Project description:We report the results NGS based of miRNA profiling in human endothelial cells under chronic and cyclic hypoxia Solid tumor microenvironments are often subjected to various levels of hypoxia. Although regulation of gene expression has been examined extensively, most studies have focused on chronic hypoxia. The tumor microenvironment, however, experience waves of hypoxia and reoxygenation that stimulates the expression of pro-angiogenic factors that promote blood vessel formation. In this study, we examined human umbilical vascular endothelial cells (HUVECs) under waves of intermittent (cyclic hypoxia) to determine how this process compares to chronic hypoxia, and more importantly, how this influences the microRNA profiles that potentially affect the posttranscriptional regulation of angiogenic genes. The rationale for these studies is that cancer cells subjected to cyclic hypoxia appear to have increased metastatic potential and endothelial cells exhibit a higher radiation resistance and greater migration potential. This indicates that the gene regulatory networks in cyclic hypoxia may be different from chronic hypoxia. Here we examined the consequences of cyclic hypoxia on miRNA gene expression and how these changes in miRNA expression could influence angiogenesis. Using Next Generation Sequencing, our results demonstrate that cyclic hypoxia has very different effects on the miRNA networks compared to chronic hypoxia, the in silico predicted effects on the certain mRNA target genes are more similar than might be expected. More importantly, these studies indicate that identifying potential miRNAs (including hsa-miR-19a-5p) as therapeutic targets for inhibiting angiogenesis and tumor progression will require this type of physiologically relevant analysis.
Project description:This work aims to characterize cycling hypoxia induced changes in metabolism related genes expression in pancreatic cancer cell line. PANC1were exposed to either 7 hours cycles of hypoxia every other day for 20 cycles cyclic acute hypoxia, or to 72 hours cycles of hypoxia once a week for 5 cycles cyclic chronic hypoxia. Gene expression changes were profiled using RT PCR and compared to cells under normoxia. Western blotting analysis confirmed upregulation of hypoxia inducible factor 1 α, glucose 6 phosphate isomerase gene, and ribokinase gene. Genes encoding glycolysis enzymes were upregulated under cyclic acute more than chronic hypoxia including hexokinase2 and phosphoglycerate kinase1. Genes encoding pentose phosphate pathway enzymes transketolase and transaldolase were upregulated similarly. Genes encoding pyruvate dehydrogenases that block pyruvate flow to TCA cycle were significantly upregulated. Exposure of PANC1 cells to acute hypoxia results in upregulation of genes that shift cells metabolism toward glycolysis and pentose phosphate pathways in adaptation to hypoxic stress
Project description:Tumor hypoxia is not a stable phenomenon but cycles between periods of deep hypoxia and reoxygenation. Cyclic hypoxia originates from heterogeneities in red blood cell flux and from the permanent remodelling of the angiogenic vascular network. Endothelial cells lining tumor blood vessels are therefore also influenced by cyclic hypoxia. The gene expression pattern promoted by cyclic hypoxia differs from those observed under normoxia and even continuous hypoxia. PTGS2 is one gene exquisitely up-regulated in endothelial cells (and tumor cells) in response to cyclic hypoxia. Elevated COX-2 (the PTGS2 gene product) expression and activity account for cyclic hypoxia-driven increase in endothelial cell survival and angiogenesis.
Project description:Major depressive disorder (MDD) is the psychiatric disorder with the highest prevalence in the world. Pharmacological antidepressant treatment (AD), such as selective serotonin reuptake inhibitors [SSRI, i.e. fluoxetine (Flx)] is the first line of treatment for MDD. Despite its efficacy, lack of AD response occurs in numerous patients characterizing Difficult-to-treat Depression. ElectroConvulsive Therapy (ECT) is a highly effective treatment inducing rapid improvement in depressive symptoms and high remission rates of ~50–63% in patients with pharmaco-resistant depression. Nevertheless, the need to develop reliable treatment response predictors to guide personalized AD strategies and supplement clinical observation is becoming a pressing clinical objective. Here, we propose to establish a proteomic peripheral biomarkers signature of ECT response in an anxio/depressive animal model of non-response to AD. Using an emotionality score based on the analysis complementary behavioral tests of anxiety/depression (Elevated Plus Maze, Novelty Suppressed Feeding, Splash Test), we showed that a 4-week corticosterone treatment (35 μg/ml, Cort model) in C57BL/6JRj male mice induced an anxiety/depressive-like behavior. A 28-days chronic fluoxetine treatment (Flx, 18 mg/kg/day) reduced corticosterone-induced increase in emotional behavior. A 50% decrease in emotionality score threshold before and after Flx, was used to separate Flx-responding mice (Flx-R, n=18), or Flx non-responder mice (Flx-NR, n=7). Then, Flx-NR mice received 7 sessions of electroconvulsive seizure (ECS, equivalent to ECT in humans) and blood was collected before and after ECS treatment. Chronic ECS normalized the elevated emotionality observed in Flx-NR mice. Then, proteins were extracted from peripheral blood mononuclear cells (PBMCs) and isolated for proteomic analysis using a high-resolution MS Orbitrap. The proteomic analysis revealed a signature of 33 peripheral proteins associated with response to ECS (7 down- and 26 upregulated). These proteins were previously associated with mental disorders and involved in regulating pathways which participate to the depressive disorder etiology. remark for sample name : ECTR=Flx-NR-ES, C=cort/Veh, CFNR= Flx-NR
Project description:Hypoxia shapes the tumor microenvironment and modulates distinct cell populations activity. Low pO2 activates pathological angiogenesis in cancer- process in which endothelial cells (ECs) are the most important players. The main goal of the study was to evidence the hypoxic tumor microenvironment influence on the global gene expression pattern characteristic for ECs and evidence the distinct and specific responses displayed by the tumor-derived ECs compared to the healthy endothelium during the endothelial to mesenchymal transition (EndMT) and its regulation by miR-200-b-3p. Immortalized lines of ECs from the same patient with diagnosed breast cancer- healthy breast tissue (HBH.MEC) and primary tumor (HBCa.MEC) were used. The experiments were performed in normoxia and hypoxia for 48 hours. By wound healing test, we investigated the migration abilities of ECs. Using the established model, global gene expression analysis with NGS was carried out to detect new pathways altered in pathological ECs and to find the most changed miRNAs. The validation of NGS data from RNA and miRNA was estimated by qPCRs. Hypoxia influences ECs migration properties in wound healing assay. In hypoxia, healthy ECs migrate slower than in normoxia, as opposed to HBCa.MEC where no decreased migration ability was induced by hypoxia, due to EndMT features. NGS data identified this process to be altered in cancer ECs through extracellular matrix (ECM) organization. The deregulated genes, confirmed by qPCR, included: SPP1, ITGB6, COL4A4, ADAMST2, LAMA1, GAS6, AGTR2, PECAM1, ELN, FBLN2, COL6A3, COL9A3. NGS also identified collagens, laminin, fibronectin and integrin, as being deregulated in tumor-derived ECs. Moreover, the analysis of ten most intensively modified miRNAs when breast tumor-derived ECs were compared to healthy ECs, put a light on miR-200b-3p which is strongly up-regulated in HBCa.MECs as compared to HBH.MECs. Microenvironment influences on ECs dedifferentiation by miR-200-b-3p and ECM. The pathological ECs differed significantly, both phenotypically and functionally, from the normal corresponding tissue, thus influencing their microenvironment cross- talk. The gene expression profile confirms the EndMT phenotype of tumor-derived ECs and migratory properties acquisition. Moreover, it indicates the role of miR-200b-3p, regulating EndMT in pathological ECs, silencing several angiogenic growth factors and their receptors by directly targeting their mRNA transcripts.
Project description:Neuronal activity-dependent gene expression plays important roles in neural plasticity. We use electroconvulsive stimulation (ECS) as an in vivo model for neuronal activation to identify genes that are regulated by neuronal activity. Dentate gyri (DG) were microdissected 4 hours after sham or ECS treatment for gene expression profiling. 4 total samples were analysed (2 for each condition). Averaged expression values between sham and ECS samples were pair-wise compared.
Project description:High altitude environments are characterized by the unique and unavoidable stress of chronic hypoxia. While much is known about gene expression responses to acute or in vitro hypoxia, less is known about the gene expression profiles of animals exposed to systemic chronic hypoxia, such as that experienced at high elevations. Here we simulated the hypoxic environment of two high altitude elevations,and a third chamber recieved ambient Reno air. Mice were housed in the hypoxic chambers for 32 days. We used microarrays to characterize the differential gene expression in the livers of mice housed in hypoxic environment of 4500 m versus 3000 and 1400 m. We used this data to draw hypotheses related to novel physiological responses to chronic systemic hypoxia
Project description:Background - Hepatocellular carcinomas (HCCs) are heterogeneous tumors with respect to etiology, cell of origin and biology. The course of the disease is unpredictable and is in part dependent on the tumor microenvironment. One of the microenvironmental factors is hypoxia, which is known to promote aggressiveness in other malignant tumors. We hypothesized that certain regions in HCC exist with chronic hypoxia and a characteristic gene expression pattern. Moreover, during the development of HCC there is an important contribution of this chronic hypoxia on prognosis via this gene expression. Until now, most research has been performed in acute hypoxic models (< 24 hours). Methods – Human hepatoblastoma cells HepG2 were cultured in either normoxic (20% O2) or hypoxic (2% O2) conditions for 72 hrs, the time it takes to adapt to chronic hypoxia. After 3 days the cells were harvested and analyzed by microarray technology. The highly significant differentially expressed genes were selected and used to assess the clinical value of our in vitro chronic hypoxia gene signature in four published patient studies. Three of these independent microarray studies on HCC patients were used as training sets to determine a minimal prognostic gene set and one study was used for validation. Gene expression analysis and correlation with clinical outcome was assessed with the bioinfomatic method of Goeman et al (). Results – In the HepG2 cells, 3592 genes were differentially expressed in cells cultured at 2% oxygen for 72 hrs. Out of these, 265 showed a high significant change (2-fold change and p=0.0001). The level of gene expression after 72 hrs was different from the acute hypoxic response (during the first 24 hours) and represented chronicity. Using computational methods we identified 7 out of the 265 highly significant genes that showed correlation with prognosis in all three different training sets and this was independently validated in a 4th dataset. With our approach we could include the largest number of HCC patients in one single study. Conclusion – We identified a 7-gene signature, which is associated with chronic hypoxia and predicts prognosis in patients with HCC. In the future this signature could be used as a diagnostic tool. In addition, chronic hypoxia gene expression information can be used in the search for new therapeutic targets. Two conditions were compared and each sample has a biological replicate. Samples are hybridized in dye-swap, resulting in 4 hybridizations.
Project description:In this study, we determined the effect of chronic consistent hypoxia (CCH) and chronic intermittent hypoxia (CIH) on global gene expression in cortical and hippocampal region of developing mouse brain using long-oligo beadschip arrays. Keywords: Gene expression profile