Gene Expression Profile of Zinc-Deficient, Homocysteine-Treated Endothelial Cells

Abstract

In the post-genome period, the technique for identifying gene expression has been progressed to high throughput screening. In the field of molecular nutrition, the use of screening techniques to clarify molecular function of specific nutrients would be very advantageous. In this study, we have evaluated Zn-regulated gene expression in Zn-deficient, homocystein-treated EA.hy926 cells, using cDNA microarray, which can be used to screen the expression of many genes simultaneously. The information obtained can be used for preliminary assessment of molecular and signaling events modulated by Zn under pro-atherogenic conditions. EA.hy926 cells derived from human umbilical vein endothelial cells were cultured in Zn-adequate (control, 15 $\mu$M Zn) or Zn-deficient (experimental, 0 $\mu$M Zn) Dulbecco's MEM media under high homocysteine level (100 $\mu$M) for 3 days of post-confluency. Cells were harvested and RNA was extracted. Total RNA was reverse-transcribed and the synthesized cDNA was labeled with Cy3 or Cy5. Fluorescent labeled cDNA probe was applied to microarray slides for hybridization, and the slide was then scanned using a fluorescence scanner. The expression of seven genes was found to be significantly decreased, and one significantly increased, in response to treatment of EA.hy926 cells with Zn-deficient medium, compared with Zn-supplemented medium. The upregulated genes were oncogenes and tumor suppressor genes, cell cycle-related genes and transporter genes. The down-regulated gene was RelB, a component of the NF-kappaB complex of transcription factors. The results of this study imply the effectiveness of cDNA microarray for expression profiling of a singly nutrient deficiency, namely Zn. Furthur study, using tailored-cDNA array and vascular endothelial cell lines, would be beneficial to clarify the molecular function of Zn in atherosclerosis, more in detail.

Keywords

• Zn-deficiency;
• endothelial cells;
• EA.hy926 cell;
• homocysteine;
• cDNA microarray;
• atherosclerosis

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