Evaluation of differentially expressed genes identified in keratoconus

Ji-Eun Lee, Boo Sup Oum, Hee Young Choi, Seung Uk Lee, Jong Soo Lee, Ji-Eun Lee, Boo Sup Oum, Hee Young Choi, Seung Uk Lee, Jong Soo Lee

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.

Figures

Figure 1
Figure 1
Annealing control primer™–base polymerase chain reaction. Annealing control primer™–base polymerase chain reaction product of eight verified differentially expressed genes shows increased or decreased levels of expression in keratoconus (arrows). Higher levels of expression in keratoconus (column 2) compared with normal cornea (column 1) were found in bone morphogenetic protein 4 (A), cofilin 1 (B), and JAW1-related protein (C), whereas lower levels of expression were found in actin, alpha 2 (D), gene rich cluster, and C 10 gene (E), tissue inhibitor of metalloproteinase 3 (F), tissue inhibitor of metalloproteinase 1 (G), and somatostatin receptor 1 (H).
Figure 2
Figure 2
Depicted are reverse transcriptase-polymerase chain reaction products of the eight verified genes and glyceraldehydes 3-phosphate dehydrogenase (GAPDH) as a housekeeping gene. Bone morphogenetic protein 4 (BMP4; A), JAW1-related protein (MRVI1; B), and cofilin 1 (CFL1; C) were significantly increased, while actin, alpha 2 (ACTA2; D), gene rich cluster, and C 10 gene (GRCC10; E), tissue inhibitor of metalloproteinase 3 (TIMP 3; F), tissue inhibitor of metalloproteinase 1 (TIMP1; G), and somatostatin receptor 1 (SSTR1; H) were significantly decreased in keratoconus (colummn 2) compared with normal cornea (column 1).
Figure 3
Figure 3
Quantitative real-time polymerase chain reaction. Quantitative real-time polymerase chain reaction of the eight verified genes using glyceraldehydes 3-phosphate dehydrogenase (GAPDH) as endogenous control showed that bone morphogenetic protein 4 (BMP4; A), cofilin 1 (CFL1; B), JAW1-related protein (MRVI1; C) were increased by 1.6, 3.3, and 11 fold, respectively, whereas actin, alpha 2 (ACTA2; D), gene rich cluster, and C 10 gene (GRCC10; E), tissue inhibitor of metalloproteinase 3 (TIMP3; F), tissue inhibitor of metalloproteinase 1 (TIMP1; G), and somatostatin receptor 1 (SSTR1; H) were decreased by 4.5, 2.7, 14, 8.5, and 1.8 fold, respectively in keratoconus relatively to norma l cornea.

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