Analysis of superoxide dismutase 1, dual-specificity phosphatase 1, and transforming growth factor, beta 1 genes expression in keratoconic and non-keratoconic corneas

Abstract

Purpose: To quantitatively assess the superoxide dismutase 1 (SOD1), transforming growth factor, beta 1 (TGF-β1), and dual-specificity phosphatase 1 (DUSP1) messenger ribonucleic acid (mRNA) expression levels as the main intracellular reactive oxygen species neutralizers, wound healing mediators, and immunomodulators (respectively) in keratoconic (KCN) and non-KCN corneas.

Methods: Total RNA was extracted from normal and keratoconic cultured corneal stromal fibroblasts. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to measure the relative expression levels of mRNAs of the SOD1, TGF-β1, and DUSP1 genes.

Results: The mRNA expression of TGF-β1 and DUSP1 was augmented in the KCN corneas (three- and fivefold, respectively; both p<0.05). The KCN and non-KCN samples showed no difference in comparative SOD1 mRNA levels.

Conclusions: This study demonstrated a higher level of DUSP1 and TGF-β1 expression as known molecules in the inflammatory process. These results may provide new insight into the complex molecular pathways underlying KCN for investigating other inflammatory molecules.

Figure 1

Primary corneal tissue culture. This figure represents primary tissue culture from sample no. 4 under light microscopy. A: The migration of cells out of the tissue started on day 7 after seeding (white arrow shows margin of tissue, 10X magnification). B: One of the cell colonies was performed after 15 days post-seeding (20X magnification). C, D: After cell culture expansion, the cultivated cells were starved for 24 h to compare the morphological difference between fibroblasts and keratocytes (C, D, respectively).

Figure 2

Determination of the exponential cycle of amplification. Semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) mixtures were taken for five alternating cycles starting at cycles 18, 21, 24, 27, and 30 for A: beta 2 microglobulin (B2M), B: superoxide dismutase 1 (SOD1), C: dual-specificity phosphatase 1 (DUSP1), and D: transforming growth factor, beta 1 (TGF-B1). Cycle 24 was selected as an exponential cycle.

Figure 3

Differential messenger ribonucleic acid expression of the target genes via semiquantitative reverse transcription–polymerase chain reaction (RT-PCR; 24^th cycle). This figure shows the results of the RT–PCR products in 2% agarose gel electrophoresis, stained with ethidium bromide. Lanes 1 to 10: Keratoconus (KCN) corneas; lane 11: pooled normal corneas and lane 12: negative control without RT. The fluorescent intensity of the bands was quantified with a GS-800 densitometer. The signal intensities were normalized to values obtained for beta 2 microglobulin (B2M). The messenger ribonucleic acid (mRNA) expression of transforming growth factor, beta 1 (TGF-β1), and dual-specificity phosphatase 1 (DUSP1) was increased in the KCN corneas (three- and fivefold, respectively; both p<0.05). The comparative superoxide dismutase 1 (SOD1) mRNA level showed no difference between the KCN and non-KCN samples.

Figure 4

The comparative mRNA expression of target genes in ten keratoconus (KCN) and pooled normal corneal tissues. This graph indicates the comparison of transcript band intensities for genes encoding superoxide dismutase 1 (SOD1), transforming growth factor, beta 1 (TGF-β1), and dual-specificity phosphatase 1 (DUSP1) between the KCN and non-KCN samples. The values were calculated relative to the beta 2 microglobulin (B2M) levels (as normalizer) and reported as mean percentages. Note that the KCN corneas showed a three- and fivefold increase in TGF-B1 and DUSP1 when compared to the pooled normal corneas. The RNA levels for SOD1 were similar for the KCN and normal corneas (p<0.05). Normal corneal samples were pooled to reduce tissue preparation errors. The error bars represent the standard deviation.