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Microarray for genes associated with signal transduction in diabetic OLETF keratocytes.

Lee JE, Lee JS, Hwang SH - Korean J Ophthalmol (2007)

Bottom Line: Real-time PCR showed a significant decrease in the IL-6 and TGF-beta2 genes and a significant increase in the Ppm1a gene.There were some differences in gene expression between normal and diabetic keratocytes related to signal transduction pathways, such as the insulin, MAPK, calcium, and TGF-beta pathways.In addition, IL-1alpha and TNF-alpha stimulating the insulin, TGF-beta, and Toll-like receptor signaling pathways may have different effects in diabetic keratocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, College of Medicine, Pusan National University, Pusan, Korea.

ABSTRACT

Purpose: The purpose of this study was to identify differences in signal transduction gene expression between normal and diabetic keratocytes stimulated with interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha).

Methods: Normal and diabetic keratocytes were primarily cultured and treated with 20 ng/ml IL-1alpha and TNF-alpha for 6 h. cDNA was hybridized to an oligonucleotide microarray. Genes identified by the microarray were further evaluated by real-time PCR.

Results: Diabetic keratocytes over-expressed components of the MAPK and Notch pathways, and under-expressed components of the insulin, calcium, and TGF-beta pathways. Cytokine treated diabetic keratocytes differentially expressed components of the TGF-beta and MAPK pathways. After IL-1alpha and TNF-alpha treatment, nine genes were under-expressed, falling in the insulin, TGF-beta, and Toll-like receptor pathways. Real-time PCR showed a significant decrease in the IL-6 and TGF-beta2 genes and a significant increase in the Ppm1a gene.

Conclusions: There were some differences in gene expression between normal and diabetic keratocytes related to signal transduction pathways, such as the insulin, MAPK, calcium, and TGF-beta pathways. In addition, IL-1alpha and TNF-alpha stimulating the insulin, TGF-beta, and Toll-like receptor signaling pathways may have different effects in diabetic keratocytes.

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Related in: MedlinePlus

Quantitative real-time PCR of three genes using GAPDH as an endogenous control. "*" indicates a significant difference between normal and diabetic rats stimulated with or without IL-1α or TNF-α IL-6 and TGF-β 2 showed no significant difference between untreated normal and diabetic rat, but the diabetic rat showed a significant down-regulation when cytokine treated. Ppm1a showed a significant up-regulation in all diabetic cases.
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Figure 1: Quantitative real-time PCR of three genes using GAPDH as an endogenous control. "*" indicates a significant difference between normal and diabetic rats stimulated with or without IL-1α or TNF-α IL-6 and TGF-β 2 showed no significant difference between untreated normal and diabetic rat, but the diabetic rat showed a significant down-regulation when cytokine treated. Ppm1a showed a significant up-regulation in all diabetic cases.

Mentions: Real-time PCR was performed on three genes that differed significantly between normal and diabetic rats, or were induced by cytokine treatment. The genes selected were IL-6, Ppm 1A, and TGF-β 2. The primer sets and annealing temperatures are shown in Table 6. These genes showed up- or down-regulation identical to the gene microarray results. For IL-6 and TGF-β 2, no significant difference between diabetic and normal cells was seen. After cytokine treatment, they showed significant down-regulation. The Ppm 1A gene showed a significant up-regulation in diabetes, as compared to the control and was up-regulated by cytokine treatment(Fig. 1).


Microarray for genes associated with signal transduction in diabetic OLETF keratocytes.

Lee JE, Lee JS, Hwang SH - Korean J Ophthalmol (2007)

Quantitative real-time PCR of three genes using GAPDH as an endogenous control. "*" indicates a significant difference between normal and diabetic rats stimulated with or without IL-1α or TNF-α IL-6 and TGF-β 2 showed no significant difference between untreated normal and diabetic rat, but the diabetic rat showed a significant down-regulation when cytokine treated. Ppm1a showed a significant up-regulation in all diabetic cases.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2629708&req=5

Figure 1: Quantitative real-time PCR of three genes using GAPDH as an endogenous control. "*" indicates a significant difference between normal and diabetic rats stimulated with or without IL-1α or TNF-α IL-6 and TGF-β 2 showed no significant difference between untreated normal and diabetic rat, but the diabetic rat showed a significant down-regulation when cytokine treated. Ppm1a showed a significant up-regulation in all diabetic cases.
Mentions: Real-time PCR was performed on three genes that differed significantly between normal and diabetic rats, or were induced by cytokine treatment. The genes selected were IL-6, Ppm 1A, and TGF-β 2. The primer sets and annealing temperatures are shown in Table 6. These genes showed up- or down-regulation identical to the gene microarray results. For IL-6 and TGF-β 2, no significant difference between diabetic and normal cells was seen. After cytokine treatment, they showed significant down-regulation. The Ppm 1A gene showed a significant up-regulation in diabetes, as compared to the control and was up-regulated by cytokine treatment(Fig. 1).

Bottom Line: Real-time PCR showed a significant decrease in the IL-6 and TGF-beta2 genes and a significant increase in the Ppm1a gene.There were some differences in gene expression between normal and diabetic keratocytes related to signal transduction pathways, such as the insulin, MAPK, calcium, and TGF-beta pathways.In addition, IL-1alpha and TNF-alpha stimulating the insulin, TGF-beta, and Toll-like receptor signaling pathways may have different effects in diabetic keratocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, College of Medicine, Pusan National University, Pusan, Korea.

ABSTRACT

Purpose: The purpose of this study was to identify differences in signal transduction gene expression between normal and diabetic keratocytes stimulated with interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha).

Methods: Normal and diabetic keratocytes were primarily cultured and treated with 20 ng/ml IL-1alpha and TNF-alpha for 6 h. cDNA was hybridized to an oligonucleotide microarray. Genes identified by the microarray were further evaluated by real-time PCR.

Results: Diabetic keratocytes over-expressed components of the MAPK and Notch pathways, and under-expressed components of the insulin, calcium, and TGF-beta pathways. Cytokine treated diabetic keratocytes differentially expressed components of the TGF-beta and MAPK pathways. After IL-1alpha and TNF-alpha treatment, nine genes were under-expressed, falling in the insulin, TGF-beta, and Toll-like receptor pathways. Real-time PCR showed a significant decrease in the IL-6 and TGF-beta2 genes and a significant increase in the Ppm1a gene.

Conclusions: There were some differences in gene expression between normal and diabetic keratocytes related to signal transduction pathways, such as the insulin, MAPK, calcium, and TGF-beta pathways. In addition, IL-1alpha and TNF-alpha stimulating the insulin, TGF-beta, and Toll-like receptor signaling pathways may have different effects in diabetic keratocytes.

Show MeSH
Related in: MedlinePlus