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miR-124及其启动子区DNA甲基化在同型半胱氨酸致动脉粥样硬化中的作用

赵丽, 焦运, 杨安宁, 曹成建, 孔繁琪, 刘现梅, 杨晓玲, 姜怡邓

宁夏医科大学1检验学院；基础学院；总医院传染科，银川 750004

摘要

本文旨在探讨miR-124在同型半胱氨酸(homocysteine, Hcy)致动脉粥样硬化中的作用及其启动子区DNA甲基化调控机制。ApoE−/−小鼠给予高蛋氨酸饮食16周复制高同型半胱氨酸血症动物模型，并设正常对照组(C57BL/6J小鼠正常饮食)及ApoE−/−对照组(ApoE−/−小鼠正常饮食)。HE及油红O染色后观察各组小鼠主动脉粥样硬化程度；生化分析仪检测各组小鼠血清Hcy及血脂水平；复制泡沫细胞模型，油红O染色确定模型是否复制成功；分别以0、50、100、200、500 μmol/L Hcy及50 μmol/L Hcy + 10 μmol/L 5-氮杂胞苷(AZC)干预细胞。实时定量PCR (RT-qPCR)检测各组小鼠主动脉及泡沫细胞中miR-124的表达变化；巢式降落式甲基化特异性PCR (nMS-PCR)检测各组小鼠主动脉及泡沫细胞中miR-124启动子区DNA甲基化水平，同时在细胞水平观察DNA甲基化抑制剂AZC对miR-124启动子区DNA甲基化水平及其表达的影响；泡沫细胞油红O染色观察miR-124甲基化改变对细胞脂质蓄积的影响。结果显示，与ApoE−/−对照组相比，高蛋氨酸组小鼠血清Hcy水平显著升高(P < 0.01)，且主动脉发生明显粥样变，miR-124的表达水平显著降低(P < 0.01)，而miR-124启动子区DNA甲基化水平显著升高(P < 0.01)。给予不同浓度Hcy刺激泡沫细胞后，miR-124的表达水平呈下降趋势，而miR-124启动子区DNA甲基化水平逐渐升高，且随Hcy浓度增加改变越明显，呈剂量依赖关系(P < 0.05, P < 0.01)，给予AZC干预后可逆转上述变化(P < 0.05)。以上结果提示，miR-124表达下调可能在Hcy致动脉粥样硬化过程中发挥重要作用，而miR-124启动子区高甲基化改变可能是其表达下调的重要机制。

关键词： 同型半胱氨酸; ApoE−; /−; 鼠; 泡沫细胞; miR-124; DNA甲基化

分类号：R363

[The effect of miR-124 on homocysteine-induced atherosclerosis via promoter region DNA methylation in ApoE−/− mice] [Article in Chinese]

ZHAO Li, JIAO Yun, YANG An-Ning, KONG Fan-Qi, LIU Xian-Mei, YANG Xiao-Ling, JIANG Yi-Deng

College of Laboratory Medicine； College of Preclinical Medicine； Department of Infectious Disease of General Hospital, Ningxia Medical University, Yinchuan 750004, China

Abstract

The aim of the present study is to explore the role of miR-124 and its promoter region DNA methylation in homocysteine (Hcy)-induced atherosclerosis. ApoE−/− mice were fed with hypermethionine diet for 16 weeks to duplicate hyperhomocysteinemia model. Meanwhile, a normal control group (C57BL/6J mice fed with normal diet, N-control) and a model control group (ApoE−/− mice fed with normal diet, A-control) were set. The degree of atherosclerosis was observed by HE and oil red O staining. Automatic biochemical analyzer was used to detect the serum levels of Hcy. Foam cell model was duplicated and oil red O staining was used to confirm whether the model was successfully established. And foam cells were stimulated with 0, 50, 100, 200, 500 μmol/L Hcy and 50 μmol/L Hcy + 10 μmol/L AZC respectively. Real-time quantitative PCR (RT-qPCR) was used to detect the expressions of miR-124 in mice aorta and foam cells; Nested landing methylation specific PCR (nMS-PCR) was used to detect the levels of miR-124 promoter DNA methylation in mice aorta and foam cells. Meanwhile, the effects of DNA methylation inhibitor AZC on miR-124 expression were observed at the cellular level. The effect of miR-124 promoter DNA methylation status on lipid accumulation in foam cells was observed by oil red O staining. The results showed that compared with model control group, the serum levels of Hcy in high methionine group were significantly increased (P < 0.01) and developed aortic atherosclerotic plaque, the expression of miR-124 was markedly decreased (P < 0.01), while the levels of miR-124 promoter DNA methylation were significantly increased (P < 0.01). Given different levels of Hcy, the expression of miR-124 in foam cells was decreased, while the levels of miR-124 promoter DNA methylation were increased in a dose-dependent manner (P < 0.05, P < 0.01). AZC reversed the results of mentioned indices as above markedly (P < 0.05). Downregulation of miR-124 may play a role in Hcy-induced atherosclerosis and its promoter DNA methylation status may be an important mechanism in this process.

Key words: Homocysteine; ApoE−;/−; mice; foam cells; miR-124; DNA methylation

收稿日期：2014-10-20　　录用日期：2014-12-02

通讯作者：姜怡邓　　E-mail: jwcjyd@163.com

引用本文：

赵丽, 焦运, 杨安宁, 曹成建, 孔繁琪, 刘现梅, 杨晓玲, 姜怡邓. miR-124及其启动子区DNA甲基化在同型半胱氨酸致动脉粥样硬化中的作用[J]. 生理学报 2015; 67 (2): 207-213.

ZHAO Li, JIAO Yun, YANG An-Ning, KONG Fan-Qi, LIU Xian-Mei, YANG Xiao-Ling, JIANG Yi-Deng. [The effect of miR-124 on homocysteine-induced atherosclerosis via promoter region DNA methylation in ApoE−/− mice] [Article in Chinese]. Acta Physiol Sin 2015; 67 (2): 207-213 (in Chinese with English abstract).