血管平滑肌细胞富集的长链非编码RNA-APPAT调控自噬及其对动脉粥样硬化斑块稳定性的作用

Autophagy is an important determinant for vascular homeostasis， as well as the atherosclerosis, especially the vulnerable plaque. By lncRNA sequencing on VSMC-derived foam cells, a down-regulated lincRNA was screened and named as linc-APPAT. The linc-APPAT was enriched in VSMC and located in the cytoplasm mainly. The previous results showed that 1) the linc-APPAT was decreased drastically in the coronary artery with grade III/IV arteriostenosis. 2) Moreover, the circulating linc-APPAT was also decreased in the plasma of AMI patients. 3) The autophagy of oxLDL-induced VSMC was changed resulting from the interference of linc-APPAT. 4) The linc-APPAT could bind with the potential target miRNA to regulate the expression of the target autophagy-related genes. Based on the previous results, the scientific hypothesis was presented as “linc-APPAT could regulated the autophagy of VSMC to influence the stability of the vulnerable plaque” in this project. The molecular mechanism of linc-APPAT regulatory network on autophagy and stability of vulnerable plaque will be elucidated by the investigations in vitro and in vivo, which will supply the new idea for the further diagnosis, prevention and treatment of AS-related diseases.

细胞自噬是血管稳态维持的关键环节，对动脉粥样硬化进程尤其是斑块稳定性有重要作用。申请人经血管平滑肌细胞（VSMC）源性泡沫细胞lncRNA测序发现显著下调的lincRNA-APPAT，该lnc富集于VSMC且定位于胞浆。预实验发现，在III/IV级动脉硬化的冠状动脉血管中linc-APPAT表达明显下降，且急性心梗患者血浆循环linc-APPAT水平显著下调；linc-APPAT干预之后oxLDL诱导的VSMC自噬发生显著变化；linc-APPAT能与相关靶miRNA结合影响自噬基因的表达。基于预实验结果，本项目提出科学假说“linc-APPAT调节VSMC自噬影响易损斑块稳定性”，经体内和体外实验阐明linc-APPAT调控网络调节自噬与斑块稳定性的分子机制，为动脉粥样硬化性疾病的诊断分型与防治提供新的思路。

本项目旨在基于“linc-APPAT-靶miRNA-靶mRNA”调控网络，研究linc-APPAT在AS斑块稳定性中的调控作用与其分子机制，提出AS诊断分型与治疗的新策略。研究发现：1）APPAT在血管壁中膜的收缩型VSMCs特异性富集，主要分布在细胞质中；APPAT表达量在管腔严重狭窄患者的冠脉样本中显著下调；在血液样本中的表达变化表现为依正常→心衰→心梗逐渐降低的趋势；在心血管患者血液内的表达改变独立于高血压和糖尿病等疾病；分析显示APPAT与心梗检测标志物cTnl在心梗发生上有相近的高预测能力，是AS疾病进展潜在的生物标记物。2）miR-647可在ox-LDL 处理过的VSMCs 中稳定扩增且表达量上调，是APPAT 的靶miRNA。MiR-647在冠脉组织样本和患者血液中表达量与APPAT 呈负相关；同时miR-647 对VSMCs 的自噬活性有促进作用。3）miR-647可以促进人VSMCs的增殖和迁移；FGF5为miR-647的靶基因，miR-647可以促进FGF5的表达；APPAT可以抑制FGF5的表达；APPAT可以与miR-647相互作用，共同调控FGF5的表达。4）APPAT可以促进HVSMCs的自噬；miR-647可以抑制HVSMCs的自噬；ATG5是miR-647的靶基因，miR-647可以抑制ATG5的表达；APPAT可以通过“APPAT-miR-647-ATG5”轴调节ATG5的表达，从而影响HVSMCs的自噬。以上研究结果表明，“linc-APPAT-miR-647-FGF5/ATG5”调控网络能够调控VSMCs自噬、增殖与迁移，从而影响动脉粥样硬化斑块的稳定性。项目研究期间，培养博士研究生1名，硕士研究生3名；参与编写专著1部；已发表SCI论文2篇，专家述评1篇；接收SCI论文1篇，专家述评1篇，另有2篇英文论文正在投稿（均标注受本项目资助）。