同型半胱氨酸对FABP 4 启动子活性的影响

Effect of homocysteine on activity of human FABP4 promoter in macrophages

摘要:

目的 通过克隆脂肪酸结合蛋白4（FABP4）基因启动子，确定其活性核心区和功能片段，分析 心血管疾病独立危险因子同型半胱氨酸（Hcy）对FABP 4 启动子活性的影响。方法 应用生物信息学预测 FABP 4 基因启动子区顺式转录作用元件和反式作用因子，以pGL3-Basic 为载体，采用基因重组法构建启动子 截取片段，转染HEK-293A 细胞，观察不同截取片段的荧光素酶活性变化，确定活性最强的片段。进一步将 核心启动子片段（-2000/-1）转染巨噬细胞，观察不同浓度Hcy 和DNA 甲基化抑制剂5- 氮杂胞苷（AZC） 对FABP 4 基因启动子活性的影响。结果 不同长度截取片段转染HEK-293A 细胞后检测荧光素酶活性结果 显示，与pGL3 对照组比较，-2000/-1 片段转录活性最强。将核心启动子片段（-2000/-1）转染巨噬细胞并 用不同浓度Hcy 干预后，与0μmol/L Hcy 组比较，100 和200μmol/L Hcy 组启动活性升高；与100μmol/L Hcy 组比较，在100μmol/L Hcy 基础上用AZC 干预后，FABP 4 基因启动活性升高（P <0.05）。结论 成功克 隆FABP 4 启动子的4 个片段，且确定FABP 4 基因核心启动子位于-2000/-1 片段；Hcy 可以促进FABP 4 基 因核心启动子片段活性，补充AZC 后可以进一步促进Hcy 引起的FABP 4 启动子活性升高。

Abstract:

Objective To clone the promoter of fatty acid-binding protein 4 (FABP4 ) and determine its core active region and functional fragment, and meanwhile to determine the effect of homocysteine (Hcy), an independent risk factor for cardiac vascular disease, on the activity of FABP4 promoter so as to provide an experimental basis for further research on the function of FABP4 . Methods Cis-acting elements and transacting factors were predicted in the promoter region of FABP4 by bioinformatics. The promoter interception fragments were constructed with pGL3-basic vector by gene recombination. The interception fragment with strongest activity was identified by observing the changes in luciferase activity after transfecting different fragments into HEK-293A cells. Meanwhile, after transfection of core promoter fragment (-2000/-1) into macrophages, the effect of different concentrations of Hcy and DNA methylation inhibitor 5-azacytidine (AZC) on the promoter activity of FABP4 gene was detected. Results After transfection of HEK-293A cells with interception fragments of different length, the detection of luciferase activity showed that the transcription activity of -2000/-1 fragment was the strongest in comparison with the pGL3 control group. After transfecting macrophages with core promoter fragment -2000/-1 and treating these cells with different concentrations of Hcy, the promoter activity was significantly increased in the 100 and 200 μmol/L Hcy groups compared with the 0 μmol/L Hcy group; compared with the 100 μmol/L Hcy group, the promoter activity of FABP4 gene was further increased after intervention with AZC on the basis of 100 μmol/L Hcy (P < 0.05). Conclusions The four fragments of FABP4 promoter have been successfully cloned and the core promoter region of FABP4 is located in the -2000/-1 fragment. Hcy can promote the activity of the core promoter fragment of FABP4 gene, supplementation of AZC can further increase the Hcy-induced activity of FABP4 promoter.