DNA Methylation Mediated And Oxidative Stress Related Genes CRYAA And GJA3in Age-related Nuclear Cataract And Its Mechanism

Purpose Age-related cataract (ARC) is the leading cause of blindness in the world. Evidence suggested that oxidative stress played an important role in the pathogenesis of ARC. This study was aim to test lens anterior capsules of nuclear ARC patients and age matched controls by DNA metylation microarray of the whole human genome and find out oxidative stress related genes which were regulated by DNA methylation for verification and further investigation.Methods Lens anterior capsules of30ARC patients were collected during cataract surgery as ARC group. Inclusion criterias were patients older than60years old with nuclear ARCs and lens with LOCS III score of NC4or more. Thirty lens anterior capsules were obtained from postmortem eyes of the eye bank as control group. Ten capsules of both groups were detected by Infinium(?) HumanMehlation450BeadChip (Illumina, USA) to find out oxidative stress related genes which were regulated by DN A methylation. Methylation status was verified by pyrosequencing of the CpG islands of promoters of CRYAA and GJA3genes in10lens anterior capsules of both groups.Results There were20cases with LOCS III score of NC4,7cases with NC5and3cases of NC6in ARC group. LOCS III scores of cases in control group were all NCI. The promoter of254genes detected by the Methylation Beadchip was hypermethylated and the promoter of60genes was hypomethylated in the lens capsules of ARC group compared to controls (p<0.001). In the genes with the top30most significant differences, CRYAA and GJA3genes were ralted to oxdative stress. CpG island was found in both the promoter of CRYAA and GJA3genes. By analyzing the pyrosequencing results of20samples, it was found that the ARC group displayed hypermethylation (47.13±3.60%) in comparison with the control group (35.89±5.88%;p<0.01)in CRYAA gene. The ARC group also displayed hypermethylation (3.21±2.40%) in comparison with the control group (1.80±1.20%; p<0.01)in GJA3gene.Conclusions Through screening and verification, we, for the first time, discovered that with aging, the methylation status of promoter of oxidative stress related genes CRYAA and GJA3which were closely related to the pathogenisis of ARC were significantly elevated. The evidence presented suggests that DNA methylation plays an important role in the pathogenisis of ARC. It builds a solid foundation for the further investigation of its mechanism. Purpose The chaperone-like activity of aA-crystallin (CRYAA) is considered to be critical for the maintenance of eye lens transparency. Intercellular communication through the extensive network of gap junctions including Cx46(GJA3) facilitates intercellular exchange of molecules including antioxidants. It is vital for the maintenance of transparency and homeostasis of the avascular lens. This study was aim to investigate the mechanism of DNA hypermethylation of promoter of CRYAA and GJA3genes in the lens epithelial of nuclear ARC patients.Methods Lens anterior capsules of12ARC patients were collected during cataract surgery as ARC group. Inclusion criterias were patients older than60years old with nuclear ARCs and lens with LOCS Ⅲ score of NC4or more.12lens anterior capsules were obtained from postmortem eyes of the eye bank as control group. Real time RT-PCR was used to analyze the expression of CRYAA and GJA3in mRNA level. Western blot was used to analyze the expression of CRYAA and GJA3in protein level. In addition, EMSA was used to analyze the impact of methylation of CpG sites on transcription factors.Results The mRNA level of both CRYAA and GJA3genes were significantly reduced in the lens epithelia of nuclear ARC cases vs. controls (p<0.01). The protein level of both CRYAA and GJA3genes were also reduced in the lens epithelia of nuclear ARC cases vs. controls. Methylation of the CpG sites of the CRYAA promoter decreases DNA-binding capacity of the transcription factor SP1detected by EMS A. While methylation of the CpG sites of the GJA3promoter does not affect DNA-binding capacity of the transcription factor. Conclusions The evidence presented suggests that CRYAA and GJA3genes undergo epigenetic repression in the lens epithelia in ARC. DNA methylation of promoter of CRYAA directly affects the binding of transcriptional factor and results in gene repression but it is negative in GJA3gene. DNA methylation of promoter of GJA3may affect gene transcription through other indirect pathways, which remains further investigation. Purpose DNA demethylating agent Zebularine is capable of inhibiting DNMTl and MeCP2in vitro. Therefore, it is able to inhibit the critical cellular events of HLECs transformation. This study was aim to determine the effect of a DNA-demethylating agent Zebularine on expression of CRYAA and GJA3genes in HLECs.Methods HLECs SRA01/04were divided into2groups treated with Zebularine for72hours. PBS was added into the control group. Cells were used for total RNA extraction. Real time RT-PCR was used to analyze the expression of CRYAA and GJA3in mRNA level. Protein was extracted from cells and Western blot was used to analyze the expression of CRYAA and GJA3in protein level.Results Zebularine was capable of regulate the expression of CRYAA and GJA3in HLECs. Zebularine treatment resulted in increases of CRYAA and GJA3in mRNA level in Zebularine group compared to controls. The difference was statisticly significant (p<0.05). Zebularine treatment also resulted in increases of CRYAA and GJA3in protein level in Zebularine group compared to controls.Conclusions Our results provide quantitative evidence that DNA demethylating agent Zebularine was able to up-regulate the expression of CRYAA and GJA3in HLECs. These suggest that Zebularine may become a therapeutic approach for the prevention and trantment of ARC.