内皮微粒包裹的miR-204-3p介导川崎病血管炎性损伤的机制探讨

赵颖; 赵欣; 娄萍; 罗英

doi:10.12125/j.chj.202303085

内皮微粒包裹的miR-204-3p介导川崎病血管炎性损伤的机制探讨

Mechanism of endothelial microparticle-encapsulated miR-204-3p induced vasculitic injury in Kawasaki disease

摘要

摘要:
目的探讨内皮微粒（EMP）包裹的miR-204-3p介导川崎病（KD）血管炎性损伤的作用机制。
方法 2016年4月至2021年3月，58例KD患者和50例对照组入选本研究。其中，18例KD患者伴有冠状动脉瘤（CAA），其余为无冠状动脉瘤（NCAA）。分离各组血清样品中EMP，并进行全基因组miRNA测序。在体外试验中，将VSMC或预转染miR-204-3p模拟物(AgomiR-204-3p)、miR-204-3p抑制剂（AntagomiR-204-3p）的VSMC与各组EMP共培养。
结果通过全基因组miRNA测序以及RT-qPCR分析证实miR-204-3p在KD EMP中显著增加，并且EMP中miR-204-3p水平根据冠状动脉病理严重程度显著降低，顺序为NCAA>SCAA>MCAA>GCAA。与对照组EMP共培养相比，VSMC与NCAA EMP共培养48 h后增殖率显著降低（P<0.05），VSMC分化标志物（ACTA2和CNN1）表达显著增加（P<0.05），去分化标志物（OPN和PDGFRβ）表达显著降低（P<0.05）。双荧光素酶报告基因检测证实，EMP中miR-204-3p在功能上靶向VSMC中的PDGFRβ。在与NCAA EMP孵育的VSMC中，使用AntagomiR-204-3p可显著降低分化标记物（ACTA2和CNN1）的表达，并增加去分化标记物（OPN和PDGFRβ）的表达。在与CAA EMP孵育的VSMC中，给予AgomiR-204-3p显著增加分化标记物的表达，并降低去分化标记物的表达。
结论 EMP将miR-204-3p转移到VSMC并通过靶向PDGFRβ部分介导KD血管炎性损伤。因此，靶向miR-204-3p-PDGFRβ轴可能为KD诱导的血管病变提供新的治疗选择。

Abstract:
AIM To investigate the mechanism of miR-204-3p encapsulated in endothelial microparticles (EMP) mediated vascular inflammatory injury in Kawasaki disease (KD).
METHODS From April 2016 to March 2021, 58 KD patients and 50 control subjects were selected for this study. By echocardiographic score, 18 KD patients were with coronary artery aneurysms (CAA) and 40 KD patients were without coronary artery aneurysm (NCAA). EMP was isolated from serum samples of each group and the whole genome miRNA was sequenced. In vitro experiments, VSMC or VSMC pre-transfected with miR-204-3p mimetic (AgomiR-204-3p) and miR-204-3p inhibitor (AntagomiR-204-3p) were co-cultured with EMP in each group.
RESULTS Whole-genome miRNA sequencing and RT-qPCR analysis confirmed that miR-204-3p was significantly increased in KD EMP and the copy number of miR-204-3p in EMP was significantly reduced dependent on the coronary pathology severity, following the order NCAA>SCAA>MCAA>GCAA. VSMC proliferation was significantly reduced (P<0.05), the expression of VSMC differentiation markers (ACTA2 and CNN1) was significantly increased (P<0.05) and the expression of dedifferentiation markers (OPN and PDGFRβ) was significantly reduced (P<0.05) after 48 hours of incubation with NCAA EMP compared with those of control EMP. Dual luciferase reporter gene detection confirmed that miR-204-3p in EMP functionally targeted PDGFRβ in VSMC. In VSMCs incubated with NCAA EMP, the administration of AntagomiR-204-3p significantly reduced the expression of differentiation markers (ACTA2 and CNN1) and increased the expression of dedifferentiation markers (OPN and PDGFRβ). In VSMC incubated with CAA EMP, the administration of AgomiR-204-3p significantly increased the expression of differentiation markers and reduced the expression of dedifferentiation markers.
CONCLUSION EMP transfers miR-204-3p to VSMC and partially mediates KD vasculitis damage by targeting PDGFRβ. Therefore, targeting the miR-204-3p-PDGFRβ axis may provide a novel therapeutic option for KD-induced vascular pathologies.

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