Identification of Iron Homeostasis Genes Dysregulation Potentially Involved in Retinopathy of Prematurity Pathogenicity by Microarray Analysis

Xian-Qiong Luo, Chun-Yi Zhang, Jia-Wen Zhang, Jing-Bo Jiang, Ai-Hua Yin, Li Guo, Chuan Nie, Xu-Zai Lu, Hua Deng, Liang Zhang, Xian-Qiong Luo, Chun-Yi Zhang, Jia-Wen Zhang, Jing-Bo Jiang, Ai-Hua Yin, Li Guo, Chuan Nie, Xu-Zai Lu, Hua Deng, Liang Zhang

Abstract

Retinopathy of prematurity (ROP) is a serious disease of preterm neonates and there are limited systematic studies of the molecular mechanisms underlying ROP. Therefore, here we performed global gene expression profiling in human fetal retinal microvascular endothelial cells (RMECs) under hypoxic conditions in vitro. Aborted fetuses were enrolled and primary RMECs were isolated from eyeballs. Cultivated cells were treated with CoCl2 to induce hypoxia. The dual-color microarray approach was adopted to compare gene expression profiling between treated RMECs and the paired untreated control. The one-class algorithm in significance analysis of microarray (SAM) software was used to screen the differentially expressed genes (DEGs) and quantitative RT-PCR (qRT-PCR) was conducted to validate the results. Gene Ontology was employed for functional enrichment analysis. There were 326 DEGs between the hypoxia-induced group and untreated group. Of these genes, 198 were upregulated in hypoxic RMECs, while the other 128 hits were downregulated. In particular, genes in the iron ion homeostasis pathway were highly enriched under hypoxic conditions. Our study indicates that dysregulation of genes involved in iron homeostasis mediating oxidative damage may be responsible for the mechanisms underlying ROP. The "oxygen plus iron" hypothesis may improve our understanding of ROP pathogenesis.

Figures

Figure 1
Figure 1
Confocal images of cultured human RMECs. (a) Immunofluorescent staining of vWF with sheep polyclonal antibody (Abcam). (b) Negative control of vWF identification. (c) Immunofluorescent staining of CD31 with mouse monoclonal antibody (Abcam). (d) Negative control of CD31 identification.
Figure 2
Figure 2
Growth curve of human RMECs treated with various concentration of CoCl2.
Figure 3
Figure 3
Heat map of gene expression pattern of five pairs of human RMEC subjects with dye-swap. The red color indicates upregulation and green color indicates downregulation. (a) Unsupervised clustering of 15,400 genes with a signal intensity >800 in matched RMEC samples. (b) A total of 326 (198 upregulated and 128 downregulated). DEGs in hypoxia-induced RMEC, compared with normoxia control (fold-change >2 and q < 0.05 with the SAM analysis).
Figure 4
Figure 4
Comparison of gene expression changes from microarray and qRT-PCR.

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