Molecular Medicine Program, University of Utah, Salt Lake City, UT, USA.
The Cardeza Foundation for Hematologic Research and the Department of Medicine, Thomas Jefferson University, Jefferson Medical College, Philadelphia, PA, USA.
J Thromb Haemost. 2019 Mar;17(3):511-524. doi: 10.1111/jth.14382. Epub 2019 Feb 25.
Essentials The action of microRNAs (miRs) in human megakaryocyte signaling is largely unknown. Cord blood-derived human megakaryocytes (MKs) were used to test the function of candidate miRs. miR-15a-5p negatively regulated MK GPVI-mediated αIIbβ3 activation and α-granule release. miR-15a-5p acts as a potential "master-miR" regulating genes in the MK GPVI signaling pathway. SUMMARY: Background Megakaryocytes (MKs) invest their progeny platelets with proteins and RNAs. MicroRNAs (miRs), which inhibit mRNA translation into protein, are abundantly expressed in MKs and platelets. Although platelet miRs have been associated with platelet reactivity and disease, there is a paucity of information on the function of miRs in human MKs. Objective To identify MK miRs that regulate the GPVI signaling pathway in the MK-platelet lineage. Methods Candidate miRs associated with GPVI-mediated platelet aggregation were tested for functionality in cultured MKs derived from cord blood. Results An unbiased, transcriptome-wide screen in 154 healthy donors identified platelet miR-15a-5p as significantly negatively associated with CRP-induced platelet aggregation. Platelet agonist dose-response curves demonstrated activation of αIIbβ3 in suspensions of cord blood-derived cultured MKs. Overexpression and knockdown of miR-15a-5p in these MKs reduced and enhanced, respectively, CRP-induced αIIbβ3 activation but did not alter thrombin or ADP stimulation. FYN, SRGN, FCER1G, MYLK. and PRKCQ, genes involved in GPVI signaling, were identified as miR-15a-5p targets and were inhibited or de-repressed in MKs with miR-15a-5p overexpression or inhibition, respectively. Lentiviral overexpression of miR-15a-5p also inhibited GPVI-FcRγ-mediated phosphorylation of Syk and PLCγ2, GPVI downstream signaling molecules, but effects of miR-15a-5p on αIIbβ3 activation did not extend to other ITAM-signaling receptors (FcγRIIa and CLEC-2). Conclusion Cord blood-derived MKs are a useful human system for studying the functional effects of candidate platelet genes. miR-15a-5p is a potential "master-miR" for specifically regulating GPVI-mediated MK-platelet signaling. Targeting miR-15a-5p may have therapeutic potential in hemostasis and thrombosis.
主要内容 微小 RNA（miRs）在人类巨核细胞信号转导中的作用在很大程度上是未知的。使用脐血来源的人类巨核细胞（MKs）来测试候选 miR 的功能。miR-15a-5p 负调控 MK GPVI 介导的αIIbβ3 活化和α-颗粒释放。miR-15a-5p 作为一种潜在的“主 miR”，调节 MK GPVI 信号通路中的基因。 背景 巨核细胞（MKs）将其后代血小板赋予蛋白质和 RNA。微小 RNA（miRs）可抑制 mRNA 翻译成蛋白质，在 MKs 和血小板中大量表达。尽管血小板 miR 与血小板反应性和疾病有关，但关于 MKs 中 miR 功能的信息很少。 目的 鉴定调节 MK-血小板谱系中 GPVI 信号通路的 MK miR。 方法 在源自脐血的培养 MK 中测试与 GPVI 介导的血小板聚集相关的候选 miR 的功能。 结果 在 154 名健康供体的无偏、全转录组筛选中，血小板 miR-15a-5p 与 CRP 诱导的血小板聚集呈显著负相关。在悬浮于来自脐血的培养 MKs 的血小板激动剂剂量反应曲线中证明了αIIbβ3 的激活。在这些 MKs 中转录物水平过表达和敲低 miR-15a-5p 分别减少和增强了 CRP 诱导的αIIbβ3 活化，但不改变凝血酶或 ADP 刺激。涉及 GPVI 信号转导的基因 FYN、SRGN、FCER1G、MYLK 和 PRKCQ 被鉴定为 miR-15a-5p 的靶基因，并且在 miR-15a-5p 过表达或抑制时分别被抑制或去抑制。miR-15a-5p 的慢病毒过表达也抑制了 GPVI-FcRγ介导的 Syk 和 PLCγ2 的磷酸化，GPVI 下游信号分子，但 miR-15a-5p 对αIIbβ3 激活的作用并未扩展到其他 ITAM 信号受体（FcγRIIa 和 CLEC-2）。 结论 源自脐血的 MKs 是研究候选血小板基因功能的有用人类系统。miR-15a-5p 是特异性调节 GPVI 介导的 MK-血小板信号的潜在“主 miR”。靶向 miR-15a-5p 在止血和血栓形成中可能具有治疗潜力。
