Department of Clinical Pharmacology Xiangya HospitalCentral South University Changsha Hunan China.
Institute of Clinical Pharmacology, Central South University Hunan Key Laboratory of PharmacogeneticsChangsha Hunan China.
J Am Heart Assoc. 2021 Nov 2;10(21):e021129. doi: 10.1161/JAHA.121.021129. Epub 2021 Oct 29.
Background 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. Methods and Results A total of 444 patients with CAD receiving dual antiplatelet therapy from 2017 to 2018 were enrolled to evaluate the effect of 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 or 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 poor metabolizers. Platelet GAS5 expression correlated positively with both platelet reactivity index and 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. Conclusions rs55829688 polymorphism might affect clopidogrel response in patients with CAD with the poor metabolizer genotypes, and GAS5 regulates P2Y12 expression and clopidogrel response by acting as a competitive endogenous RNA for miR-223-3p.
基于阿司匹林和 P2Y12 受体拮抗剂（如氯吡格雷）的双联抗血小板治疗目前是冠状动脉疾病（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抵抗，其中遗传因素起着重要作用。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。
共纳入 2017 年至 2018 年间接受双联抗血小板治疗的 444 例 CAD 患者，以评估单核苷酸多态性 rs55829688 对血小板反应指数的影响。从这些患者中的 37 名患者中用微珠纯化血小板，以检测 GAS5 和 microRNA-223-3p（miR-223-3p）表达。从另外 17 名健康志愿者和 46 名新诊断的 CAD 患者中分离富含血小板的血浆，以检测 GAS5 和 miR-223-3p 表达。进行双荧光素酶报告基因实验，以探讨 miR-223-3p 与在（表达 SV40 大 T 抗原突变体的人胚肾 293 细胞系）HEK 293T 和（1983 年从慢性髓性白血病伴巨核细胞危象患者的骨髓中衍生的巨核细胞系）MEG-01 细胞中或 3'-UTR 的相互作用。进行失活和功能获得实验，以揭示 miR-223-3p 在 MEG-01 细胞中对 GAS5 向 P2Y12 的调节作用。我们观察到 rs55829688 CC 纯合子在代谢不良者中的血小板反应指数明显低于 TT 纯合子。血小板 GAS5 表达与血小板反应指数和 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，我们在血小板中观察到 GAS5 和 miR-223-3p 表达之间存在负相关。miR-223-3p 模拟物减少，而 miR-223-3p 抑制剂增加了 MEG-01 细胞中的 GAS5 和 P2Y12 表达。siRNA 敲低 GAS5 增加了 miR-223-3p 的表达，降低了 P2Y12 的表达，而 miR-223-3p 抑制剂可逆转这一作用。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 miR-223-3p 模拟物可逆转这一作用。
rs55829688 多态性可能会影响代谢不良的 CAD 患者对氯吡格雷的反应，而 GAS5 可能通过作为 miR-223-3p 的竞争性内源性 RNA 来调节 P2Y12 表达和氯吡格雷反应。
