miRNAs靶向调控己糖激酶-‖对肺动脉高压Warburg效应的影响及机制

Pulmonary hypertension (PH) is characterized by progressively elevated pulmonary vascular resistance resulting in increased pulmonary blood pressure. This disorder eventually leads to right ventricular failure and death. Although there have been an increasingly large number of therapeutic drugs , PH is still a disease with poor prognosis and high mortality. The glycolytic shift lies on the central part of interaction among the resistance to apoptosis and increased vascular cell proliferation, which are hallmarks of PH. Hexokinase-Ⅱ(HK-Ⅱ), which is one of the rate-limiting enzymes of glycolysis and involved in the mitochondrial apoptotic pathway, is a crucial factor connecting energy metabolism with cell apoptosis. Basing on our ongoing National Natural Science Foundation, it has been found that HK-Ⅱplays a key role in the glycolysis and vascular remodeling of PH. Therefore, a hypothesis is proposed that the metabolic intervention targeting on HK-Ⅱmay be a potential and powerful pathway leading to the inhibition of vascular cell proliferation by capturing cells energy. In this study, we will select the key microRNAs (miRNAs) of HK-Ⅱby bioinformatics methods, then try to validate aberrant expression of miRNAs in PH being a new molecular marker of upregulated HK-Ⅱexpression, and investigate the possible underling mechanism. In addition, in vitro and in vivo experiments will be used to test the effects of miRNAs on the vascular energy metabolism and cell proliferation, examine the correlation between the miRNA expressions and the severity of PH, as well as explore the possibility and feasibility reducing PH by the application of miRNA.

肺动脉高压(PH)可导致右心肥大、衰竭甚至死亡，是一种预后差、病死率高的疾病。肺血管壁增生和重构是PH的重要标志，并与糖酵解密切相关。己糖激酶-Ⅱ(HK-Ⅱ) 是糖酵解的限速酶，参与线粒体凋亡途径，是连接能量代谢与细胞凋亡重要的交汇点。我们在前项国家自然科学基金资助下证实HK-Ⅱ参与PH糖酵解和血管重构，并发挥关键作用。因此, 我们提出假设：针对HK-Ⅱ的靶向代谢干预可能从根源上夺取细胞的能量，抑制血管壁细胞增生。本项目以前期工作为基础，以HK-Ⅱ为靶标，通过生物信息学方法预测可能作用于HK-Ⅱ的关键miRNA，试图证明PH中某些特异性的miRNA表达变化可能是细胞HK-Ⅱ表达上调的新分子机制，并期望找出miRNA分子异常表达的可能机制。同时，通过体外和动物实验，观察miRNA分子对血管细胞能量代谢和增殖能力的影响，以及与PH严重程度的相关性，探索应用miRNA分子抗PH的可能性和可行性。

肺血管平滑肌细胞（pulmonary arterial smooth muscular cells，PASMCs）异常增殖引起的肺血管重构是肺动脉高压发生发展的重要病理过程。PASMCs能量代谢异常是其增殖的重要原因。本项目围绕调控能量代谢的关键酶——己糖激酶2（Hexokinase 2，HK2）在肺动脉高压（pulmonary arterial hypertension，PAH）中的作用及其机制进行了深入的研究。我们取得了以下关键研究结果：①缺氧时HK2表达增加，葡萄糖消耗与乳酸生成增多（Warburg效应），细胞增殖明显及凋亡减少。②构建过表达HK2慢病毒，体外转染PASMCs，应用生物信息学分析，发现HK2 mRNA 3’-UTR 端是miR143-3p 的识别序列，荧光素酶实验证实HK2是miR143-3p的直接靶点。③目前多项研究表明HK2与细胞增殖时的能量代谢异常密切相关，抑制HK2蛋白表达可以使细胞恢复正常的代谢模式，从而抑制细胞增殖。我们通过miR143-3p mimics使缺氧状态下的PASMCs过表达miR143-3p，发现miR143-3p会抑制HK2引起的细胞增殖，提示缺氧后miR143-3p的下调在PAH中的重要作用。同时我们检测了miR143-3p过表达后PASMCs的糖酵解水平，发现葡萄糖消耗和乳酸生成均下降，提示miR143-3p也许是通过调控能量代谢异常继而抑制PASMCs的增殖。④动物实验中，我们成功制作野百合碱（MCT）诱导的PAH的动物模型，发现肺组织中miR143-3p表达减少，HK2蛋白表达增加，PAH大鼠肺动脉重构明显，肺动脉环张力下降，心脏结构及功能均受到损害，PAH大鼠呈现糖酵解为主的代谢模式（Warburg效应）。⑤我们脂联素修饰的脂肪干细胞移植入PAH大鼠体内，结果显示其能恢复异常的氧化供能途径，降低PAH大鼠平均肺动脉压，改善肺部血管重构，同步超声心动图显示，右室结构和功能也有不同程度的改善。通过本次研究，为肺动脉高压的机制提供了新的理论。基于以上的研究发现，发表SCI 和CSCD论文各1篇。