Atg7-p53介导lncRNA APF促进自噬减轻心肌缺血损伤机制研究

Previous studies have shown that the upregulation of autophagy during ischemia can reduce myocardial injury, and long non-coding RNA APF could upregulate autophagy by modulating Atg7. In our preliminary study, we have demonstrated that Atg7 binds to p53 in the neonatal rat cardiomyocytes（NRCs）hypoxia model, both the amount of Atg7-p53 and the level of autophagy are upregulated. But whether Atg7-p53 regulates autophagy during ischemia is unknown. To further explore its mechanism, we found in myocardial hypoxia/ischemia model lncRNA APF expression was significantly upregulated; the knock-down of APF by shRNA-APF interference increased hypoxic myocardial injury. we hypothesize that APF can up-regulate Atg7-p53, which further enhances autophagy and reduces myocardial ischemic damage. To verify this hypothesis, we intend to further in vitro and in vivo experiments: ① using deletion mutation of Atg7, CO-IP and other methods to illustrate the Atg7-p53 binding domain; ② constructing the Atg7-p53 binding domain mutation of ATG7 vector, combined with morphological and molecular biological methods to reveal the role of Atg7-p53 in the regulation of autophagy; ③ the overexpression or knock down of APF in Atg7-p53 binding zone mutant mice to reveal that APF/Atg7-p53 could increase autophagy level and reduce myocardial ischemia injury. This project is expected to reveal the specific mechanism of autophagy regulated by APF/Atg7-p53, and provide a new target for the treatment of myocardial ischemic disease.

研究显示，自噬上调可减轻心肌缺血损伤，长链非编码RNA APF上调自噬与Atg7相关。我们前期在心肌细胞缺氧模型中发现Atg7可与p53结合（Atg7-p53），其结合量与自噬水平均上调，但Atg7-p53是否参与缺血心肌自噬水平的调控尚不清楚。此外，我们在心肌缺氧/缺血模型中发现lncRNA APF表达显著上调；使用shRNA下调APF表达后，缺氧心肌损伤加重。因此，我们提出假设：APF可能通过上调Atg7-p53增强自噬，从而减轻心肌缺血损伤。本项目拟进行以下研究：①利用缺失突变、CO-IP等方法明确Atg7与p53结合区域；②构建结合区突变的Atg7载体病毒，结合形态学、分子生物学等方法阐明Atg7-p53对自噬的调控机制；③在Atg7结合区突变小鼠中过表达或敲低APF，揭示APF在Atg7-p53介导下可增强自噬水平最终减轻心肌缺血损伤。本项目有望为改善心肌缺血损伤提供新思路。

本研究拟通过建立小鼠心肌梗死(MI)及缺血再灌注(MIRI)模型和SD乳鼠/c57小鼠乳鼠心肌细胞缺氧及缺氧复氧模型，探索在心肌缺血及再灌注期间基因的差异表达，筛选除表达差异显著的基因，并探究其对小鼠心功能的影响及其可能机制，主要从细胞死亡包括自噬、凋亡、铁死亡等方面阐述。采用基因敲除小鼠、腺相关病毒载体和RNA干扰技术实现基因敲除、过表达及基因沉默，以研究心肌缺血及再灌注期间KLF5、ATF3的表达上调与下调对心肌细胞凋亡及梗死面积的影响，并探究其抑制或加重心肌再灌注期间细胞死亡的可能机制。最后我们还在细胞死亡机制探讨的基础上，探索了在调控自噬和铁死亡的角度的一些药理学研究，证明了在心肌缺血再灌注期间，维持正常的自噬流水平、抑制铁死亡可有效改善心肌损伤。我们的研究从自噬和铁死亡的角度阐明了心肌缺血再灌注过程中的可能机制，有望为缺血性心脏病和心肌缺血再灌注损伤的临床防治提供新的靶点。