Identification of Hypoxia-Induced Genes in Human SGBS Adipocytes by Microarray Analysis

Hypoxia in adipose tissue is suggested to be involved in the development of a chronic mild inflammation, which in obesity can further lead to insulin resistance. The effect of hypoxia on gene expression in adipocytes appears to play a central role in this inflammatory response observed in obesity. However, the global impact of hypoxia on transcriptional changes in human adipocytes is unclear. Therefore, we compared gene expression profiles of human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes under normoxic or hypoxic conditions to detect hypoxia-responsive genes in adipocytes by using whole human genome microarrays. Microarray analysis showed more than 500 significantly differentially regulated mRNAs after incubation of the cells under low oxygen levels. To gain further insight into the biological processes, hypoxia-regulated genes after 16 hours of hypoxia were classified according to their function. We identified an enrichment of genes involved in important biological processes such as glycolysis, response to hypoxia, regulation of cellular component movement, response to nutrient levels, regulation of cell migration, and transcription regulator activity. Real-time PCR confirmed eight genes to be consistently upregulated in response to 3, 6 and 16 hours of hypoxia. For adipocytes the hypoxia-induced regulation of these genes is shown here for the first time. Moreover in six of these eight genes we identified HIF response elements in the proximal promoters, specific for the HIF transcription factor family members HIF1A and HIF2A. In the present study, we demonstrated that hypoxia has an extensive effect on gene expression of SGBS adipocytes. In addition, the identified hypoxia-regulated genes are likely involved in the regulation of obesity, the incidence of type 2 diabetes, and the metabolic syndrome

A common variant of the MACC1 gene is significantly associated with overall survival in colorectal cancer patients

<p>Abstract</p> <p>Background</p> <p>The newly discovered metastasis-associated in colon cancer-1 (MACC1) gene is a key regulator of the HGF/MET pathway. Deregulation of HGF/MET signaling is reported as a prognostic marker for tumorigenesis, early stage invasion, and metastasis. High expression levels of MACC1 have been associated with colon cancer metastasis and reduced survival. Potential links between the genetic diversity of the MACC1 locus and overall survival are unknown. We therefore investigated the association between MACC1 tagging single nucleotide polymorphisms (SNPs) and overall survival in a large cohort of colorectal cancer patients.</p> <p>Methods</p> <p>The study included 318 subjects with histopathologically proven colorectal cancer at the Academic Teaching Hospital Feldkirch, Austria. Survival data were provided by the federal agency for statistics in Austria. Genomic DNA was isolated from formalin-fixed paraffin-embedded specimens; six tagging SNPs (rs1990172, rs3114446, rs10275612, rs3095007, rs3095009, and rs7780032), capturing most of the common variants of the MACC1 locus, were genotyped by SNaPshot assays.</p> <p>Results</p> <p>Over a mean follow up period of 5.3 (± 1.0) years, 94 deaths were recorded. Carriers of the G-allele of SNP rs1990172 showed a significantly decreased overall survival (additive HR = 1.38 [1.05-1.82]; <it>p </it>= 0.023). Multivariate analysis adjusted for age and UICC tumor stage confirmed this result (HR = 1.49 [1.12-1.98]; <it>p </it>= 0.007). Other investigated genetic variants of the MACC1 gene were not significantly associated with overall survival (<it>p</it>-values > 0.05).</p> <p>Conclusions</p> <p>For the first time, our study investigated the influence of MACC1 tagging polymorphisms on overall survival suggesting SNP rs1990172 as a predictor for reduced overall survival in colorectal cancer patients. Further studies will be required to validate our findings.</p

Single nucleotide polymorphisms of TCF7L2 are linked to diabetic coronary atherosclerosis.

BackgroundCoronary artery disease (CAD) shares common risk factors with type 2 diabetes (T2DM). Variations in the transcription factor 7-like 2 (TCF7L2) gene, particularly rs7903146, increase T2DM risk. Potential links between genetic variants of the TCF7L2 locus and coronary atherosclerosis are uncertain. We therefore investigated the association between TCF7L2 polymorphisms and angiographically determined CAD in diabetic and non-diabetic patients.Methodology/principal findingsWe genotyped TCF7L2 variants rs7903146, rs12255372, and rs11196205 in a cross-sectional study including 1,650 consecutive patients undergoing coronary angiography for the evaluation of established or suspected stable CAD. Significant CAD was diagnosed in the presence of coronary stenoses ≥50%. Variant rs7903146 in the total study cohort was significantly associated with significant CAD (adjusted additive OR = 1.29 [1.09-1.53]; p = 0.003). This association was strong and significant in T2DM patients (n = 393; OR = 1.91 [1.32-2.75]; p = 0.001) but not in non-diabetic subjects (OR = 1.09 [0.90-1.33]; p = 0.370). The interaction risk allele by T2DM was significant (p(interaction) = 0.002), indicating a significantly stronger impact of the polymorphism on CAD in T2DM patients than in non-diabetic subjects. TCF7L2 polymorphisms rs12255372 and rs11196205 were also significantly associated with CAD in diabetic patients (adjusted additive OR = 1.90 [1.31-2.74]; p = 0.001 and OR = 1.75 [1.22-2.50]; p = 0.002, respectively). Further, haplotype analysis demonstrated that haplotypes including the rare alleles of all investigated variants were significantly associated with CAD in the whole cohort as well as in diabetic subjects (OR = 1.22 [1.04-1.43]; p = 0.013 and OR = 1.67 [1.19-2.22]; p = 0.003, respectively).Conclusions/significanceThese results suggest that TCF7L2 variants rs7903146 rs12255372, and rs11196205 are significantly associated with angiographically diagnosed CAD, specifically in patients with T2DM. TCF7L2 therefore appears as a genetic link between diabetes and atherosclerosis

Transcription factors binding sites identified within the promoter regions.

<p>The figure displays the sequences of matched position weight matrices (PWMs) together with the corresponding gene symbols of the transcription factors, which are overrepresented in the promoters of the 9 upregulated genes. The PWMs are ranked according to their “Yes/No” ratio, which is defined as the ratio of the average number of putative binding sites per 1000 bp, given for the query (Yes) and the background (No) sets. Overrepresentation is defined as a Yes/No ratio greater than one. Significance of the representation value is measured by the p-value derived from a binomial distribution. Matched promoters p-value assesses the statistical significance of the number of promoters in the query set that have at least one predicted site compared to that of promoters in the background set. Matrix similarity score (mss) and core similarity score (css) are indicated for comparison.</p

Binding sites for transcription factors within promoter regions.

<p>(A) Tabulated view of all matched matrix sequences for transcription factors (TF) as displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026465#pone-0026465-g003" target="_blank">figure 3</a>. Hits for transcription factors which were generated according to different matrices representing the same transcription factor were summed up. (B) Schematic representation of matched binding sites for transcription factors (arrows) within proximal promoters of the eight verified hypoxia-induced genes and WDR73. Overlapping binding sites of different matrices which represent the same transcription factor (family) are indicated as single site in the figure. Binding sites of the unknown factor were omitted.</p

Venn diagram of the hypoxia-regulated genes in human adipocytes.

<p>Microarray analysis resulted in 10 differentially regulated genes after 3 hours, 52 differentially regulated genes after 6 hours and 514 differentially regulated genes after 16 hours cultivation of the cells in a hypoxic environment (1% O₂). In the intersection of the circles the number of genes commonly regulated to the corresponding time points is indicated. The genes included in this analysis showed at least a 2-fold change in the expression compared to the control with a p value <0.05.</p

Functional classification of 10 genes upregulated after 3 h of hypoxia by GO annotation.

<p>The 10 genes upregulated after 3 h of hypoxia were classified according to their biological processes and gene ontology (GO) IDs (<a href="http://www.geneontology.org" target="_blank">www.geneontology.org</a>) by GO annotation, using manual curated GO groups provided by the Biobase software package Explain.</p

Transcriptional induction according to real-time PCR analysis.

<p>Real-time PCR analysis was performed analysing mRNA levels of the 9 genes differentially regulated after 3, 6 and 16 hours treatment under hypoxic conditions (1% O2). Results of 4–6 independent experiments each performed in triplicate are expressed as mean values ± SE. Values are depicted relative to the untreated control. *** p<0.001; ** p<0.01; * p<0.05.</p