Unknown,Transcriptomics,Genomics,Proteomics

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Hypoxia induced gene expression in lung adenocarcinoma cells

ABSTRACT: Two lung adenocarcinoma cell lines, A549 and SPC-A1, were cultured under two different oxygen concentrations, 1% and 20%, respectively. After 24 hours cultured, cells were collected and total RNA was extracted. Affymetrix GeneChip® Gene 1.0 ST Array System for Human was applied to detect the differences of gene expression of lung adenocarcinoma cell lines cultured under different oxygen concentrations (total RNA of the two cells was pooled with equal quantity). The experiments were repeated with the same procedures except culturing tumor cell lines in matrigel.

ORGANISM(S): Homo sapiens

SUBMITTER: Guichun Huang

PROVIDER: E-MTAB-3512 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:- Background and Aims: Oxygen can fall to low concentrations within plant tissues, either because of environmental factors that decrease the external oxygen concentration or because the movement of oxygen through the plant tissues cannot keep pace with the rate of oxygen consumption. Recent studies document that plants can decrease their oxygen consumption in response to relative small changes in oxygen concentrations to avoid internal anoxia. The molecular mechanisms underlying this response have not been identified yet. The aim of this study was to use transcript and metabolite profiling to investigate the genomic response of Arabidopsis roots to a mild decrease in oxygen concentrations. - Methods: Arabidopsis seedlings were grown on vertical agar plates at 21, 8, 4 and 1% (v/v) external oxygen for 0.5, 2 and 48h. Roots were analyzed for changes in transcript levels using Affymetrix whole genome DNA microarrays, and for changes in metabolite levels using routine GC-MS based metabolite profiling. Root extension rates were monitored in parallel to investigate adaptive changes in growth. - Key results: Results show that root growth was inhibited and transcript and metabolite profiles were significantly altered in response to a moderate decrease in oxygen concentrations. Low oxygen leads to a preferential up-regulation of genes that might be important to trigger adaptive responses in the plant. A small but highly specific set of genes is induced very early in response to a moderate decrease in oxygen concentrations. Genes that were down-regulated mainly encoded proteins involved in energy-consuming processes. In line with this, root extension growth was significantly decreased which will ultimately save ATP and decrease oxygen consumption. This was accompanied by a differential regulation of metabolite levels at short and long term incubation at low oxygen. - Conclusions: Results show that there are adaptive changes in root extension involving large-scale reprogramming of gene expression and metabolism when oxygen concentration is decreased in a very narrow range. Experiment Overall Design: Arabidopsis seedlings were grown on vertical agar plates and treated with various oxygen concentrations (1%, 4%, 8%, and 21% as a control), 350ppm CO2 and N2 (rest) for different time periods (0.5 hours, 2 hours and 2 days. At the end of the experiment, the roots of the seedlings were immediately frozen in liquid nitrogen and used for gene expression analysis.

Project description:Background: Genes upregulated by low oxygen have been suggested as endogenous markers for tumor hypoxia. Yet, most of the genes investigated have shown inconsistent results, which have led to concerns about their ability to be true hypoxia markers. Previous studies have demonstrated that expression of hypoxia induced genes can be affected by extracellular pH (pH e ). Methods: Five different human cell lines (SiHa, FaDu DD, UTSCC5, UTSCC14 and UTSCC15) were exposed to different oxygen concentrations and pH (7.5 or 6.3), and gene expression analyzed with microarray (Affymetrix - Human Genome U133 Plus 2.0 Array). Results: An analysis of two of the cell lines using SAM identified 461 probesets that were able to separate the four groups “ Normal oxygen, normal pH ” , “ Low oxygen, normal pH ” , “ Normal oxygen, low pH ” and “ Low oxygen, low pH ” . From here it was possible to identify a fraction of probesets induced at low oxygen independent of pH in these two cell lines, this fraction included HIG2, NDRG1, PAI1 and RORA. Further verifi cation by qPCR highlighted the necessity of using more cell lines to obtain a robust gene expression profi les. To specifi cally select pH independent hypoxia regulated genes across more cell lines, data for FaDu DD, UTSCC5, UTSCC14 and UTSCC15 were analyzed to identify genes that were induced by hypoxia in each cell line, where the induction was not affected by low pH, and where the gene was not signifi cantly induced by low pH alone. Each cell line had 65 – 122 probesets meeting these criteria. For genes to be considered as target genes (hypoxia inducible pH independent), genes had to be present in three of four cell lines. Conclusion: The result is a robust hypoxia profile unaffected by pH across cell lines consisting of 27 genes. This study demonstrates a way to identify hypoxia markers by microarray, where other factors in the tumor microenvironment are taken into account. Five cell lines, four HNSCC (FaDuDD, UTSCC5, UTSCC14 and UTSCC15) and one cervical (SiHa). Hypoxia was achieved by continually gassing the cells in an airtight chamber for 24 hours with either atmo- spheric air, 5%, 1%, 0.1%, 0.01% or 0% oxygen, sup- plemented with 5% CO 2 and nitrogen, at 37ºC. To simulate acidosis, pH of the medium (without sodium bicarbonate) was buffered with 20 mM tris (Hydroxym- ethyl) aminomethane (TRIS) base (Sigma), 20 mM 2-(N-Morpholino)ethanesulfonic acid (MES) (Sigma) and 0.52 g/l NaHCO 3 (Sigma) and titrated to 6.3 or 7.4. The pH adjusted media was applied immediately before the induction of hypoxia. No replicates.

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Hypoxia induced gene expression in lung adenocarcinoma cells

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