Evaluation of differentially expressed genes identified in keratoconus

Published on Nov 28, 2009in Molecular Vision2.202
Ji Eun Lee19
Estimated H-index: 19
(PNU: Pusan National University),
Boo Sup Oum19
Estimated H-index: 19
+ 2 AuthorsJong-Soo Lee31
Estimated H-index: 31
Sources
Abstract
Purpose: To identify the differentially expressed genes (DEGs) in the human keratocytes in keratoconus. Methods: Total RNA extracted from cultured corneal stromal fibroblasts from normal and keratoconic corneas were used for the synthesis of cDNA. DEGs were screened by an annealing control primer TM -based PCR method using GeneFishing ™ DEG kits. The differentially expressed bands were sequenced and analyzed. The genes identified were further evaluated by reverse transcriptase PCR and quantitative real-time PCR. Results: Overexpression of bone morphogenetic protein 4 (BMP4), cofilin 1 (CFL1), and JAW1-related protein (MRVI1) and underexpression of actin, alpha 2 (ACTA2), gene rich cluster, and C 10 gene (GRCC10), tissue inhibitor of metalloproteinase 3 (TIMP3), tissue inhibitor of metalloproteinase 1 (TIMP1), and somatostatin receptor 1 (SSTR1) were verified, and these results were confirmed by reverse transcriptase PCR and quantitative real-time PCR. Conclusions: Eight genes were identified to be differentially expressed in keratoconus and related with apoptosis, the cytoskeleton, wound healing, and nerve fibers. The genes identified may be involved in the mechanism underlying stromal thinning; thus, they could be important and deserve further investigation. Keratoconus is characterized by thinning of the corneal stroma, but its pathological mechanism has not been fully elucidated. Clinical studies have suggested that the disease has a high incidence among long-term users of contact lenses, often attacks those who have a history of rubbing their eyes, and is related to atopy of the eye [1-4]. Therefore, it is believed that the long-term damage to and stimulation of the corneal epithelium play a key role in the pathogenesis of keratoconus. Furthermore, it has been proven that when the corneal epithelium is damaged, surrounding stromal keratocytes disappear due to apoptosis, and that an imbalance between cell death and proliferation is involved in the pathological mechanism of keratoconus. The apoptosis of keratocytes plays an important role in the corneal thinning in keratoconus [5]. Hence, it is valuable to investigate the genes of the keratocytes that are involved in the thinning of the cornea because the it is important to grasp the fundamental pathogenesis in order to understand and treat the disease of keratoconus. Even though there have been studies on the morphologic or ultrastructural differences between normal and keratoconic cornea, there are few reports on their differentially expressed genes (DEGs) [6-9]. Kim et al. [6-8] compared the differential gene expression in the keratoconic and normal corneal epithelium using microarray focusing on the epithelium; their study had the advantage of early
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