Wang Yanjun, Zhang Xin, Ren Mengmeng, He Shu, Bie Hengjie, Duan Mengyang, Chen Zhiyuan, Jia Qiaowei, Chi Boyu, Gan Xiongkang, Li Chengcheng, Fu Yahong, Zhou Hanxiao, Zhang Sheng, Zhang Qian, An Fenghui, Chen Xiumei, Jia Enzhi
Department of Cardiovascular Medicine, The First Affiliated Hospital of Nanjing Medical University, Guangzhou Road 300, Nanjing, 210029, Jiangsu, China.
Department of Critical Care Medicine, The Friendship Hospital of Ili Kazakh Autonomous Prefecture, Yining, 835000, Xinjiang, China.
J Transl Med. 2024 Dec 31;22(1):1171. doi: 10.1186/s12967-024-05966-2.
Coronary artery disease (CAD) has become a dominant economic and health burden worldwide, and the role of autophagy in CAD requires further clarification. In this study, we comprehensively revealed the association between autophagy flux and CAD from multiple hierarchies. We explored autophagy-associated long noncoding RNA (lncRNA) and the mechanisms underlying oxidative stress-induced human coronary artery endothelial cells (HCAECs) injury.
(1) Autophagy-related proteins including LC3, p62, Beclin1, ATG5, and ATG7 were immunohistochemical stained in coronary specimens; (2) The levels and function of autophagy in the HCAEC oxidative stress model were evaluated using western blot (WB), transmission electron microscopy (TEM), and mRFP-GFP-LC3 adenovirus transfection experiments; (3) The competing endogenous RNA (ceRNA) network of lncRNA LUCAT1/hsa-miR-6776-5p/LRRC25 axis was constructed and validated; (4) The expression levels of above autophagy-related RNAs in peripheral blood mononuclear cells (PBMCs) were verified by qPCR, and their diagnostic performance was subsequently analyzed using receiver operating characteristic (ROC) analysis.
(1) The expression of LC3, Beclin1, ATG5, and ATG7 demonstrated a consistent decline whereas p62 expression exhibited an opposite increase as atherosclerosis progressed; (2) Autophagy levels was significantly elevated in HCAECs under oxidative stress, while inhibition of the initial stage of autophagy with 3-MA exacerbated cellular damage; (3) The lncRNA LUCAT1/hsa-miR-6776-5p/LRRC25 axis was established through bioinformatic prediction and validated by dual-luciferase reporter assay, which resulted in a significant decrease in autophagy levels in HCAECs; (4) In total, p62, ATG7, lncRNA LUCAT1 and LRRC25 were validated as robust diagnostic biomarkers for CAD.
Our results delineated the dynamic disruption of the autophagy landscape during the progression of human coronary atherosclerosis and identified the lncRNA LUCAT1/hsa-miR-6776-5p/LRRC25 axis, uncovered through transcriptomic profiling, as a protective mechanism against endothelial cell injury through autophagy activation. Furthermore, we recognized p62, ATG7, lncRNA LUCAT1, and LRRC25 as dependable autophagy-related diagnostic biomarkers in circulating PBMCs, correlating with CAD severity. Collectively, Our findings furnish novel insights into the intricate autophagy landscape at various levels of coronary atherosclerosis and propose potential diagnostic biomarkers, and a theoretical foundation for managing CAD patients.
冠状动脉疾病（CAD）已成为全球主要的经济和健康负担，自噬在CAD中的作用需要进一步阐明。在本研究中，我们从多个层面全面揭示了自噬通量与CAD之间的关联。我们探索了自噬相关的长链非编码RNA（lncRNA）以及氧化应激诱导人冠状动脉内皮细胞（HCAECs）损伤的潜在机制。
（1）对冠状动脉标本进行免疫组织化学染色，检测包括LC3、p62、Beclin1、ATG5和ATG7在内的自噬相关蛋白；（2）利用蛋白质免疫印迹法（WB）、透射电子显微镜（TEM）和mRFP-GFP-LC3腺病毒转染实验，评估HCAEC氧化应激模型中的自噬水平和功能；（3）构建并验证lncRNA LUCAT1/hsa-miR-6776-5p/LRRC25轴的竞争性内源RNA（ceRNA）网络；（4）通过qPCR验证外周血单个核细胞（PBMCs）中上述自噬相关RNA的表达水平，随后使用受试者工作特征（ROC）分析评估其诊断性能。
（1）随着动脉粥样硬化的进展，LC3、Beclin1、ATG5和ATG7的表达呈一致下降，而p62表达则呈相反增加；（2）氧化应激下HCAECs的自噬水平显著升高，而用3-MA抑制自噬起始阶段会加剧细胞损伤；（3）通过生物信息学预测建立lncRNA LUCAT1/hsa-miR-6776-5p/LRRC25轴，并通过双荧光素酶报告基因检测进行验证，该轴导致HCAECs自噬水平显著降低；（4）总体而言，p62、ATG7、lncRNA LUCAT1和LRRC25被验证为CAD的可靠诊断生物标志物。
我们的结果描绘了人类冠状动脉粥样硬化进展过程中自噬格局的动态破坏，并确定了通过转录组分析发现的lncRNA LUCAT1/hsa-miR-6776-5p/LRRC25轴是一种通过自噬激活来保护内皮细胞免受损伤的机制。此外，我们将p62、ATG7、lncRNA LUCAT1和LRRC25识别为循环PBMCs中与自噬相关的可靠诊断生物标志物，与CAD严重程度相关。总的来说，我们的研究结果为冠状动脉粥样硬化不同层面复杂的自噬格局提供了新见解，提出了潜在的诊断生物标志物，并为CAD患者的管理提供了理论基础。
