核孔蛋白Nup93通过线粒体钙通道蛋白MCU调控心肌细胞凋亡的研究

According to the research published by the applicant, Nup is closely associated with myocardial ischemia; however, the pathological mechanism still remains unknown. Apoptosis is an important pathological process of myocardial ischemia injury, but so far there haven’t been any reports of Nup regulating effect on apoptosis or specific mechanisms. During preliminary experiments of the research project, the Nup of differentially expressed myocardial ischemia was screened and the down-regulation of Nup93 and others were discovered. In view of previous studies and reports by the applicant on mitochondrial calcium channel MCU, the Nup , which could regulate MCU, was further screened. It was found that the mere knock-down of Nup93 can give rise to up-regulated MCU expression and induce myocardial apoptosis. We speculate that this is because myocardial ischemia results in down-regulated Nup93 expression, which gives rise to up-regulated MCU expression and thus facilitates apoptosis. This project is designed to, from both vivo and vitro perspectives, systematically probe into the expression characteristics of Nup93 and MCU when ischemia occurs. By way of separate or combined intervention of Nup93 and MCU, it aims to verify that Nup93 regulates apoptosis through MCU. With breakthrough point focusing on its potential regulating of MCU mRNA nuclear export, the project will probe into the molecular mechanism of Nup93. The project will reveal, for the first time, that Nup regulates myocardial apoptosis. It will break through tradition cognitions and gain deeper insight into the functions of Nup.

核孔蛋白（Nup）是控制RNA等大分子出入细胞核的核心组件，申请人已发表的研究提示Nup与心肌缺血密切相关，但病理意义尚不清楚。细胞凋亡是心肌缺血损伤的重要病理过程，Nup对其调控作用及机制何在，目前未见报道。本项目预实验筛选了缺血心肌差异表达的Nup，发现Nup93等下调；基于申请人前期对线粒体钙通道MCU的研究和报道，进一步筛选了可调控MCU的Nup，发现单纯敲低Nup93可引起MCU表达上调、并诱导心肌细胞凋亡。我们推测：心肌缺血引起Nup93表达下调，Nup93的减少使得MCU表达上调，继而促进细胞凋亡。本项目拟从整体和离体水平，研究缺血时Nup93和MCU的表达特征；通过单独或联合干预Nup93和MCU，明确Nup93经MCU调控凋亡；以调控MCU mRNA出核为切入点，探讨Nup93作用的分子机制。本项目将首次揭示Nup调控心肌细胞凋亡，突破认知，赋予其新的功能意义。

研究背景：心肌梗死仍然是引起我国人口死亡的主要原因之一。核孔复合体（NPC）是细胞核和细胞质之间唯一的通道。核孔蛋白在心肌梗死中的表达、功能和机制尚不清楚。.方法：用siRNA减少心肌细胞核孔蛋白，用重组腺病毒增加心肌细胞Nup93和YAP1。采用细胞活力、乳酸脱氢酶释放和膜联蛋白V-FITC凋亡测定来评估缺氧对心肌细胞的影响。RNA-seq和生物信息学分析用于评估Nup93在调节mRNA转录和运输中的功能。.主要结果：我们探讨NUP93在缺氧诱发的心肌细胞凋亡中的功能。我们发现NUP93在心梗诱发的心衰中表达降低；在培养的心肌细胞，敲低Nup93 引起细胞凋亡增多；过表达Nup93减轻缺氧诱发的心肌细胞凋亡。在机制方面，我们敲低Nup93后，进行核质分离，对细胞质和细胞核的每一部分进行了RNA二代测序，发现敲低Nup93对mRNA的出核转运基本没有影响，然而敲低Nup93会导致很多基因的表达改变。我们推测Nup93的功能并不是影响mRNA的出核转运，而是影响mRNA的转录。Nup93的DNA结合实验显示许多Nup93结合基因与Nup93敲低异常表达的基因是一致的。针对这些差异表达的基因进行功能富集分析，显示异常表达的基因主要集中在线粒体能量代谢方面，其中YAP1等在Nup93敲低的心肌细胞中表达降低。挽救实验显示，将YAP1在心肌细胞中过表达能够挽救Nup93基因敲低导致心肌细胞死亡的功能，并减弱了缺氧对细胞损伤的影响。.科学意义：本项目揭示了Nup93 全新的生物学作用及分子机制，不仅对认知核孔蛋白具有创新性意义，而且对于理解心肌细胞凋亡的机制具有学术价值，可望为心肌梗死的诊断和治疗提供新的分子标志和干预靶点。