Circ_0003575 敲低通过 miR-637/TRAF6 轴减轻 ox-LDL 诱导的动脉粥样硬化人主动脉内皮细胞功能障碍。

Circ_0003575 knockdown alleviates ox-LDL-induced human aortic endothelial cell dysfunction in atherosclerosis by miR-637/TRAF6 axis.

机构信息

Department of Cardiovascular Medicine, The First Affiliated Hospital of Hebei North University, Zhangjiakou City, Hebei, China.

出版信息

Clin Hemorheol Microcirc. 2023;85(2):173-187. doi: 10.3233/CH-231858.

DOI:10.3233/CH-231858

PMID:37599528

Abstract

BACKGROUND

Circular RNAs (circRNAs) are involved in the progression of atherosclerosis (AS). The present study aimed to determine the functions and mechanism of circ_0003575 in AS.

METHODS

Oxidized low-density lipoprotein (ox-LDL) was used to induce human aortic endothelial cells (HAECs) to establish an AS cell model. Cell Counting Kit-8 (CCK-8) assay and 5'-ethynyl-2'-deoxyuridine (EdU) assay were conducted to assess cell proliferation. Flow cytometry analysis was utilized to quantify cell apoptosis. Tube formation assay was performed to analyze angiogenesis ability. Enzyme linked immunosorbent assay (ELISA) was used to examine the concentrations of inflammatory factors. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were manipulated for the expression of circ_0003575, microRNA-637 (miR-637) and TNF receptor associated factor 6 (TRAF6). Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were adopted to estimate the downstream targets of circ_0003575.

RESULTS

Ox-LDL treatment repressed the proliferation and angiogenesis and promoted the apoptosis and inflammation in HAECs. Circ_0003575 knockdown ameliorated ox-LDL-induced injury of HAECs. Circ_0003575 interacted with mi-R-637, which directly targeted TRAF6. Inhibition of miR-637 reversed the impacts of circ_0003575 knockdown on HAEC injury. Moreover, miR-637 overexpression promoted cell proliferation and angiogenesis and inhibited cell apoptosis and inflammation by targeting TRAF6 in ox-LDL-treated HAECs. Further, circ_0003575 silencing inhibited the activation of NF-κB pathway.

CONCLUSION

Circ_0003575 knockdown alleviated ox-LDL-induced HAEC damage by regulating miR-637/TRAF6 and NF-κB pathways.

摘要

背景

环状 RNA（circRNAs）参与动脉粥样硬化（AS）的进展。本研究旨在确定 circ_0003575 在 AS 中的功能和机制。

方法

使用氧化低密度脂蛋白（ox-LDL）诱导人主动脉内皮细胞（HAECs）建立 AS 细胞模型。通过细胞计数试剂盒-8（CCK-8）测定和 5'-乙炔基-2'-脱氧尿苷（EdU）测定评估细胞增殖。通过流式细胞术分析定量细胞凋亡。通过管形成测定分析血管生成能力。酶联免疫吸附测定（ELISA）用于检测炎症因子的浓度。采用定量实时聚合酶链反应（qRT-PCR）和蛋白质印迹法检测 circ_0003575、微小 RNA-637（miR-637）和肿瘤坏死因子受体相关因子 6（TRAF6）的表达。采用双荧光素酶报告基因和 RNA 免疫沉淀（RIP）测定来评估 circ_0003575 的下游靶标。

结果

ox-LDL 处理抑制 HAEC 增殖和血管生成，并促进其凋亡和炎症。circ_0003575 敲低可改善 ox-LDL 诱导的 HAEC 损伤。circ_0003575 与 miR-637 相互作用，miR-637 可直接靶向 TRAF6。抑制 miR-637 逆转了 circ_0003575 敲低对 ox-LDL 处理的 HAEC 损伤的影响。此外，miR-637 过表达通过靶向 TRAF6 促进 ox-LDL 处理的 HAEC 中的细胞增殖和血管生成，并抑制细胞凋亡和炎症。进一步，circ_0003575 沉默抑制 NF-κB 通路的激活。