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. 2016 Dec 9;11(12):e0167744.
doi: 10.1371/journal.pone.0167744. eCollection 2016.

Hyperhomocysteinemia in ApoE-/- Mice Leads to Overexpression of Enhancer of Zeste Homolog 2 via miR-92a Regulation

Yang Xiaoling  1   2 , Zhao Li  1 , Li ShuQiang  1 , Ma Shengchao  1 , Yang Anning  1 , Ding Ning  1 , Li Nan  1 , Jia Yuexia  1 , Yang Xiaoming  1 , Li Guizhong  1 , Jiang Yideng  1
Affiliations

Hyperhomocysteinemia in ApoE-/- Mice Leads to Overexpression of Enhancer of Zeste Homolog 2 via miR-92a Regulation

Yang Xiaoling et al. PLoS One. .

Erratum in

Abstract

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular diseases, such as atherosclerosis. HHcy promotes atherogenesis by modifying the histone methylation patterns and miRNA regulation. In this study, we investigated the effects of homocysteine (Hcy) on the expression of enhancer of zeste homolog 2 (EZH2), and tested our hypothesis that Hcy-induced atherosclerosis is mediated by increased EZH2 expression, which is regulated by miR-92a. The levels of EZH2 and H3K27me3 were increased in the aorta of ApoE-/- mice fed a high-methionine diet for 16 weeks, whereas miR-92a expression was decreased. Over-expression of EZH2 increased H3K27me3 level and the accumulation of total cholesterol and triglycerides in the foam cells. Furthermore, upregulation of miR-92a reduced EZH2 expression in the foam cells. These data suggested that EZH2 plays a key role in Hcy-mediated lipid metabolism disorders, and that miR-92a may be a novel therapeutic target in Hcy-related atherosclerosis.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Hcy promoted atherosclerosis progression in ApoE-/- mice.
ApoE-/- and control mice were fed a regular, methionine or folate diet, and after 16 weeks, serum and thoracic aorta were isolated. (A) Hematoxylin and eosin (H&E) and Oil Red O staining of the thoracic aorta sections (10×10). The original magnification was × 400. The red arrow indicates a plaque region. (B) Quantification of the average plaqe area was performed using the Histolab software. Representative Oil red O staining of the thoracic aorta sections of the mice (n = 5 to 7 sections per mice) were shown and the plaque area was presented as lesion % (% of whole aorta). Each symbol represented one animal and the horizontal bar indicated the mean value. (C) The serum from ApoE-/- and control mice were analyzed for total Hcy levels. (D) Positive correlation of the atherosclerotic plaque area with Hcy levels was examined by Pearson correlation analysis. All results were presented as mean ± SD. * P < 0.05, ** P < 0.01, vs. CON. #P < 0.05, ##P< 0.01, vs. HLP. ΔP < 0.05, ΔΔP < 0.01, vs. HHcy. CON: WT C57BL/6J mice were fed with a regular mouse diet; HLP: ApoE-/-mice were fed standard mouse diet; HHcy: ApoE-/- mice were fed standard mouse diet plus methionine; HHcy+FA+VB: mice were fed standard mouse diet with methionine, folate and vitamin B12 supplements.
Fig 2
Fig 2. Hcy increased lipid accumulation of macrophage foam cell.
Human macrophage foam cells were incubated with 100 μM Hcy or folate supplements for 24, 48 or 72 h. TC and TG levels in cells were quantified. Lipid droplets were observed by oil red O stain. The results were derived from triplicate experiments. Data were presented as mean ± SD. *P < 0.05, **P < 0.01, vs. CON. #P < 0.05, ##P< 0.01, vs. 100 μM Hcy. CON: WT C57BL/6J mice were fed with a regular mouse diet; HLP: ApoE-/-mice were fed standard mouse diet; HHcy: ApoE-/- mice were fed standard mouse diet plus methionine; HHcy+FA+VB: mice were fed standard mouse diet with methionine, folate and vitamin B12 supplements.
Fig 3
Fig 3. Hcy increased H3K27me3 level.
(A) H3K27me1, 2, 3 levels in the mice were detected by Western blot. (B) H3K27me3 level was detected in macrophage foam cells by Western blot. Signal intensity of H3K27me1, 2, 3 was quantified by densitometric analysis and normalized to pan H3 control. Data were presented as mean ± SD. *P < 0.05, **P < 0.01, vs. CON. ##P< 0.01, vs. HLP. ΔP < 0.05, ΔΔP < 0.01, vs. HHcy or 100 μM Hcy. CON: WT C57BL/6J mice were fed with a regular mouse diet; HLP: ApoE-/-mice were fed standard mouse diet; HHcy: ApoE-/- mice were fed standard mouse diet plus methionine; HHcy+FA+VB: mice were fed standard mouse diet with methionine, folate and vitamin B12 supplements.
Fig 4
Fig 4. Upregulated EZH2 increased H3K27me3 level and lipid accumulation.
(A) RNA and protein were isolated from the artery of ApoE-/- and control mice, and the expression of EZH2 was detected by qRT-PCR and western blot. (B) Macrophage foam cells were treated with 100 μM Hcy or folate for 48 h, RNA and protein were isolated from the cells, and the expression of EZH2 was quantified. (C) EZH2 mRNA and protein expression in macrophage foam cells transfected with Ad-EZH2 or Ad-EZH2-siRNA. (D) Western blot analysis for H3K27Me3 and pan H3 control in macrophage foam cells transfected with Ad-EZH2 or Ad-EZH2-siRNA. (E, F) After 48 h incubation with 100 μM Hcy and folate, macrophage foam cells were lysed, and intracellular TC and TG were measured. The results were derived from triplicate experiments. Data were presented as mean ± SD. **P < 0.01, vs. CON. ΔΔP < 0.01, vs. HHcy or 100 μM Hcy.
Fig 5
Fig 5. miR-92a was involved in the atherosclerosis process and targeted the 3’-UTR of EZH2.
(A, B) Total RNA was isolated from the artery of mice and macrophage foam cells, and miR-92a expression was assessed by qRT-PCR. (C) Ad-EZH2 was transfected into macrophage foam cells, and the expression of miR-92a was assessed by qRT-PCR. (D) Predicted binding site for miR-92a in the 3’UTR region of human EZH2 mRNA. Mutant-type pMIR vector was inserted with mutated seed sequence (from TGCAATA to GTAATAC) for miR-92a. (E, F) Lv-miR-92a or Lv-miR-92a-siRNA was transfected into macrophage foam cells, and the expression of EZH2 mRNA and protein were assessed by qRT-PCR and western blot analysis, respectively. (G) Luciferase activities after co-transfection with control or miR-92a, and wild-type or mutant-type pMIR vectors with the indicated 3’UTR of EZH2. The results were derived from triplicate experiments. Data were presented as mean ± SD. **P < 0.01, vs. CON. ΔΔP < 0.01, vs. HHcy or 100 μM Hcy.
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Grants and funding

This work was supported in part by grants from the National Natural Science Foundation of China (81360027, 81360052, 81360053, 81460080), the Provincial Natural Science Foundation of Ningxia (NZ15209), the Education Department Foundation of Ningxia (NGY2015092, NGY2015084) and the Ningxia Medical University Foundation (XY201505). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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