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Xin Liu, Yi Luo, Peng Zhou, Yi Lu; DNA methylation mediated and oxidative stress related genes CRYAA and GJA3 in nuclear age-related cataract (ARC) and its mechanism. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5877.
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Evidence suggested that DNA methylation and oxidative stress played a role in the pathogenesis of ARC but their relationship and mechanism remained unknown. We tested lens anterior capsules of nuclear ARC patients versus age-matched controls to find out oxidative stress related genes that were regulated by DNA methylation for further investigation.
Lens anterior capsules were collected during cataract surgery or obtained from postmortem eyes of the eye bank as ARC or control group respectively. Ten capsules of both groups ere detected by InfiniumR HumanMehlation 450 BeadChip. Methylation status was further verified by pyrosequencing. Real time RT- PCR and western blot were used to analyze the expression of genes. EMSA was used to analyze the impact of methylation of CpG sites on transcription factors. HLECs SRA01/04 were treated with a DNA-demethylating agent Zebularine or PBS for 72 hours. Real time RT- PCR and Western blot were used to analyze the expression of genes.
The promoters of 254 genes detected by Methylation Beadchip were hypermethylated and 60 genes were hypomethylated in ARC group compared to controls (p<0.001). In the top 30 most significantly different genes, CRYAA and GJA3 were related to oxdative stress. Pyrosequencing results of 20 samples indicated that ARC group displayed hypermethylation in comparison with the control group (47.13±3.60% vs 35.89±5.88%; p<0.01; in CRYAA and 3.21±2.40% vs 1.80±1.20%; p<0.01, in GJA3). The mRNA and protein level of both genes were significantly reduced in ARC group vs. controls (p<0.01). Methylation of the CpG sites of CRYAA promoter decreased DNA-binding capacity of the transcription factor SP1 detected by EMSA. While methylation of the CpG sites of GJA3 promoter did not affect DNA-binding capacity of the transcription factor. Zebularine treatment resulted in increase of CRYAA and GJA3 in mRNA and protein level in Zebularine group compared to controls.
DNA methylation plays an important role in the pathogenisis of ARC. Oxidative related CRYAA and GJA3 genes undergo epigenetic repression in the lens epithelia in ARC. DNA methylation of promoter of CRYAA directly affected the binding of transcriptional factor and resulted in gene repression but it was negative in GJA3 gene. DNA demethylating agent Zebularine was able to up-regulate the expression of CRYAA and GJA3 in HLECs.
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