HMGB1- and GSNOR-dependent mechanism and suggest that HMGB1 and GSNOR might be effective therapeutic targets for reducing vasculopathy and chronic lung disease in humans with SCD.
Assuntos
Anemia Falciforme , Benzamidas , Proteína HMGB1 , Pneumopatias , Lesão Pulmonar , Pirróis , Doenças Vasculares , Humanos , Animais , Camundongos , Lesão Pulmonar/etiologia , Proteína HMGB1/genética , Células Endoteliais/metabolismo , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/genética , Inflamação , Doenças Vasculares/etiologiaRESUMO
Despite their importance as members of the Roundabout (Robo) family in the control of axonal and vascular patterning, the transcriptional regulation of these genes is poorly understood. In this study, we show that members of the Sry-related high mobility box (Sox) transcription factor family as being transcriptional regulators of roundabout4 (robo4), a Robo gene family member that participates in sprouting angiogenesis in vivo, in zebrafish. Double whole mount in situ hybridization analysis in zebrafish embryos revealed co-localization of the vascular relevant Sox factors sox7 or sox18 mRNA with robo4 transcripts in developing intersomitic vessels. A 3-kb human ROBO4 promoter element was able to drive reporter expression in zebrafish to recapitulate the endogenous temporal intersomitic vessel expression pattern of robo4. EMSA analysis confirmed binding of Sox18 to a canonical Sox binding site (from -1170 bp to -1176 bp) in the ROBO4 promoter (3 kb), and mutation analysis indicated that this site was partially responsible for ROBO4 promoter activity in ECs. A combination of gain- and loss-of-function analysis identified Sox7 and Sox18 co-regulation of robo4 but not fli1a transcripts in zebrafish. Finally, Sox-mediated robo4 transcriptional regulation is conserved across evolution. These studies imply Sox-mediated transcriptional regulation of Robo4 in the developing embryonic vasculature.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Receptores de Superfície Celular/biossíntese , Proteínas de Peixe-Zebra/biossíntese , Animais , Movimento Celular , Análise Mutacional de DNA , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Camundongos , Mutação , Neovascularização Patológica , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Receptores de Superfície Celular/fisiologia , Fatores de Transcrição SOXF/metabolismo , Transcrição Gênica , Peixe-Zebra , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Endothelial cell-specific chemotaxis receptor (ECSCR) is a cell surface protein expressed by blood endothelial cells with roles in endothelial cell migration and signal transduction. We investigated the function of ecscr in the development of the zebrafish vasculature. Zebrafish ecscr is expressed in angioblasts and in axial vessels during angioblast migration and vasculogenesis. Morpholino-directed ecscr knockdown resulted in defective angioblast migration in the posterior lateral plate mesoderm, a process known to depend on vascular endothelial-derived growth factor (VEGF). In cultured cells, transfected ECSCR localized to actin-rich membrane protrusions, colocalizing with kinase insert domain protein receptor (KDR)/VEGF receptor 2 in these regions. ECSCR-silenced cells show reduced VEGF-induced phosphorylation of KDR but not of FMS-like tyrosine kinase 1 (FLT1)/VEGF receptor 1. Finally, chemical inhibition of VEGF receptor activity in zebrafish resulted in angioblast deficiencies that partially overlap with those seen in ecscr morphants. We propose that ecscr promotes migration of zebrafish angioblasts by enhancing endothelial kdr sensitivity to VEGF.
Assuntos
Vasos Sanguíneos/embriologia , Vasos Sanguíneos/metabolismo , Quimiotaxia/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose , Sequência de Bases , Movimento Celular , Proliferação de Células , Células Cultivadas , Primers do DNA/genética , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Transfecção , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Recently, messenger RNAs in eukaryotes have shown to associate with antisense (AS) transcript partners that are often referred to as long noncoding RNAs (lncRNAs) whose function is largely unknown. Here, we have identified a natural AS transcript for tyrosine kinase containing immunoglobulin and epidermal growth factor homology domain-1 (tie-1), tie-1AS lncRNA in zebrafish, mouse, and humans. In embryonic zebrafish, tie-1AS lncRNA transcript is expressed temporally and spatially in vivo with its native target, the tie-1 coding transcript and in additional locations (ear and brain). The tie-1AS lncRNA selectively binds tie-1 mRNA in vivo and regulates tie-1 transcript levels, resulting in specific defects in endothelial cell contact junctions in vivo and in vitro. The ratio of tie-1 versus tie-1AS lncRNA is altered in human vascular anomaly samples. These results directly implicate noncoding RNA-mediated transcriptional regulation of gene expression as a fundamental control mechanism for physiologic processes, such as vascular development.
Assuntos
Loci Gênicos/genética , RNA Antissenso/genética , RNA não Traduzido/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Animais , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Endotélio/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/metabolismo , Junções Intercelulares/patologia , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Fenótipo , RNA Antissenso/metabolismo , RNA não Traduzido/metabolismo , Receptor de TIE-1/genética , Receptor de TIE-1/metabolismo , Especificidade da Espécie , Doenças Vasculares/genética , Doenças Vasculares/patologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Proteínas de Peixe-Zebra/genéticaRESUMO
Mitogen-activated protein kinases play an integral role in several cellular processes. To regulate mitogen-activated protein kinases, cells express members of a counteracting group of proteins called phosphatases. In this study, we have identified a specific role that one member of this family of phosphatases, dual-specific phosphatase-5 (Dusp-5) plays in vascular development in vivo. We have determined that dusp-5 is expressed in angioblasts and in established vasculature and that it counteracts the function of a serine threonine kinase, Snrk-1, which also plays a functional role in angioblast development. Together, Dusp-5 and Snrk-1 control angioblast populations in the lateral plate mesoderm with Dusp-5 functioning downstream of Snrk-1. Importantly, mutations in dusp-5 and snrk-1 have been identified in affected tissues of patients with vascular anomalies, implicating the Snrk-1-Dusp-5 signaling pathway in human disease.
Assuntos
Vasos Sanguíneos/embriologia , Fosfatases de Especificidade Dupla/biossíntese , Regulação da Expressão Gênica , Hemangioma/enzimologia , Mesoderma/irrigação sanguínea , Proteínas de Neoplasias/biossíntese , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas de Peixe-Zebra/biossíntese , Peixe-Zebra/embriologia , Animais , Vasos Sanguíneos/patologia , Fosfatases de Especificidade Dupla/genética , Regulação da Expressão Gênica/genética , Hemangioma/genética , Hemangioma/patologia , Humanos , Mesoderma/embriologia , Mesoderma/patologia , Mutação , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
In vertebrates, molecular mechanisms dictate angioblasts' migration and subsequent differentiation into arteries and veins. In this study, we used a microarray screen to identify a novel member of the sucrose nonfermenting related kinase (snrk-1) family of serine/threonine kinases expressed specifically in the embryonic zebrafish vasculature and investigated its function in vivo. Using gain- and loss-of-function studies in vivo, we show that Snrk-1 plays an essential role in the migration, maintenance, and differentiation of angioblasts. The kinase function of Snrk-1 is critical for migration and maintenance, but not for the differentiation of angioblasts. In vitro, snrk-1 knockdown endothelial cells show only defects in migration. The snrk-1 gene acts downstream or parallel to notch and upstream of gridlock during artery-vein specification, and the human gene compensates for zebrafish snrk-1 knockdown, suggesting evolutionary conservation of function.
Assuntos
Artérias/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas Serina-Treonina Quinases/biossíntese , Veias/embriologia , Peixe-Zebra/embriologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Movimento Celular/fisiologia , Células Endoteliais/enzimologia , Técnicas de Silenciamento de Genes , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
A major hurdle preventing effective interventions for patients with mild traumatic brain injury (mTBI) is the lack of known mechanisms for the long-term cognitive impairment that follows mTBI. The closed head impact model of repeated engineered rotational acceleration (rCHIMERA), a non-surgical animal model of repeated mTBI (rmTBI), mimics key features of rmTBI in humans. Using the rCHIMERA in rats, this study was designed to characterize rmTBI-induced behavioral disruption, underlying electrophysiological changes in the medial prefrontal cortex (mPFC), and associated mitochondrial dysfunction. Rats received 6 closed-head impacts over 2 days at 2 Joules of energy. Behavioral testing included automated analysis of behavior in open field and home-cage environments, rotarod test for motor skills, novel object recognition, and fear conditioning. Following rmTBI, rats spent less time grooming and less time in the center of the open field arena. Rats in their home cage had reduced inactivity time 1 week after mTBI and increased exploration time 1 month after injury. Impaired associative fear learning and memory in fear conditioning test, and reduced short-term memory in novel object recognition test were found 4 weeks after rmTBI. Single-unit in vivo recordings showed increased neuronal activity in the mPFC after rmTBI, partially attributable to neuronal disinhibition from reduced inhibitory synaptic transmission, possibly secondary to impaired mitochondrial function. These findings help validate this rat rmTBI model as replicating clinical features, and point to impaired mitochondrial functions after injury as causing imbalanced synaptic transmission and consequent impaired long-term cognitive dysfunction.
RESUMO
Rho GTPases play an important and versatile role in several biological processes. In this study, we identified the zebrafish ortholog of the mammalian Rho A guanine exchange factor, synectin-binding guanine exchange factor (Syx), and determined its in vivo function in the zebrafish and the mouse. We found that Syx is expressed specifically in the vasculature of these organisms. Loss-of-function studies in the zebrafish and mouse point to a specific role for Syx in angiogenic sprouting in the developing vascular bed. Importantly, vasculogenesis and angioblast differentiation steps were unaffected in syx knockdown zebrafish embryos, and the vascular sprouting defects were partially rescued by the mouse ortholog. Syx knockdown in vitro impairs vascular endothelial growth factor-A-induced endothelial cell migration and angiogenesis. We have also uncovered a potential mechanism of endothelial sprout guidance in which angiomotin, a component of endothelial cell junctions, plays an additive role with Syx in directing endothelial sprouts. These results identify Syx as an essential contributor to angiogenesis in vivo.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neovascularização Fisiológica , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Proteína rhoA de Ligação ao GTP/metabolismo , Angiomotinas , Animais , Movimento Celular/fisiologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Junções Intercelulares/genética , Junções Intercelulares/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Pervasive transcription is a feature of the human genome that requires better understanding. Over the last decade or so, RNA species longer than 200 nucleotides-dubbed long non-coding RNA (lncRNAs)-had been found in sense or anti-sense orientation within or outside of genes that encode proteins. Importantly, lncRNA-mediated gene regulation and the elements that control lncRNA expression are a source of fascination among molecular biologists. In vascular biology, a dozen or so lncRNAs had been identified, and progress occurs each day. In this review, we highlighted our laboratories' contribution to the lncRNA field by discussing lessons learned from two lncRNAs in the tyrosine kinase containing immunoglobulin and epidermal growth factor homology1 (Tie1) and delta-like 4 (Dll4) loci. These genes are responsible for basic vascular patterning and pathophysiological remodeling in angiogenesis.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/genética , Neovascularização Fisiológica/genética , RNA Longo não Codificante/genética , Receptor de TIE-1/genética , Remodelação Vascular/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Padronização Corporal , Proteínas de Ligação ao Cálcio , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Animais , RNA Longo não Codificante/metabolismo , Receptor de TIE-1/metabolismo , Transdução de Sinais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The plasmacytoma variant translocation 1 gene (PVT1) is an lncRNA that has been designated as an oncogene due to its contribution to the phenotype of multiple cancers. Although the mechanism by which PVT1 influences disease processes has been studied in multiple cancer types, its role in cervical tumorigenesis remains unknown. Thus, the present study was designed to investigate the role of PVT1 in cervical cancer in vitro and in vivo. PVT1 expression was measured by quantitative PCR (qPCR) in 121 invasive cervical carcinoma (ICC) samples, 30 normal cervix samples, and cervical cell lines. Functional assays were carried out using both siRNA and LNA-mediated knockdown to examine PVT1's effects on cervical cancer cell proliferation, migration and invasion, apoptosis, and cisplatin resistance. Our results demonstrate that PVT1 expression is significantly increased in ICC tissue versus normal cervix and that higher expression of PVT1 correlates with poorer overall survival. In cervical cancer cell lines, PVT1 knockdown resulted in significantly decreased cell proliferation, migration and invasion, while apoptosis and cisplatin cytotoxicity were significantly increased in these cells. Finally, we show that PVT1 expression is augmented in response to hypoxia and immune response stimulation and that this lncRNA associates with the multifunctional and stress-responsive protein, Nucleolin. Collectively, our results provide strong evidence for an oncogenic role of PVT1 in cervical cancer and lend insight into potential mechanisms by which PVT1 overexpression helps drive cervical carcinogenesis.
Assuntos
Fenótipo , RNA Longo não Codificante/genética , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/genética , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Inativação Gênica , Humanos , Invasividade Neoplásica , Fosfoproteínas/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Neoplasias do Colo do Útero/patologia , NucleolinaRESUMO
We first investigated the cytotoxicity of three cycloartane triterpenoids isolated from the aerial part of C. dahurica. Their cytotoxic activity was investigated on several cancer cell lines including solid tumor (HepG2), blood tumor (HL-60), drug resistant tumor (R-HepG2) and primary cultured normal mouse and rat hepatocytes in order to find efficient anti-tumor agents against both parental and drug-resistant tumor with reduced toxicity. Evident cytotoxicity of these compounds on all tested neoplastic cell lines revealed that they are efficient on both drug-resistant tumor and parental tumor. Furthermore, they all showed relatively selective cytotoxicity on cancerous cells based on the higher IC(50) values of them on normal cells than that on tumor cells. Morphological observation and cell cycle analysis were employed to elucidate the cytotoxicity of the tested compounds. They brought out similar apoptotic morphological changes and G(2)/M cell cycle arrest in HepG2, R-HepG2 and HL-60 cells. Moreover, they suppressed the expression of cdc2 and COX-2 protein. These results imply that the three compounds possess potential anti-tumor activities and they exert their cytotoxicity via apoptosis and G(2)/M arrest. In addition, inhibition of cdc2 protein expression correlates with mechanism of G(2)/M arrest.
Assuntos
Ciclo Celular/efeitos dos fármacos , Cimicifuga/química , Glicosídeos/farmacologia , Triterpenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Citometria de Fluxo , Hepatócitos/efeitos dos fármacos , Humanos , Camundongos , Ratos , Células Tumorais CultivadasRESUMO
Alginate (Alg), chitosan (Chi), collagen (Col), gelatin (Gel), and the mixtures of every two of them were screened to determine their suitability for hepatocyte culture. The test materials were fabricated as films and then evaluated on the basis of their abilities to promote the attachment and functions of rat hepatocytes cultured on them. Cellular attachment on Col and Gel was favorable. However, cellular viability, cytoskeleton organization and function, as evaluated by albumin production, ureagenesis, and enzyme activity of cytochrome P450 as well as expression levels of hepatocyte nuclear factor 4 alpha deteriorated. Reverse cellular behavior was observed on Alg and Chi. Two blends, composed of Chi and Col or Gel, were found to be superior to other materials and sustained viability and differentiated functions of hepatocyte.
Assuntos
Materiais Biocompatíveis/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Polímeros/metabolismo , Alginatos/química , Alginatos/metabolismo , Animais , Materiais Biocompatíveis/química , Adesão Celular , Forma Celular , Sobrevivência Celular , Quitosana/química , Quitosana/metabolismo , Colágeno/química , Colágeno/metabolismo , Citoesqueleto/metabolismo , Gelatina/química , Gelatina/metabolismo , Ácido Glucurônico/química , Ácido Glucurônico/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Ácidos Hexurônicos/química , Ácidos Hexurônicos/metabolismo , Teste de Materiais , Polímeros/química , Ratos , Ratos Sprague-Dawley , Propriedades de Superfície , Engenharia TecidualRESUMO
BACKGROUND: Recent evidence suggests that a majority of RNAs in the genome do not code for proteins. They are located in the sense (S) or antisense (AS) orientation and, to date, the functional significance of these non-coding RNAs (ncRNAs) is poorly understood. Here, we examined the relationship between S and AS transcripts in the regulation of a key angiogenesis gene, Delta-like 4 (Dll4). METHODS: Rapid Amplification of cDNA Ends (RACE) method was used to identify natural antisense transcripts in the Dll4 gene locus in murine and human endothelial cells, referred to as Dll4 Anti-Sense (Dll4-AS). Messenger RNA (mRNA) levels of Dll4 and Dll4-AS were quantified by real-time PCR. The function of Dll4-AS was investigated by overexpression and knocking down of Dll4-AS. RESULTS: Dll4-AS comprises of three isoforms that map proximal to the Dll4 promoter region. Expression patterns of Dll4-AS isoforms vary among different endothelial cell lines, but are always congruent with those of Dll4. A dual promoter element in the Dll4 locus has been identified that controls the expression of both transcripts. Both Dll4-AS and Dll4 are sensitive to cellular density in that higher cellular density favors their expression. Exogenous Dll4 stimuli such as VEGF, FGF and Notch signaling inhibitor altered both DLL4-AS and DLL4 expression suggesting co-regulation of the transcripts. Also, knocking down of Dll4-AS results in down-regulation of Dll4 expression. As a consequence, endothelial cell proliferation and migration increases in vitro, and sprout formation increases. The regulation of Dll4 by Dll4-AS was also conserved in vivo. CONCLUSION: A novel form of non-coding RNA-mediated regulation at the Dll4 locus contributes to vascular developmental processes such as cell proliferation, migration and sprouting.
RESUMO
Necrotizing enterocolitis (NEC) is a severe form of bowel disease that develops in premature infants. Although animal data and human studies suggest that aberrant activation of the intestinal immune system contributes to NEC, the pathogenesis remains unclear. We hypothesized that inherited defects in the regulation of Toll-like receptor signaling can contribute to NEC susceptibility in premature infants. A forward genetic screen done in an infant with lethal NEC using exome sequencing identified a novel stop mutation (p.Y168X) and a rare missense variant (p.S80Y) in SIGIRR, a gene that inhibits intestinal Toll-like receptor signaling. Functional studies carried out in human embryonic kidney cells and intestinal epithelial cells demonstrated that SIGIRR inhibited inflammation induced by lipopolysaccharide, a cell wall component of Gram-negative bacteria implicated in NEC. The genetic variants identified in the infant with NEC resulted in loss of SIGIRR function and exaggerated inflammation in response to lipopolysaccharide. Additionally, Sanger sequencing identified missense, stop, or splice region SIGIRR variants in 10 of 17 premature infants with stage II+ NEC. To the best of our knowledge, this is one of the first reports of a phenotype associated with SIGIRR in humans. Our data provide novel mechanistic insight into the probable causation of NEC and support additional investigation of the hypothesis that inherited defects in the regulation of innate immune signaling can contribute to NEC susceptibility in premature infants.
Assuntos
Enterocolite Necrosante/genética , Variação Genética , Receptores de Interleucina-1/genética , Feminino , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Masculino , MutaçãoRESUMO
OBJECTIVES: To establish a surveillance system for antituberculous drug resistance according to WHO and IUATLD guidelines, to collect the data of initial and acquired drug resistance, and to evaluate the efficacy of ongoing tuberculosis control strategies. METHODS: Thirty counties (or cities or districts) were randomly selected as surveillance points by cluster sampling. Thirty new cases of smear positive pulmonary tuberculosis in each cluster were enrolled consecutively. Bacterial typing and susceptibility tests (proportion method for SM, INH, RFP and EMB) were done in all culture positive samples. Laboratory quality control was implemented by Korean Super-country Reference Laboratory. Data were analyzed by WHO SDRTB2 software. RESULTS: 1,136 patients with pulmonary tuberculosis were enrolled in 30 clusters, among them 1,097 were culture positive. Smear-positive culture-positive rate was 96.6% and the contamination rate was 0.4%. The total drug resistance rate was 23.3%, the initial drug resistance being 17.5% and the acquired drug resistance being 44.5%. The initial multi-drug resistance rate (H + R) was 2.1% and the acquired multi-drug resistance rate (H + R) was 21.8%. CONCLUSIONS: Laboratory methods for surveillance of drug resistant tuberculosis and quality control were established, and reliable data on the status of antituberculous drug resistance in Hubei province were collected. The results showed that implementation of DOTS and regular surveillance were very important.
Assuntos
Antituberculosos/farmacologia , Resistência a Múltiplos Medicamentos/fisiologia , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Distribuição por Idade , Fatores Etários , Antituberculosos/uso terapêutico , China/epidemiologia , Farmacorresistência Bacteriana , Humanos , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologiaRESUMO
In this study, we have identified a novel member of the AMPK family, namely Sucrose non-fermenting related kinase (Snrk), that is responsible for maintaining cardiac metabolism in mammals. SNRK is expressed in the heart, and brain, and in cell types such as endothelial cells, smooth muscle cells and cardiomyocytes (CMs). Snrk knockout (KO) mice display enlarged hearts, and die at postnatal day 0. Microarray analysis of embryonic day 17.5 Snrk hearts, and blood profile of neonates display defect in lipid metabolic pathways. SNRK knockdown CMs showed altered phospho-acetyl-coA carboxylase and phospho-AMPK levels similar to global and endothelial conditional KO mouse. Finally, adult cardiac conditional KO mouse displays severe cardiac functional defects and lethality. Our results suggest that Snrk is essential for maintaining cardiac metabolic homeostasis, and shows an autonomous role for SNRK during mammalian development.
RESUMO
The extracellular matrix plays a critical role in neural crest (NC) cell migration. In this study, we characterize the contribution of the novel GPI-linked matrix metalloproteinase (MMP) zebrafish mmp17b. Mmp17b is expressed post-gastrulation in the developing NC. Morpholino inactivation of mmp17b function, or chemical inhibition of MMP activity results in aberrant NC cell migration with minimal change in NC proliferation or apoptosis. Intriguingly, a GPI anchored protein with metalloproteinase inhibitor properties, Reversion-inducing-Cysteine-rich protein with Kazal motifs (RECK), which has previously been implicated in NC development, is expressed in close apposition to NC cells expressing mmp17b, raising the possibility that these two gene products interact. Consistent with this possibility, embryos silenced for mmp17b show defective development of the dorsal root ganglia (DRG), a crest-derived structure affected in RECK mutant fish sensory deprived (sdp). Taken together, this study has identified the first pair of MMP, and their putative MMP inhibitor RECK that functions together in NC cell migration.
Assuntos
Movimento Celular/genética , Metaloproteinase 17 da Matriz/genética , Metaloproteinase 17 da Matriz/metabolismo , Crista Neural/citologia , Crista Neural/metabolismo , Sequência de Aminoácidos , Animais , Padronização Corporal/genética , Desenvolvimento Embrionário/genética , Ativação Enzimática , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Perfilação da Expressão Gênica , Metaloproteinase 17 da Matriz/química , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Alinhamento de Sequência , Peixe-ZebraRESUMO
The vertebrate genome contains large spans of non-coding RNA, which for the most part were considered of little functional value to the organism. Recent studies have indicated that vertebrate genomes may have stored hidden secrets in this large span of non-coding RNA, which we refer to here as "Natural Antisense Transcripts (NATs)." NATs can be found in introns, exons, promoters, enhancers, intergenic sequences, and untranslated regions of the genome. They can be located in either the plus or minus DNA strand. NATs utilize several mechanisms that include DNA replication interference, chromatin remodeling, transcriptional interference, RNA masking, double-stranded RNA (dsRNA)-dependent mechanisms and translation interference to mechanistically regulate gene expression. Recently, NAT levels have been identified as dysregulated in various disease states. This review presents an overview of the current state of NAT biology and highlights the main points with specific examples.