Zhao Li, Jiao Yun, Yang An-Ning, Cao Cheng-Jian, Kong Fan-Qi, Liu Xian-Mei, Yang Xiao-Ling, Jiang Yi-Deng
College of Laboratory Medicine, Ningxia Medical University, Yinchuan 750004, China.
Department of Infectious Disease of General Hospital, Ningxia Medical University, Yinchuan 750004, China.
Sheng Li Xue Bao. 2015 Apr 25;67(2):207-13.
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.
本研究旨在探讨miR-124及其启动子区域DNA甲基化在同型半胱氨酸（Hcy）诱导的动脉粥样硬化中的作用。给载脂蛋白E基因敲除（ApoE(-/-)）小鼠喂食高甲硫氨酸饮食16周以复制高同型半胱氨酸血症模型。同时，设置正常对照组（喂食正常饮食的C57BL/6J小鼠，N-对照组）和模型对照组（喂食正常饮食的ApoE(-/-)小鼠，A-对照组）。通过苏木精-伊红（HE）染色和油红O染色观察动脉粥样硬化程度。使用自动生化分析仪检测血清Hcy水平。复制泡沫细胞模型并使用油红O染色确认模型是否成功建立。分别用0、50、100、200、500 μmol/L Hcy和50 μmol/L Hcy + 10 μmol/L 5-氮杂胞苷（AZC）刺激泡沫细胞。采用实时定量聚合酶链反应（RT-qPCR）检测小鼠主动脉和泡沫细胞中miR-124的表达；采用巢式降落甲基化特异性聚合酶链反应（nMS-PCR）检测小鼠主动脉和泡沫细胞中miR-124启动子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表达的影响。通过油红O染色观察miR-124启动子DNA甲基化状态对泡沫细胞脂质蓄积的影响。结果显示，与模型对照组相比，高甲硫氨酸组血清Hcy水平显著升高（P < 0.01），出现主动脉粥样硬化斑块，miR-124表达明显降低（P < 0.01），而miR-124启动子DNA甲基化水平显著升高（P < 0.01）。给予不同水平的Hcy，泡沫细胞中miR-124表达降低，而miR-124启动子DNA甲基化水平呈剂量依赖性升高（P < 0.05，P < 0.01）。AZC显著逆转上述指标的结果（P < 0.05）。miR-124的下调可能在Hcy诱导的动脉粥样硬化中起作用，其启动子DNA甲基化状态可能是这一过程中的重要机制。
