Influence of GAS5/MicroRNA-223-3p/P2Y12 Axis on Clopidogrel Response in Coronary Artery Disease.
Influence of GAS5/MicroRNA-223-3p/P2Y12 Axis on Clopidogrel Response in Coronary Artery Disease.
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DOI：
10.1161/jaha.121.021129
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发表时间：
2021-11-02
影响因子：
5.4
通讯作者：
Chen XP
中科院分区：
文献类型：
--
作者：
Liu YL;Hu XL;Song PY;Li H;Li MP;Du YX;Li MY;Ma QL;Peng LM;Song MY;Chen XP
基于阿司匹林和 P2Y12 受体拮抗剂（如氯吡格雷）的双重抗血小板治疗是目前冠状动脉疾病 (CAD) 的主要治疗方法。然而，有一定比例的患者表现出氯吡格雷耐药，其中遗传因素起着至关重要的作用。本研究旨在探讨 GAS5（生长停滞特异性 5）及其 rs55829688 多态性在 CAD 患者氯吡格雷反应中的作用。纳入2017年至2018年接受双重抗血小板治疗的444例CAD患者，评价GAS5单核苷酸多态性rs55829688对血小板反应性指数的影响。使用微珠纯化来自这些患者中的 37 名患者的血小板，以检测 GAS5 和 microRNA-223-3p (miR-223-3p) 的表达。从另外 17 名健康志愿者和 46 名新诊断的 CAD 患者中分离出富含血小板的血浆，以检测 GAS5 和 miR-223-3p 的表达。进行双荧光素酶报告基因测定，以探索 miR-223-3p 与 GAS5 或 P2Y12 3'-UTR（表达 SV40 大 T 抗原突变版本的人胚胎肾 293 细胞系）HEK 293T 和（源自 1983 年患有巨核细胞危机的慢性粒细胞白血病患者骨髓的巨核细胞系）之间的相互作用MEG-01 细胞。进行功能丧失和功能获得实验以揭示 MEG-01 细胞中 GAS5 通过 miR-223-3p 对 P2Y12 的调节。我们观察到，在CYP2C19弱代谢者中，rs55829688 CC纯合子表现出比TT纯合子显着降低的血小板反应性指数。血小板 GAS5 表达与血小板反应指数和 P2Y12 mRNA 表达呈正相关，而血小板 miR-223-3p 表达与血小板反应指数呈负相关。同时，血小板中 GAS5 和 miR-223-3p 表达呈负相关。 MEG-01 细胞中 miR-223-3p 模拟物减少，而 miR-223-3p 抑制剂增加 GAS5 和 P2Y12 的表达。 siRNA 敲低 GAS5 会增加 miR-223-3p 表达并减少 P2Y12 表达，而 miR-223-3p 抑制剂可以逆转这一现象。同时，GAS5 的过表达降低了 miR-223-3p 的表达并增加了 P2Y12 的表达，而这种情况可以被 miR-223-3p 模拟物逆转。 GAS5 rs55829688 多态性可能影响 CYP2C19 弱代谢基因型 CAD 患者的氯吡格雷反应，并且 GAS5 通过充当 miR-223-3p 的竞争性内源 RNA 来调节 P2Y12 表达和氯吡格雷反应。
Dual antiplatelet therapy based on aspirin and P2Y12 receptor antagonists such as clopidogrel is currently the primary treatment for coronary artery disease (CAD). However, a percentage of patients exhibit clopidogrel resistance, in which genetic factors play vital roles. This study aimed to investigate the roles of GAS5 (growth arrest‐specific 5) and its rs55829688 polymorphism in clopidogrel response in patients with CAD. A total of 444 patients with CAD receiving dual antiplatelet therapy from 2017 to 2018 were enrolled to evaluate the effect of GAS5 single nucleotide polymorphism rs55829688 on platelet reactivity index. Platelets from 37 patients of these patients were purified with microbeads to detect GAS5 and microRNA‐223‐3p (miR‐223‐3p) expression. Platelet‐rich plasma was isolated from another 17 healthy volunteers and 46 newly diagnosed patients with CAD to detect GAS5 and miR‐223‐3p expression. A dual‐luciferase reporter assay was performed to explore the interaction between miR‐223‐3p and GAS5 or P2Y12 3′‐UTR in (human embryonic kidney 293 cell line that expresses a mutant version of the SV40 large T antigen) HEK 293T and (megakaryoblastic cell line derived in 1983 from the bone marrow of a chronic myeloid leukemia patient with megakaryoblastic crisis) MEG‐01 cells. Loss‐of‐function and gain‐of‐function experiments were performed to reveal the regulation of GAS5 toward P2Y12 via miR‐223‐3p in MEG‐01 cells. We observed that rs55829688 CC homozygotes showed significantly decreased platelet reactivity index than TT homozygotes in CYP2C19 poor metabolizers. Platelet GAS5 expression correlated positively with both platelet reactivity index and P2Y12 mRNA expressions, whereas platelet miR‐223‐3p expression negatively correlated with platelet reactivity index. Meanwhile, a negative correlation between GAS5 and miR‐223‐3p expressions was observed in platelets. MiR‐223‐3p mimic reduced while the miR‐223‐3p inhibitor increased the expression of GAS5 and P2Y12 in MEG‐01 cells. Knockdown of GAS5 by siRNA increased miR‐223‐3p expression and decreased P2Y12 expression, which could be reversed by the miR‐223‐3p inhibitor. Meanwhile, overexpression of GAS5 reduced miR‐223‐3p expression and increased P2Y12 expression, which could be reversed by miR‐223‐3p mimic. GAS5 rs55829688 polymorphism might affect clopidogrel response in patients with CAD with the CYP2C19 poor metabolizer genotypes, and GAS5 regulates P2Y12 expression and clopidogrel response by acting as a competitive endogenous RNA for miR‐223‐3p.