A functional and transcriptomic analysis of NET1 bioactivity in gastric cancer

<p>Abstract</p> <p>Background</p> <p>NET1, a RhoA guanine exchange factor, is up-regulated in gastric cancer (GC) tissue and drives the invasive phenotype of this disease. In this study, we aimed to determine the role of NET1 in GC by monitoring the proliferation, motility and invasion of GC cells in which NET1 has been stably knocked down. Additionally, we aimed to determine NET1-dependent transcriptomic events that occur in GC.</p> <p>Methods</p> <p>An in vitro model of stable knockdown of NET1 was achieved in AGS human gastric adenocarcinoma cells via lentiviral mediated transduction of short-hairpin (sh) RNA targeting NET1. Knockdown was assessed using quantitative PCR. Cell proliferation was assessed using an MTS assay and cell migration was assessed using a wound healing scratch assay. Cell invasion was assessed using a transwell matrigel invasion assay. Gene expression profiles were examined using affymetrix oligonucleotide U133A expression arrays. A student's t test was used to determine changes of statistical significance.</p> <p>Results</p> <p>GC cells were transduced with NET1 shRNA resulting in a 97% reduction in NET1 mRNA (p < 0.0001). NET1 knockdown significantly reduced the invasion and migration of GC cells by 94% (p < 0.05) and 24% (p < 0.001) respectively, while cell proliferation was not significantly altered following NET1 knockdown. Microarray analysis was performed on non-target and knockdown cell lines, treated with and without 10 μM lysophosphatidic acid (LPA) allowing us to identify NET1-dependent, LPA-dependent and NET1-mediated LPA-induced gene transcription. Differential gene expression was confirmed by quantitative PCR. Shortlisted NET1-dependent genes included STAT1, TSPAN1, TGFBi and CCL5 all of which were downregulatd upon NET1 downregulation. Shortlisted LPA-dependent genes included EGFR and PPARD where EGFR was upregulated and PPARD was downregulated upon LPA stimulation. Shortlisted NET1 and LPA dependent genes included IGFR1 and PIP5K3. These LPA induced genes were downregulated in NET1 knockdown cells.</p> <p>Conclusions</p> <p>NET1 plays an important role in GC cell migration and invasion, key aspects of GC progression. Furthermore, the gene expression profile further elucidates the molecular mechanisms underpinning NET1-mediated aggressive GC cell behaviour.</p

Intronic Cis-Regulatory Modules Mediate Tissue-Specific and Microbial Control of angptl4/fiaf Transcription

The intestinal microbiota enhances dietary energy harvest leading to increased fat storage in adipose tissues. This effect is caused in part by the microbial suppression of intestinal epithelial expression of a circulating inhibitor of lipoprotein lipase called Angiopoietin-like 4 (Angptl4/Fiaf). To define the cis-regulatory mechanisms underlying intestine-specific and microbial control of Angptl4 transcription, we utilized the zebrafish system in which host regulatory DNA can be rapidly analyzed in a live, transparent, and gnotobiotic vertebrate. We found that zebrafish angptl4 is transcribed in multiple tissues including the liver, pancreatic islet, and intestinal epithelium, which is similar to its mammalian homologs. Zebrafish angptl4 is also specifically suppressed in the intestinal epithelium upon colonization with a microbiota. In vivo transgenic reporter assays identified discrete tissue-specific regulatory modules within angptl4 intron 3 sufficient to drive expression in the liver, pancreatic islet β-cells, or intestinal enterocytes. Comparative sequence analyses and heterologous functional assays of angptl4 intron 3 sequences from 12 teleost fish species revealed differential evolution of the islet and intestinal regulatory modules. High-resolution functional mapping and site-directed mutagenesis defined the minimal set of regulatory sequences required for intestinal activity. Strikingly, the microbiota suppressed the transcriptional activity of the intestine-specific regulatory module similar to the endogenous angptl4 gene. These results suggest that the microbiota might regulate host intestinal Angptl4 protein expression and peripheral fat storage by suppressing the activity of an intestine-specific transcriptional enhancer. This study provides a useful paradigm for understanding how microbial signals interact with tissue-specific regulatory networks to control the activity and evolution of host gene transcription

Remoção de introdutor arterial pós-intervenção coronária percutânea: médico residente versus enfermeiro especializado Arterial sheath removal after percutaneous coronary intervention: resident versus specialized nurse

OBJETIVO: Comparar os resultados da retirada de introdutor arterial pelo enfermeiro especializado em Unidade de Hemodinâmica e pelo médico residente em Cardiologia Intervencionista em pacientes submetidos à intervenção coronária percutânea. MÉTODOS: Trata-se de registro prospectivo em 100 pacientes submetidos à intervenção coronária percutânea, no período de setembro a outubro de 2004, divididos em dois grupos: Grupo A (GA) - enfermeiro (n = 48 pacientes) - e Grupo B (GB) - médico residente (n = 52 pacientes). Hematoma pequeno foi definido como inchaço palpável no local da punção menor que 2 cm; hematoma moderado, com 2 a 6 cm de diâmetro; e hematoma grande, maior que 6 cm de diâmetro. A dose de heparina foi de 100 UI/kg. Os introdutores foram retirados após controle do tempo de coagulação ativado (TCA < 180 segundos), e foi realizada compressão manual por 15 minutos. RESULTADOS: A idade dos pacientes foi de 59,54 &plusmn; 11,1 anos (GA) e 61,7 &plusmn; 10,4 anos (GB), com predomínio do sexo masculino (GA = 75% e GB = 58%). Os introdutores foram 7 French. O tempo de compressão manual foi de 19,4 &plusmn; 3,1 minutos no GA e 19,6 &plusmn; 3,1 minutos no GB (P = 0,76). Ocorreram oito hematomas no GA (sete pequenos e um moderado) e nove hematomas no GB (sete pequenos e dois moderados), P = não-significante. Os hematomas foram tratados clinicamente, sem complicações. CONCLUSÃO: A retirada de introdutor arterial, após intervenções coronárias percutâneas, pode ser realizada pelo enfermeiro especializado em Unidade de Hemodinâmica ou pelo médico residente em Cardiologia Intervencionista com segurança e sem complicações maiores.<br>OBJECTIVE: To compare the results of sheath removal by the catheterization lab specialist nurse and by the interventional cardiology resident in patients submitted to a percutaneous coronary intervention. METHODS: Prospective study with 100 patients submitted to percutaneous coronary intervention, from September to October 2004, who were divided into two groups: Group A (GA) - nurse (n = 48) and Group B (GB) - resident (n = 52). Small hematoma was defined as a palpable swelling at the access site measuring less than 2 cm; mild hematoma, from 2 to 6 cm in diameter; and large hematoma when it was larger than 6 cm in diameter. The heparin dose was 100 IU/kg. The sheaths were removed after activated coagulation time control (ACT < 180 seconds) and a 15-minute manual compression was used. RESULTS: Patients' age was 59.54 &plusmn; 11.1 (GA) and 61.7 &plusmn; 10.4 (GB) years with a predominance of male patients (GA = 75% and GB = 58%). 7F sheaths were used. Manual compression time was 19.4 &plusmn; 3.1 min for GA and 19.6 &plusmn; 3.1 min for GB (P = 0.76). There were eight hematomas in GA (seven small and one mild) and nine hematomas in GB (seven small and two mild), P = nonsignificant. The hematomas were clinically treated, with no complications. CONCLUSION: Arterial sheath removal, after percutaneous coronary interventions, can be made by the catheterization lab specialist nurse or interventional cardiology resident safely and without major complications