心肌肥厚发生的新机制—lncRNA Gm15834/miR-30b/ULK1/自噬调控网络参与心肌肥厚的作用及机制

The exact mechanism of pathological cardiac hypertrophy remains unclear. In recent years, there is increasing concern about the role of lncRNA in cardiac hypertrophy. Our previous work found that the expression of lncRNA Gm15834 was increased, miR-30b was decreased, ULK1 was enhanced, and the autophagy was activated in the mouse hypertrophic hearts. At the same time, we found that silencing lncRNA Gm15834 could inhibit myocardial hypertrophy and autophagy activation. Combining our previous work, we will employ mice cardiac hypertrophy models and the serum of hypertrophic cardiomyopathy patients as study objects to explore the pathogenesis of myocardial hypertrophy from a completely new angle. We will solve the following problems through this project: Are lncRNA Gm15834, miR-30b and ULK1 related to the occurrence of cardiac hypertrophy? Do the three have regulatory effect on the occurrence of cardiac hypertrophy? Is there a regulatory relationship between the three? Is there a lncRNA Gm15834/miR-30b/ULK1 regulatory network? Does the regulation network participate in myocardial hypertrophy by mediating autophagy? The resolution of the above questions would elucidate a novel mechanism of lncRNA Gm15834/miR-30b/ULK1/autophagy in cardiac hypertrophy, and provide a new idea for the therapy of pathological cardiac hypertrophy.

心肌肥厚的发病机制不清。近年来lncRNA在心肌肥厚中的作用日益受到关注。我们前期工作发现，心肌肥厚小鼠伴有lncRNA Gm15834的增多、miR-30b的减少、ULK1的增多和自噬的激活；同时发现敲减lncRNA Gm15834能抑制心肌肥厚的发生和自噬的激活。结合前期工作，本课题将以小鼠心肌肥厚模型和肥厚型心肌病人血清为研究对象，从全新的角度探讨心肌肥厚的发病机制。通过本课题我们将解决：lncRNA Gm15834、miR-30b和ULK1是否与心肌肥厚的发生有关？三者对心肌肥厚的发生是否有调控作用？三者之间是否有调控关系？是否存在lncRNA Gm15834/miR-30b/ULK1调控网络？该调控网络是否通过介导自噬参与心肌肥厚的发生？这些问题的解决将会揭示心肌肥厚发病的新机制：lncRNA Gm15834/miR-30b/ULK1/自噬/心肌肥厚，将会为临床治疗提供新思路。

心肌肥厚的发病机制不清，据文献报道lncRNA参与心肌肥厚的发生，我们预测到lncRNA Gm15834（Gm15834）与心肌肥厚高度相关。本课题以缩窄主动脉弓诱导小鼠心肌肥厚和Ang II诱导心肌细胞肥大为模型，采用透射电镜检测、RT-PCR、western blot、免疫荧光染色、自噬双标腺病毒转染、荧光素酶报告基因等方法，对Gm15834在心肌肥厚中的作用及机制进行了研究。结果显示：（1）心肌肥厚时Gm15834表达增加、miR-30b-3p表达降低、ULK1表达增加，心肌细胞自噬活性增强。过表达Gm15834能降低miR-30b-3p表达、增加ULK1蛋白表达、增强心肌细胞自噬、加重心肌肥厚，敲降则相反；抑制自噬能减轻Gm15834诱导的心肌肥厚。过表达miR-30b-3p能降低ULK1蛋白表达、抑制心肌细胞自噬、减轻心肌肥厚，敲降则相反。miR-30b-3p能与Gm15834结合，是Gm15834下游靶点；ULK1是miR-30b-3p的直接作用靶点；Gm15834、miR-30b-3p和ULK1之间有直接作用关系。过表达miR-30b-3p能改善Gm15834诱导的心肌肥厚和心肌细胞自噬活性增强。（2）心肌肥厚时Sam68、NF-κB及炎症相关因子（TNF-α、IL-1β和IL-6）蛋白表达增加。过表达Gm15834能增加Sam68、NF-κB及炎症相关因子蛋白表达，敲降则相反。过表达Sam68能增加NF-κB及炎症相关因子蛋白表达，加重心肌肥厚。Sam68与Gm15834之间有直接绑定关系，是Gm15834的下游蛋白。过表达Sam68能逆转敲降Gm15834对炎症信号通路和心肌肥厚的抑制。NF-κB与Gm15834存在正反馈调节作用，敲降NF-κB能减缓Gm15834诱导的心肌肥厚以及Sam68、NF-κB和炎症相关因子表达的上调。（3）在体敲降Gm15834对小鼠心肌细胞自噬、炎症和心肌肥厚具有抑制作用；过表达Gm15834则对炎症和心肌肥厚具有促进作用。以上结果说明心肌肥厚时，存在Gm15834/miR-30b-3p/ULK1/自噬调控轴和Gm15834/Sam68/NF-κB/炎症调控轴，通过增强自噬和激活炎症，导致心肌肥厚。通过本研究，我们发现了一个新的心肌肥厚致病lncRNA，并初步阐明了其致病机制，将会为心肌肥厚的治疗提供新的靶点。