环境因素诱导miR-133a在血管内皮细胞异位表达的机制及其在高血压病中的作用

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 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 the ectopic expression of muscle-specific microRNA-133a (miR-133a) in endothelial cells, downregulates GTPCH1 gene expression post-transcriptionally and contributes to endothelial dysfunction in rats with cardiovascular risk factors. How miR-133a, as a novel epigenetic factor, was induced by risk factors in endothelial cells remains unknown. To identify the upstream regulator of miR-133a, 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 miR-133a which locates the region between -842 to -364 bp. In this area, we also identified a common binding site (-665 to -674 bp) of transcriptional factor activator protein 2 alpha (AP-2α) was in miR-133a promoter, which is 5’-GCCCTGCGGC-3’. Our exciting preliminary experimental data also indicated that AP-2α overexpression increased the levels of miR-133a, while knockdown of AP-2α reduced miR-133a expression in endothelial cells. Based on these evidence, we propose that risk factors may activate AP-2α to upregulate miR-133a, resulting in the repression of GTPCH1 gene expression and consequent endothelial dysfunction. In this way, risk factors induces the initiation of hypertension. In order to test this hypothesis, we plan to identity AP-2α as a new transactional factor of miR-133a, to illuminate the molecular mechanism how risk factors induced endothelial dysfunction, and to determine the AP-2α/miR-133a/GTPCH1 axis in hypertension. This multidisciplinary, integrative, and translational approach to testify the hypothesis that the AP-2α/miR-133a signaling is critical in GTPCH1 gene expression is technically and conceptually innovative. The proposed studies will provide new insights into how high risk factors induce hypertension via miRNA-133a ectopic expression in endothelial cells and prove that specific modulation of endothelial function such as inhibition of miRNA-133a/b might be a novel approach to prevent high blood pressure. We believe that the outcome of our investigation will have an immediate impact in providing new choice in patients with hypertension, the most common disease affecting about 160 million people in China.

血管内皮功能异常是环境因素引起高血压病的始动因素和关键环节。我们发现环境因素能诱导肌肉特异性miR-133a异位表达于血管内皮细胞，再通过抑制GTP环化水解酶1（GTPCH1）的表达而引起内皮功能异常，但miR-133a异位表达的机制不明。生物信息学发现miR-133a的启动子含有转录因子AP-2α的结合位点。预实验发现过表达AP-2α上调miR-133a的表达，而沉默AP-2α则抑制了miR-133a的表达。据此，我们推测“环境因素通过AP-2α上调miR-133a的表达而降低GTPCH1，最终引起血管内皮功能异常和高血压”。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miR-133a的转录因子，明确AP-2α对miR-133a的转录调控作用是环境因素致血管内皮功能异常及高血压的关键机制。该课题将解析血管内皮功能异常的新机制，旨在为开发降压药提供新靶点。

血管内皮功能异常是环境因素引起高血压病的始动因素和关键环节。环境因素能诱导肌肉特异性miR-133a异位表达于血管内皮细胞，再通过抑制GTP环化水解酶1（GTPCH1）的表达而引起内皮功能异常，但miR-133a异位表达的机制不明。我们用先进的生物学实验方法，体外体内研究相结合，确定miR-133a/b直接作用于GTPCH1-mRNA的3′-UTR的长（1-2011）和短（1-1033，即长3′-UTR的979-2011部分）序列均可结合。miR-133a/b通过影响GTPCH1-mRNA的稳定性而影响基因表达。miR-133a/b通过影响GTPCH1的基因表达而调控血管内皮细胞的功能。在高血糖和高血脂小鼠模型，将miR-133a/b抑制物注射给小鼠，发现抑制miR-133a/b能上调GTPCH1的基因表达、BH4的合成、eNOS的功能以及改善血管内皮功能，过表达miR-133a/b能模拟环境因素，诱导血管内皮损伤。在DOCA和SHR等两种高血压模型，miR-133a/b抑制物具有内皮保护作用和降压作用。通过该研究，不仅阐明高血压的发病机制和探索新防治措施，也为本课题的转化医学前景提供了基础。通过这些研究，我们阐明了GTPCH1基因表达的转录后水平的调控机制，确立miR-133a/b在血管内皮细胞功能异常中的重要作用，为开发防治高血压的药物提供靶点。