Rac1/LncRNA-MEG3/FUNDC1调控线粒体自噬在糖尿病缺血性脑损伤中的机制研究

Diabetes mellitus is one of high risk factors of ischemic brain injury, but the clinical interventions of ischemic brain damage in patients with diabetes are poorly reported. In our previous study, we demonstrated that inhibition of Rac1 played an important role not only in the treatment of ischemic brain damage in rats with diabetes mellitus, but also in the protection against cerebral ischemic injury by promoting mitophagy. In this progress, we confirmed the expression of LncRNA-MEG3 is up-regulated in the mitochondria and the inhibition of LncRNA-MEG3 could abrogate neuroprotection induced by Rac1 lentivirus. Our results also proved FUNDC1, a key factor involved in mitophagy, was the mitochondrial protein interacting with LncRNA-MEG3. Based on these findings, we assume Rac1-LncRNA-MEG3-FUNDC1 signaling pathway may mediate the mitophagy during cerebral ischemic injury, but the precise mechanism is not clearly defined. Therefore, this proposal is aimed to expound the exact function and molecular pathway of LncRNA-MEG3 in mitophagy regulation and the possible molecular mechanisms of the interaction between LncRNA-MEG3 and FUNDC1 in mitochondria by the methods of diabetic ischemic injury models both in vivo and in vitro, LncRNA mimic/inhibitor transfection, bioinformatics analysis and signaling transduction examination. Ultimately we intend to provide novel theoretical basis and strategy for diabetic cerebral ischemic injury.

糖尿病是缺血性脑损伤一个高危因素，但是临床中能减少糖尿病患者缺血性脑损伤的治疗措施很少。我们的前期研究表明抑制Rac1活性在治疗糖尿病缺血性脑损伤中发挥关键作用。抑制Rac1活性也可以促进线粒体自噬而发挥抗缺血性脑损伤作用。前期研究还显示抑制Rac1后，引发线粒体内LncRNA-MEG3水平上调，且LncRNA-MEG3介导了抑制Rac1活性的神经保护作用。此外，明确了与LncRNA-MEG3互作的线粒体蛋白为FUNDC1，而FUNDC1是调控线粒体自噬的关键因子。因此Rac1-LncRNA-MEG3通路可能与FUNDC1相互作用调控线粒体自噬，但具体机制目前尚不清楚。故本项目的目标是阐明LncRNA-MEG3参与调控糖尿病缺血性脑损伤后线粒体自噬以及Rac1-LncRNA-MEG3与FUNDC1相互作用的分子机制。为临床糖尿病缺血性脑损伤的治疗提供新的靶点和理论依据。

1. 背景：研究表明糖尿病是导致围术期脑损伤一个高危因素，糖尿病患者围术期脑损伤的危险性相较于非糖尿病患者显著增高。.2. 主要研究内容：LncRNA-MEG3介导糖尿病或高糖所致的神经细胞损伤涉及调控线粒体源性的细胞凋亡及线粒体自噬，以及LncRNA-MEG3-Rac1信号分子通路与FUNDC1相互作用的分子机制。.3. 重要结果及关键数据：.(1)LncRNA-MEG3通过抑制Rac1的激活减轻糖尿病大鼠海马细胞凋亡；.(2)过表达LncRNA-MEG3抑制Rac1激活.(3)LncRNA-MEG3信号通路促进FUNDC1活化；.(4)高糖下调MEG3表达及LncRNA-MEG3调控线粒体介导的凋亡；.(5)LncRNA-MEG3通过抑制Rac1-ROS通路减少高糖诱导的神经细胞凋亡；.(6)高糖干扰神经细胞的线粒体自噬过程；.(7)LncRNA-MEG3通过促进FUNDC1介导的线粒体自噬减轻高糖诱导的神经细胞线粒体功能障碍；.(8)高糖条件下Rac1和FUNDC1在线粒体上形成复合物，表明Rac1对FUNDC1激活具有直接调节作用，LncRNA-MEG3的过表达显著抑制线粒体中Rac1和FUNDC1复合物的形成；.(9)LncRNA-MEG3通过改善原代海马神经元的线粒体功能缓解高糖诱导的神经损伤.(10)RNA Pull-down和RIP实验检测了lncRNA MEG3相关的线粒体蛋白，Plectin介导了lncRNA MEG3在原代海马神经元中的线粒体定位.(11)LncRNA MEG3通过抑制原代海马神经元中HSP90A的磷酸化来增强HSP90A的线粒体易位.(12)LncRNA MEG3通过HSP90A改善原代海马神经元的线粒体功能，从而减轻高糖诱导的损伤.结论：选择性过表达 LncRNA-MEG3能有效减轻糖尿病导致的脑损伤， LncRNA-MEG3 一方面通过抑制Rac1的激活减少活性氧的生成抑制线粒体源性的细胞凋亡，另一方面通过干扰Rac1与自噬因子-FUNDC1 在线粒体上的的相互结合启动FUNDC1-依耐性的线粒体自噬而实现。 此外，LncRNA MEG3通过介导HSP90A的线粒体易位及其磷酸化，改善高糖处理的原代海马神经元线粒体功能。.4.科学意义.为临床糖尿病所致的脑损伤的治疗提供新的靶点和理论依据。