NPPB基因rs3753581位点多态性与RAS系统失调诱发高血压的相关性及机制

The pathogenesis of hypertension is far from been fully elucidated. We previously found that polymorphism of NPPB gene rs3753581 locus within the promoter region may be associated with essential hypertension, but the mechanism involved is not clear. In the present study, to explore the relationship between NPPB gene rs3753581 locus polymorphism and abnormal metabolism of BNP-RAS system induced hypertension, the correlation between NPPB and gene rs3753581 polymorphism and serum NT-proBNP and BNP levels was analyzed through expanding the subjects of EH patients and healthy control, and the metabolisms of renin-angiotensin system (RAS) and endothelium-depended Vasodilation in EH patients were investigated. Furthermore, the NPPB gene promoter was cloned, the effect of G/T variation in rs3753581 sites on NPPB gene transcriptional activity was evaluated, and the transcription factors binding to rs3753581 sites were screened and determined by assays of DNA-pulldown, EMSA and ChIP respectively. In conclusion, this study explore the correlation between the single nucleotide polymorphism of NPPB gene promoter and pathogenesis of hypertension, the results obtained in this study will contribute to a better understanding of the pathogenesis of hypertension.

原发性高血压的发病机制尚未完全明确。我们前期发现位于启动子区的NPPB基因rs3753581位点多态性可能与原发性高血压相关，但机制不明。本课题拟通过扩大临床病例-对照样本，观察患者rs3753581位点多态性与血清NT-proBNP和BNP的含量的相关性，同时检测RAS系统的代谢情况以及患者内皮依赖性血管舒张功能，阐明NPPB基因rs3753581位点多态性与BNP-RAS系统代谢异常诱发高血压的相关性；进一步克隆NPPB基因启动子，探讨启动子rs3753581位点G/T碱基差异对其转录活性的影响，并以DNA-pulldown、EMSA及ChIP等实验筛查与确定和该位点结合的转录因子，阐明rs3753581位点多态性致NPPB基因转录差异的机制。总之，本研究从影响基因转录及蛋白水平的角度，探讨NPPB基因启动子区基因多态性与高血压的相关性，所获结果有助于深入对高血压发病机制的认知。

背景 本研究旨在明确NPPA-NPPB基因多态性位点与脉压高血压的相关性以及其主要作用机制。.方法 选取898例高血压患者以分析NPPA (rs5063、rs5065)和NPPB (rs3753581、rs198388、rs198389)基因位点与脉压、钠尿肽、RAAS系统的相关性。构建NPPB基因启动子rs3753581位点G/T，荧光素酶检测其转录活性。利用TESS和染色质免疫沉淀预测和验证潜在的转录因子。构建过表达NPPB和沉默NPPB基因载体并转染，RT-qPCR和Western Blot检测利钠肽和RAAS相关指标的表达。.结果 NPPB rs3753581与脉压增大密切相关，其中rs3753581的TT基因型与高血压脉压增大具有显著关联性，其基因型分布对脉压及血清NT-proBNP含量产生显著影响，而对ACE、ACE2、AngⅡ、Ang(1-7)等表达未见明显影响。暂未发现NPPB rs198388和rs198389位点，以及NPPA rs5063和rs5065位点与高血压发病风险及脉压有关。荧光素酶的活性检测显示pGL3-NPPB-G组明显高于pGL3-NPPB-T组。ChIP实验证实NPPB启动子特定区域可与IRF1和ZNF263、PRDM1相结合，而与MEIS1无交联反应。慢病毒转染获得pLVX-NPPB组、pLVX-siNPP组、空载组，结果显示pLVX-NPPB组人血管内皮细胞NPPB过表达约4倍，而pLVX-siNPPB组NPPB几乎不表达；pLVX-NPPB组NT-proBNP、BNP、Ang（1-7）、AngⅡ、NOS、Mas1的蛋白表达水平较空载组升高，而AT1受体和AT2受体的蛋白表达水平下降。反之，pLVX-siNPPB组NT-proBNP、BNP、Ang（1-7）、AngⅡ、NOS、Mas1表达水平下降，而ACE2、AT1受体的蛋白水平升高。mRNA表达水平检测发现pLVX-NPPB组ACE2、AT1受体mRNA的表达下降，而Mas1的mRNA表达上升； pLVX-siNPPB组ACE2、AT1受体的mRNA表达增加，而Mas1的mRNA表达下降。.结论 NPPB rs3753581可能是通过影响启动子区域与转录因子IRF1、PRDM1和ZNF263的结合影响BNP及RAS系统相关指标的表达参与脉压高血压的调节。