Identification of Differentially Expressed Genes in Keratoconus Keratocyte By Microarray Analysis

Abstract

Abstract: : Purpose: To identify differentially expressed genes in keratoconus keratocyte by cDNA microarray. Methods: Normal and keratoconus corneas were cultured for keratocyte in the same standard conditions. Total RNA, then Poly A+ RNA was extracted from keratocytes of second passage. cDNA probe labeled with fluorescent nucleotide Cy3 or Cy5 was prepared in duplicate with vice versa labeling, then hybridized with microarray slide containing 164 human apoptosis-related genes. Hybridization signal intensity was measured by laser Array Scanner. Expression ratio between keratoconus and normal keratocyte (Cy3/Cy5 ratio) was determined using ImaGene Ver.3.0 software. Differentially expressed genes were further evaluated by RT-PCR and quantitative real-time PCR. Results: Microarray analysis was performed with 4 pairs of keratoconus and normal samples, each in duplicate. Although the change in expression was variable throughout 8 hybridizations, 9 genes including 5 over-expressed: tumor necrosis factor alpha-induced protein 6 (TNFAIP6), P450 (Cytochrome) oxidoredutase, mitogen-activated protein kinase 3, LPS-induced TNF-alpha factor and 4 under-expressed: human insulin-like growth factor binding protein 5 (IGFBP5), insulin-like growth factor 2 (somatomedin A), insulin-like growth factor binding protein 3, syndecan 1 were identified as most frequent differentially expressed in keratoconus compared with normal keratocyte. Of these, TNFAIP6, IGFBP5 and syndecan 1 were selected for further verification. RT-PCR confirmed the change in expression for these three genes, consistent with microarray findings. Quantitative real-time PCR showed TNFAIP6 over-expressed by 3.3 folds, IGFBP5 under-expressed by 13.9 folds and syndecan 1 under-expressed by 1.6 folds in keratoconus compared with normal keratocyte. Conclusions: Microarray analysis of 164 apoptosis-related genes identified 9 differentially expressed genes in keratoconus keratocytes, those could be potentially important in molecular mechanism underlying keratoconus and deserve further investigation. In mRNA level, significant over-expression of TNFAIP6, especially remarkable under-expression of IGFBP5 in keratoconus relatively to normal keratocyte confirmed by RT-PCR and real-time PCR suggest an important role of these genes in keratocyte apoptosis that may lead to stromal thinning in keratoconus.

Keywords: keratoconus • cornea: stroma and keratocytes • gene/expression