原发性高血压患者线粒体融合基因2启动子及转录调控元件变异引发的转录调控异常与机制研究

The mobidity of essential hypertension (EH) is estimated about 20% in Chinese, which is a heavy burden for patients and the government. Millions of people die of EH and its complications every year. Mitofusion 2 (Mfn2) is a novel gene with the function of negatively regulating the hyperplasia of vasculare smooth muscle cells (VSMCs), which is the basic pathophysiology character of EH. We have reported that the expression of mfn2 is down-regulated in VSMCs of EH. However, the potential mechanism is remaining undiscovered. In the previous study, we have found several new single nucleotide polymorphsims in EH patients. Particularly, a new variation in the 5'-uncoding region of Mfn2 is the binding site of activator protein-1 (AP-1). Our preliminary study has confirmed that the transcript activity of this variated promoter down-regulated about 35% (the study is accepted for publication by YMJ, a SCI journal). In the present study, firstly, we will screen new EH patients and normotensive subjects on the basis of previous study. The scanning range of mfn2 promoter region will be enlarged to the whole 5'-uncoding region. After designing the primers and cloning the variated promoter by PCR in vitro, combined with SNP database and site directed mutation technology, we will analyse the 5'-uncoding region of Mfn2 by bioinformatics method. The transcript activity of the normal and veriated promoters will be tested by Dual luciferase reporter gene system. Then, transgenic mouse is used for indentifiction of the function of the promoters. Secondly, CHiP,footprinting and EMSA will be used to identify the transcript factors and their binding sites, in vivo and vitro. Then, RNA interference and over expression of transcription factors will be used to test the function of transcription factors by the measurement of Mfn2 expression in VSMCs. At last, the structure-activity relationship between promoter structure and its function will be studied by bioinformatics methods. This will lead to the screening of variated promoter as well as related transcript factors. The potential mechanism will also be investigated. Since this study may uncover the unrevealed mechanism of Mfn2 low expression in VSMCs, it will be valuable in the prevention, morbidity and motality of EH, as well as in the development of antihyperplasia drugs. Therefore, it has a prospective future on the aspects of basic study and clinical practice as well.

原发性高血压(EH)发病率极高，危害巨大。线粒体融合基因2(Mfn2)是一个新发现的血管平滑肌细胞（VSMC）增殖负调控基因，在EH患者VSMC中表达极低，但其分子机制还不明确。我们前期研究发现新的Mfn2启动子元件变异与EH高度相关，且可致其转录活性下降35%。本课题拟以此为切入点，对Mfn2的转录调控进行深入研究。首先在前期研究基础上扩大EH患者样本量和启动子区扫描范围，体外克隆后，结合定点突变技术，通过双荧光素酶报告基因系统检测启动子的转录活性，转基因鼠验证其功能；其次应用CHiP、足迹法和EMSA研究变异调控元件及与其互作的转录因子，通过RNA干扰和基因过表达再现转录因子的调控功能；最后研究变异启动子的构效机制，从而确定mfn2变异启动子、调控元件及转录调控因子，并阐明其调控机制。这对于从分子遗传学角度解决EH患者VSMC中Mfn2的低表达机制问题，降低该病致残致死率等，意义深远。

通过对Mfn2基因进行生物信息学方法分析研究，筛选出15种启动子上与顺式作用元件结合的转录因子。通过CHiP、足迹法和EMSA在体内和体外研究了6种转录因子，除已知的SP1转录因子外，最终验证4个新的确实存在并且具有转录调控功能的转录因子，这4种转录因子结合于Mfn2的核心启动子区。通过双荧光素酶基因表达系统进一步验证了这4种转录因子对该基因的表达及对VSMC增殖的调控作用。这几种转录因子是我们首次发现对Mfn2具有重要的调控作用，目前未见相关的报道（由于正在进行相关的专利申请，相关数据未列出）。. 同时，双荧光素酶基因表达系统研究发现其中2个已验证的转录因子会影响Mfn2的表达。进一步我们对这二种转录因子在启动子区的结合位点分别进行了人工突变，确定了这二个转录因子的结合位点及其功能特征。为了进一步验证这二个转录因子对VSMC的调节功能，我们还通过RNA干扰技术和RNA过表达技术，观察这二个转录因子在高表达和低表达的情况下对VSMC增殖的影响情况。结果发现这二个转录因子确实对VSMC的表达存在着重要的调控作用。. 另外，我们先后筛选NT组和EH患者组各1200例二组人群，测序发现在二组人群中的Mfn2启动子区有18个变异位点存在着明显的差别，如Mfn2基因单核苷酸多态性位点5’端非编码区-2786（位于Mfn2 5’端非编码区中，Chromosome 1 - NC_000001.11区域的11977395 C/T）与高血压密切相关，而且发现了它的新功能：该位点基因型的改变将导致高血压发病风险升高。即Mfn2基因5’端非编码区-2786 C/T在病例组和对照组中的分布存在显著性差异（p<0.05），即在EH患者中该基因型主要表现为C碱基，而在NT组中主要为T碱基，该SNP变化可改变转录因子结合位点。进一步挑选上述高血压病例-照组样本各300例进行了验证，结果是一致的。. 基于此，可以明确这二个转录因子可能通过对Mfn2的表达调控来对EH患者的血管增殖发挥着非常重要的作用，因而是高血压患者动脉硬化和血管稳态维持的非常重要转录调控因子。这对于EH等血管增殖性疾病的VSMC增殖的预防和治疗等，具有非常重要的意义。