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RMECs: hypoxia-induced group vs. paired untreated group

ABSTRACT: Objective To explore the molecular mechanisms of retinopathy of prematurity (ROP) at whole genome scale using cultivated human fetal retinal microvascular endothelial cells (RMECs) under hypoxic condition. Study design Aborted fetuses with mean gestational age of 20-28 weeks at Guangdong Women and Children’ Hospital were enrolled, 3 males and 2 females, with mean weight 610-2205 g. Primary RMECs were isolated by mechanical morcel and trypsin-collagenase digestion of eyeballs. Cultivated cells were treated with or without 150 μM CoCl2 for 72 h. The dual-color microarray approach was adopted to compare gene expression profiling between treated RMECs and the paired untreated control using Agilent SurePrint G3 Human GE 8x60K Microarray Kit. We applied average-linkage unsupervised hierarchical clustering and TreeView to visualize the results. The one class algorithm in the SAM software was used to screen the differentially expressed genes (DEGs) with a threshold set at fold-change >2.0 and q-value <0.05. Gene Ontology was employed for functional enrichment analysis. Results There were 326 DEGS between the hypoxia-induced group and untreated group. Of these genes, 198 were up-regulated in hypoxic RMECs, while other 128 hits were down-regulated. More importantly, altered expression genes in iron ion homeostasis pathway were highly enriched under hypoxic condition. Quantitative RT-PCR (qRT-PCR) was performed to validate the results obtained from microarray analysis. Conclusions Dysregulation of genes involved in iron homeostasis mediating oxidative damage may contribute to the ROP pathogenesis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE59462 | GEO | 2014/07/17

SECONDARY ACCESSION(S): PRJNA255374

REPOSITORIES: GEO

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Project description:Iron is an important trace element that affects the growth and development of animals and regulates oxygen transport, hematopoiesis, and hypoxia adaptations. Wujin pig has unique hypoxic adaptability and iron homeostasis; however, the specific regulatory mechanisms have rarely been reported. This study randomly divided 18 healthy Wujin piglets into three groups: the control group, supplemented with 100 mg/kg iron (as iron glycinate); the low-iron group, no iron supplementation; and the high-iron group, supplemented with 200 mg/kg iron (as iron glycinate). The pre-feeding period was 5 days, and the formal period was 30 days. Serum was collected from empty stomachs before slaughter and at slaughter to detect changes in the serum iron metabolism parameters. Gene expression in the liver was analyzed via transcriptome analysis to determine the effects of low- and high- iron diets on transcriptome levels. Correlation analysis was performed for apparent serum parameters, and transcriptome sequencing was performed using weighted gene co-expression network analysis to reveal the key pathways underlying hypoxia regulation and iron metabolism. The main results are as follows. (1) Except for the hypoxia-inducible factor 1 (HIF-1) content (between the low- and high-iron groups), significant differences were not observed among the serum iron metabolic parameters. The serum HIF-1 content of the low-iron group was significantly higher than that of the high-iron group (P<0.05). (2) Sequencing analysis of the liver transcriptome revealed 155 differentially expressed genes (DEGs) between the low-iron and control groups, 229 DEGs between the high-iron and control groups, and 279 DEGs between the low- and high-iron groups . Bioinformatics analysis showed that the HIF-1 and transforming growth factor-beta (TGF-β) signaling pathways were the key pathways for hypoxia regulation and iron metabolism. Four genes were selected for qPCR validation, and the results were consistent with the transcriptome sequencing data. In summary, the serum iron metabolism parameter results showed that under the influence of low- and high-iron diets, Wujin piglets maintain a steady state of physiological and biochemical indices via complex metabolic regulation of the body, which reflects their stress resistance and adaptability. The transcriptome results revealed the effects of low-iron and high-iron diets on the gene expression level in the liver and showed that the HIF-1 and TGF-β signaling pathways were key for regulating hypoxia adaptability and iron metabolism homeostasis under low-iron and high-iron diets. Moreover, HIF-1α and HEPC were the key genes. The findings provide a theoretical foundation for exploring the regulatory pathways and characteristics of iron metabolism in Wujin pigs.

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RMECs: hypoxia-induced group vs. paired untreated group

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