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. 2023 Jan 9:13:1079455.
doi: 10.3389/fgene.2022.1079455. eCollection 2022.

DNA methylation and mRNA expression of glutathione S-transferase alpha 4 are associated with intracranial aneurysms in a gender-dependent manner

Tianqi Xu  1   2 , Xi Yu  3 , Shenjun Zhou  2   4 , Yiwen Wu  4 , Xinpeng Deng  4 , Yuefei Wu  1 , Shiyi Wang  1   5 , Xiang Gao  4 , Sheng Nie  4 , Chenhui Zhou  2   4 , Jie Sun  4 , Yi Huang  2   4
Affiliations

DNA methylation and mRNA expression of glutathione S-transferase alpha 4 are associated with intracranial aneurysms in a gender-dependent manner

Tianqi Xu et al. Front Genet. .

Abstract

Objective: We performed a case-control study to investigate the correlation between DNA methylation and mRNA expression of the glutathione S-transferase alpha 4 (GSTA4) gene and the risk of intracranial aneurysm (IA) in the Chinese Han population. Methods: After propensity score matching, 44 pairs of cases and controls were collected in this study. Fasting blood samples were collected for DNA and RNA extraction within 24 h of admission. Nine CpG dinucleotides were selected from the GSTA4 promoter region for DNA methylation pyrosequencing. mRNA expression of GSTA4 was measured by quantitative real-time polymerase chain reaction (RT-qPCR). In vitro cell experiments were conducted to verify the association between 5-aza-2'-deoxycytidine induced DNA hypomethylation and GSTA4 mRNA expression. Results: The mean methylation level of GSTA4 was much lower in IA patients, especially in IA patients, especially in unruptured IA (UIA), than that in controls (IA vs. Control, p < .001; ruptured IA (RIA) vs. Control, p = .005; UIA vs. Control, p < .001). With sex stratification, we further found that the association between GSTA4 methylation and IA risk presented only in women (mean methylation level: IA vs. Control, p < .001; RIA vs. Control, p = .009; UIA vs. Control, p < .001). GSTA4 mRNA expression was significantly higher in the IA group than in the control group (p < .01) and negatively correlated with DNA methylation in all individuals (r = -.746, p < .001). DNA hypomethylation can increase GSTA4 mRNA expression in human primary artery smooth muscle cells. The receiver operating characteristic (ROC) curve showed that GSTA4 mean methylation (AUC = .80, p < .001) was a reliable predictor of women intracranial aneurysm, among which CpG 1 exhibited the best predictive value (AUC = .89, p < .001). In addition, GSTA4 expression levels could also predict the risk of IA in women (AUC = .87, p = .005). Conclusion: Decreased DNA methylation and increased mRNA expression of the GSTA4 gene are associated with the risk of IA in women.

Keywords: 5-aza-2'-deoxycytidine; DNA methylation; glutathione S-tranferase alpha 4; intracranial aneurysm; mRNA expression.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Locations and analysis of nine CpGs in GSTA4. (A) The locations of the nine CpGs in the GSTA4 gene. (B) Representative sequencing analysis of nine methylation sites.
FIGURE 2
FIGURE 2
Pearson correlation analysis involving GSTA4 methylation status and clinical characters. (A) In men and women: methylation levels of all nine CpGs were significantly correlated. (B) In men: methylation levels of all nine CpGs were significantly correlated. (C) In women: methylation levels of all nine CpGs were significantly correlated.
FIGURE 3
FIGURE 3
Significant association between GSTA4 mRNA expression and DNA methylation. (A) mRNA expression levels in the IA group were higher than that in controls. (B) mRNA expression levels in women IAs were higher than that in women controls. (C) No statistical significance was observed between men IAs and men controls. (D) GSTA4 mRNA expression was correlated with its DNA methylation levels in all participants. (E) DNA hypomethylation induced by AZA increased the mRNA expression of the GSTA4 gene in HBVSMCs. (F,G) GSTA4 methylation levels in between smoker and non-smoker group. (H) GSTA4 mRNA expression between smoker and non-smoker groups.
FIGURE 4
FIGURE 4
ROC curves for prediction of IAs in men and women. (A) GSTA4 mean methylation is a predictor of women IA. (B) GSTA4 CpG1 methylation is a predictor of women IA. (C) GSTA4 expression is a predictor of women IA.
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