Department of Vascular Surgery, the Affiliated People's Hospital of Ningbo University, Ningbo City, Zhejiang, China.
Vasc Med. 2023 Apr;28(2):104-112. doi: 10.1177/1358863X221132591. Epub 2023 Feb 27.
Abdominal aortic aneurysms (AAA) are an important cause of cardiovascular deaths. The loss of vascular smooth muscle cells (VSMCs) has been reported to be related to the pathology of AAA. This study focused on investigating the function of circ_0002168 in VSMC apoptosis.
Levels of genes and proteins were measured by quantitative real-time-polymerase chain reaction (qRT-PCR) and Western blot. The growth of VSMCs was determined by using cell counting kit-8 assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry and the evaluation of caspase-3 activity analysis, reactive oxygen species (ROS) production as well as lactate dehydrogenase (LDH) activity. The binding between miR-545-3p and circ_0002168 or Cytoskeleton-associated protein 4 (CKAP4) was confirmed by bioinformatics analysis, dual-luciferase reporter, RNA immunoprecipitation, and pull-down assays.
Circ_0002168 decreased in the aortic tissues of patients with AAA. Functionally, ectopic overexpression of circ_0002168 dramatically induced proliferation and suppressed apoptosis in VSMCs. Mechanistically, circ_0002168 sequestered miR-545-3p to release CKAP4 expression via the ceRNA mechanism, indicating the circ_0002168/miR-545-3p/CKAP4 feedback loop in VSMCs. Increased miR-545-3p and a decreased CKAP4 expression were observed in patients with AAA. Rescue experiments showed that miR-545-3p reversed the protective effects of circ_0002168 on VSMC proliferation. Moreover, inhibition of miR-545-3p could restrain the apoptosis of VSMCs, which was abolished by CKAP4 silencing.
Circ_0002168 has a protective effect on VSMC proliferation by regulating the miR-545-3p/CKAP4 axis, adding further understanding of the pathogenesis of AAA and a potential therapeutic approach in AAA management.
腹主动脉瘤（AAA）是心血管死亡的一个重要原因。血管平滑肌细胞（VSMCs）的损失已被报道与 AAA 的病理学有关。本研究专注于研究 circ_0002168 在 VSMC 凋亡中的功能。
通过定量实时聚合酶链反应（qRT-PCR）和 Western blot 测量基因和蛋白质的水平。通过细胞计数试剂盒-8 测定法、5-乙炔基-2'-脱氧尿苷（EdU）测定法、流式细胞术以及 caspase-3 活性分析、活性氧（ROS）产生和乳酸脱氢酶（LDH）活性评估来测定 VSMC 的生长。通过生物信息学分析、双荧光素酶报告、RNA 免疫沉淀和下拉测定来验证 miR-545-3p 与 circ_0002168 或细胞骨架相关蛋白 4（CKAP4）之间的结合。
AAA 患者的主动脉组织中 circ_0002168 减少。功能上，circ_0002168 的异位过表达可显著诱导 VSMCs增殖并抑制凋亡。机制上，circ_0002168 通过 ceRNA 机制与 miR-545-3p 结合，释放 CKAP4 表达，表明在 VSMCs 中存在 circ_0002168/miR-545-3p/CKAP4 反馈环。AAA 患者中观察到 miR-545-3p 增加和 CKAP4 表达降低。挽救实验表明，miR-545-3p 逆转了 circ_0002168 对 VSMC 增殖的保护作用。此外，抑制 miR-545-3p 可以抑制 VSMCs 的凋亡，而 CKAP4 沉默则消除了这种抑制作用。
circ_0002168 通过调节 miR-545-3p/CKAP4 轴对 VSMC 增殖具有保护作用，为 AAA 的发病机制提供了进一步的了解，并为 AAA 管理提供了潜在的治疗方法。
