Comprehensive Gene Expression Analysis of Diabetic Mouse Retina with In–house cDNA Microarray System

Abstract

Abstract: : Purpose: Changes in retinal gene expression in a mouse model of diabetic retinopathy (DR) was investigated by cDNA microarray analysis to clarify the molecular pathophysiology of DR. Methods: We developed a system named the widely–applicable cDNA/cRNA system to construct and analyze an in–house microarray from a single mouse retina. Diabetes was induced in male C57BL/6 mice by an intraperitoneal injection of streptozotocin (STZ), and the changes in retinal mRNA levels were examined in three pairs of diabetic and age–matched control mice at 1 and 3 months after the injection of STZ. Hybridization was carried out with the two–fluorescent dye method, and the significance analysis of microarrays (SAM) was used to identify genes with significant changes in expression levels in the diabetic state. Northern blot analysis with amplified cRNA was performed to validate the results of the microarray analysis and SAM. Results: Most of the significantly up–regulated genes could be classified into two functional categories: oxidative phosphorylation and protein turnover. All mitochondrial DNA– and most of nuclear DNA–encoded genes for oxidative phosphorylation were up–regulated in the diabetic retina. This was in sharp contrast to the recently reported down–regulation of these genes in skeletal muscles of STZ–induced diabetic mice and type 2 diabetic humans. A number of the genes for protein synthesis and ubiquitin–proteasome system were also up–regulated in the diabetic retina. Northern blot analysis confirmed the increase in mRNA levels of cytochrome c oxidase 1 and ubiquitin. Conclusions:Up–regulation of genes for oxidative phosphorylation and protein turnover suggests that the diabetic retina appears to be in a potentially activated state for intermediary metabolism, presumably because of an increase in insulin–independent glucose influx. This gives insights into possible preventive and therapeutic intervention in diabetic retinopathy.

Keywords: diabetic retinopathy • gene microarray • gene/expression