DNA hypermethylation-mediated downregulation of antioxidant genes contributes to the early onset of cataracts in highly myopic eyes

Abstract

High myopia is recognized as a risk factor for earlier onset of nuclear cataracts. One possible explanation for this is that lenses in highly myopic eyes are exposed to higher levels of oxygen than normal eyes owing to earlier vitreous liquefaction and, hence, are subjected to oxidative insults. Here, we first compared the methylation levels of six essential antioxidant genes (GSTP1, NRF2, OGG1, TXN, TXNRD1 and TXNRD2) between highly myopic cataract (HMC) and age-related cataract (ARC) lens epithelial samples via Sequenom MassARRAY. We found that specific CpG units in the promoters of GSTP1 and TXNRD2 were hypermethylated and that the expression levels of these two genes were lower in the HMC group than in the ARC group. A luciferase reporter assay confirmed the significance of differentially methylated fragments in the activation of transcription. The importance of GSTP1 and TXNRD2 in antioxidant capacity was confirmed by overexpression or knockdown experiments on cultured lens epithelial cells (LECs). In addition, the expression of DNA methyl transferase 1 (DNMT1) was higher in the lens epithelium of HMC patients than that of ARC patients, and the expression of GSTP1 and TXNRD2 was upregulated by use of a DNMT inhibitor in cultured LECs. Finally, we mimicked the intraocular environment of highly myopic eyes by treating LECs with hydrogen peroxide (H₂O₂) and observed both alterations in the methylation status of the GSTP1 and TXNRD2 promoters and time-dependent altered expression levels. Therefore, we propose that in an environment with high oxygen, in which lenses in highly myopic eyes are immersed, there exists a vicious cycle composed of increased oxidative stress and decreased enzymatic antioxidants via the hypermethylation of antioxidant genes.

Keywords: Cataract; DNA methylation; GSTP1; High myopia; Oxidative stress; TXNRD2.

Graphical abstract

Fig. 1

Higher indexes of oxidative stress in HMC. (A) Representative slit lamp images of lenses with increasing nuclear color of cataract. (B) The total antioxidant capacity in the anterior capsular membrane of the HMC group was significantly lower than that of the ARC group. (C) The malonaldehyde concentration was significantly higher in HMC. In both assays, n = 3 independent experiments, mean ± SD, unpaired two-tailed Student's t-test, *P < 0.05.

Fig. 2

Methylations status of six antioxidant genes. The methylation levels of GSTP1, TXNRD2, OGG1, TXN, NRF2 and TXNRD1 were measured in ARC and HMC lens epithelium using Sequenom MassARRAY. n = 3 individual patients, mean ± SD, unpaired two-tailed Student's t-test, *P < 0.05, **P < 0.01, ***P <0.001.

Fig. 3

Hypermethylation in promoter and decreased expression of GSTP1 and TXNRD2 in HMC. (A-B) Diagram of the positions of tested CpG units within the promoters of GSTP1 and TXNRD2. (C) Sequenom MassARRAY experiments using expanded sample size confirmed that one CpG unit in GSTP1 and one in the TXNRD2 CpG island promoter were statistically hypermethylated in the HMC group compared to the ARC group. n = 12 individual patients. (D) Relative mRNA levels of GSTP1, TXNRD2 in ARC and HMC lens epithelium. n = 8 independent experiments. (E) Western blot assay results revealed that the protein levels of both GSTP1 and TXNRD2 were decreased in HMC compared to ARC. mean ± SD, unpaired two-tailed Student's t-test, *P < 0.05, ****P < 0.0001.

Fig. 4

Immunofluorescence staining of both GSTP1 and TXNRD2 in ARC and HMC lens epithelium. Representative images of GSTP1 (green) or TXNRD2 (red) with nuclei (blue) suggest a reduced expression of these two genes in HMC. scale bar = 50 µm. The results of the quantified fluorescence intensity analysis are presented next to the images. n = 5 individual patients, mean ± SD, unpaired two-tailed Student's t-test, ****P < 0.0001.

Fig. 5

GSTP1 and TXNRD2 affect T-AOC in cultured LECs. A specific assay kit was used to measure T-AOC in LECs with overexpressed (OE) or knocked down (KD) GSTP1 and TXNRD2. The relative activities were calculated using untreated cells (NC) as control. n = 3 independent experiments, mean ± SD, unpaired two-tailed Student's t-test, *P < 0.05, ***P < 0.001.

Fig. 6

Significant decrease in promoter activity without hypermethylated fragments in GSTP1/ TXNRD2 genes. (A) Diagrams of the full-length promoter and shortened promoter without hypermethylated fragments of GSTP1 and TXNRD2. (B) Luciferase reporter assay results showed that transcription activity of GSTP1 and TXNRD2 promoter was significantly decreased without DNA fragments, including hypermethylated CpG units found in Sequenom MassARRAY. n = 3 independent experiments, mean ± SD, unpaired two-tailed Student's t-test, ****P < 0.0001.

Fig. 7

Higher expression of DNMT1 in the lens epithelium of HMC and increased expression of GSTP1 and TXNRD2 in LECs after treatment with 5-Aza. (A) qPCR results showed that the mRNA level of DNMT1 was higher in HMC lens epithelium than in the ARC group. n = 8 independent experiments. (B) ELISA results showed that protein level of DNMT1 was higher in HMC lens epithelium than in ARC samples. n = 5 independent experiments. (C) qPCR results showed that mRNA levels of both GSTP1 and TXNRD2 in cultured LECs increased significantly after treatment with 5-Aza. n = 3 independent experiments. (D) Western blot assay results showed that protein levels of both GSTP1 and TXNRD2 increased in cultured LECs after treatment with 5-Aza. mean ± SD, unpaired two-tailed Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 8

Upregulation of methylation by H₂O₂ treatment in a time-dependent manner. (A) Cell image before H₂O₂ treatment. (B) Cell image after 24 h of treatment with 200 μM H₂O₂. (C) CCK-8 assay revealed that the proliferation of LECs was significantly decreased after 24 h of treatment with 200 μM H₂O₂. n = 4 independent experiments. (D) The methylation levels of selected CpG units in the GSTP1 and TXNRD2 promoters (CpG units 21–25 of GSTP1; CpG units 34 of TXNRD2) increased in a time-dependent pattern under H₂O₂ treatment. (E) Under H₂O₂ treatment, the relative mRNA levels of GSTP1 and TXNRD2 gradually increased in the first six days and declined at day 8. The expression of DNMT1 remained stable until day 6, when it increased to about 2-fold relative to day 0. n = 3 independent experiments, mean ± SD, unpaired two-tailed Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001 (each sample was compared to day 0 when statistically analyzed).

Fig. S1

Pyrosequencing result of GSTP1 #21–25. n = 7 individual patients, mean ± SD, unpaired two-tailed Student’s t-test, *P < 0.05.

Fig. S2

Immunofluorescence staining of GSTP1 in ARC lens epithelia. Scale bar 50 µm.

Fig. S3

Immunofluorescence staining of GSTP1 in HMC lens epithelia. Scale bar 50 µm.

Fig. S4

Immunofluorescence staining of TXNRD2 in ARC lens epithelia. Scale bar 50 µm.

Fig. S5

Immunofluorescence staining of TXNRD2 in HMC lens epithelia. Scale bar 50 µm.

Fig. S6

No significant differences were observed between ARC and HMC lens epithelial samples in the expression of OGG1, TXN, NRF2 or TXNRD1 at the mRNA level. β-actin was used as the internal control in all qPCR assays. n = 3 independent experiments, mean ± SD, unpaired two-tailed Student’s t-test.

Fig. S7

qPCR experiments confirmed the overexpression and knockdown of GSTP1 and TXNRD2 genes in cultured LECs. OE.G: overexpression of GSTP1; OE.T: overexpression of TXNRD2; KD.G: knockdown of GSTP1; KD.T: knockdown of TXNRD2; Con: control. β-actin was used as the internal control in all qPCR assays. n = 3 independent experiments, mean ± SD, unpaired two-tailed Student’s t-test, ****P < 0.0001.

Fig. S8

EMSA experiments suggested no direct binding of GSTP1 or TXNRD2 DNA probes with nuclear extracts of the lens epithelium.

Fig. S9

Protein levels of GSTP1 and TXNRD2 in lenses of the lens-induced myopia model mice were determined by western blot analyses. Ctrl: control, untreated eye.