转录因子AP-2α调控circRNA_102979表达在他汀抑制miR-133a改善血管内皮功能中的作用

Endothelial dysfunction, caused by risk factors and characterized by loss of nitric oxide (NO) from endothelial NO synthase (eNOS), is an early marker for cardiovascular anomalies including atherosclerosis and hypertension. Deficiency of GTP cyclohydrolase 1 (GTPCH1), the rate-limiting enzyme for de novo tetrahydrobiopterin (BH4) synthesis, is an important mechanism of eNOS dysfunction. We have previously reported that statins inhibit the ectopic expression of muscle-specific microRNA-133a (miR-133a) in endothelial cells to upregulate GTPCH1 gene expression post-transcriptionally and prevent endothelial dysfunction induced by multiple cardiovascular risk factors. However, how miR-133a as a novel epigenetic factor in endothelial cells was inhibited by statins remains unknown. Statins have been reported to activate transcriptional factor activator protein 2 alpha (AP-2α). We also observed that statins increased the levels of circRNA_102979 by screening the expressional profiles of circRNAs in endothelial cells, which may function as a sponge of miR-133a. To identify the upstream regulator of circRNA_102979, we have made a binding site prediction in its promoter sequence by using bioinformatics website (Core-Promoter Prediction Program) and found the promoter sequence of circRNA_102979 which locates the region between -842 to -364 bp. In this area, we also identified a common binding site (-665 to -674 bp) of AP-2α was in circRNA_102979 promoter, which is 5’-GCCCTGCGGC-3’. This binding was further confirmed by chromatin immunoprecipitation (ChIP) assay. Our exciting preliminary experimental data also indicated that silence of AP-2α or circRNA_102979 abolished statins-repressed miR-133a ectopic expression in endothelial cells. Based on these evidence, we propose that statins may activate AP-2α to upregulate circRNA_102979, resulting in the inhibition of miR-133a and consequent improvement of endothelial dysfunction. In order to test this hypothesis, we plan to identity AP-2α as a new transactional factor of circRNA_102979, to illuminate the molecular mechanism how statins inhibit miR-133a and prevent endothelial dysfunction, and to determine the roles of AP-2α/circRNA_102979 axis in statins-alleviated atherosclerosis and hypertension. The proposed studies will provide new insights into how statins prevent endothelial dysfunction via AP-2α/circRNA_102979/miRNA-133a/GTPCH1/BH4/eNOS in endothelial cells and prove that specific modulation of endothelial function such as activation of AP-2α or inhibition of miRNA-133a/b might be a novel approach to prevent cardiovascular diseases. We believe that the outcomes of our investigations will have an immediate impact in providing new choice in patients with cardiovascular diseases, the most common disease affecting about 290 million people in China.

我们发现他汀通过抑制血管内皮细胞异位表达miR-133a而改善众多环境因素所致的内皮功能异常，但机制不明。他汀能激活AMPK，上调转录因子AP-2α的活性。环形RNA能吸附miRNA，解除其对靶基因表达的抑制作用。生物信息学发现AP-2α能结合到circRNA_102979的基因启动子，且该环形RNA上存在miR-133a的结合位点。预实验发现沉默AP-2α和circRNA_102979阻断了他汀对miR-133a的抑制作用。据此，我们推测“他汀通过AP-2α/circRNA_102979途径抑制miR-133a的表达”。本课题拟用相应的生物学实验方法，体外体内研究相结合，确定AP-2α是circRNA_102979的调控因子，明确AP-2α对该环形RNA的转录调控作用是他汀抑制miR-133a及改善血管内皮功能的关键。本研究将解析他汀保护血管内皮的新机制，为开发心血管保护药物提供新靶点。

我们既往发现他汀通过抑制血管内皮细胞异位表达miR-133a而改善众多环境因素所致的内皮功能异常。他汀能激活AMPK，上调转录因子AP-2α的活性。环形RNA能吸附miRNA，解除其对靶基因表达的抑制作用。生物信息学发现AP-2α能结合到circRNA_102979的基因启动子，且该环形RNA上存在miR-133a的结合位点。据此，我们提出“他汀通过AP-2α/circRNA_102979途径抑制miR-133a的表达”的研究假说。本课题运用分子生物学、细胞生物学、模式生物学和药理学等多种先进的实验方法，体外体内研究相结合，发现AP-2α是circRNA_102979的调控因子，AP-2α对circRNA_102979转录的调控作用是他汀抑制miR-133a及改善血管内皮功能的关键。本研究结果解析了他汀保护血管内皮的新机制，为开发心血管保护药物提供新靶点。