氧化低密度脂蛋白刺激的巨噬细胞外泌体通过传递miR-186-5p，然后使SHIP2介导的PI3K/AKT/mTOR通路失活，从而促进动脉粥样硬化性血管平滑肌细胞的存活和侵袭。
OxLDL-stimulated macrophage exosomes promote proatherogenic vascular smooth muscle cell viability and invasion via delivering miR-186-5p then inactivating SHIP2 mediated PI3K/AKT/mTOR pathway.
作者信息
Ren Lingyun, Chen Shanshan, Yao Dan, Yan Hong
机构信息
Anesthesiology Department, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430013, China.
Key Laboratory for Molecular Diagnosis of Hubei Province, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430013, China.
出版信息
Mol Immunol. 2022 Jun;146:27-37. doi: 10.1016/j.molimm.2022.02.018. Epub 2022 Apr 11.
The current study aimed to investigate the implication of microRNA (miRNA) profile in the linkage between oxidized low-density-lipoprotein (oxLDL)-stimulated-macrophages (MФ) exosomes and vascular smooth muscle cells (VSMCs) during atherosclerosis. VSMCs were treated by oxLDL-stimulated-MФ with/without GW4869. MiRNA profile in oxLDL-stimulated-MФ and untreated-MФ was detected by microarray, then candidate miRNAs were proposed to RT-qPCR and functional validation in VSMCs. MiR-186-5p mimic/inhibitor was transfected into oxLDL-stimulated-MФ, then its exosomes were used to VSMCs. Subsequently, miR-186-5p, SHIP2 and PI3K/AKT/mTOR pathway were modified alone or in combination in VSMCs. VSMCs viability, invasion and apoptosis were detected. OxLDL-stimulated-MФ induced VSMCs viability, invasion, but repressed apoptosis (all P < 0.01); while after GW4869 treatment to delete exosomes, its effect was weakened (all P < 0.05). Totally 45 dysregulated miRNAs were identified in oxLDL-stimulated-MФ versus untreated-MФ. The top-three dysregulated miRNAs (miR-186-5p, miR-21-5p, miR-320b) were elevated in VSMCs after oxLDL-stimulated-MФ treatment (all P < 0.001); while only miR-186-5p mimic greatly enhanced VSMCs viability and invasion (both P < 0.01). Furthermore, miR-186-5p-overexpressed oxLDL-stimulated-MФ exosomes promoted VSMCs viability, invasion, repressed apoptosis, while miR-186-5p-knockdown oxLDL-stimulated-MФ exosomes exhibited opposite effect (all P < 0.05). MiR-186-5p negatively regulated SHIP2 in VSMCs and bound SHIP2 via luciferase-reporter-gene assay (all P < 0.05). SHIP2 overexpression decreased VSMCs viability, invasion, PI3K/AKT/mTOR pathway, increased apoptosis, and attenuated miR-186-5p-overexpression's effect on these functions (all P < 0.05). Besides, PI3K activator (740 Y-P) weakened SHIP2-overexpression's effect on VSMCs viability, invasion and apoptosis (all P < 0.05). In conclusion, oxLDL-stimulated-MФ exosomes deliver miR-186-5p to inactivate SHIP2 mediated PI3K/AKT/mTOR pathway, then promote cell viability and invasion in VSMCs to accelerate atherosclerosis.
当前研究旨在探讨微小RNA（miRNA）谱在动脉粥样硬化过程中氧化型低密度脂蛋白（oxLDL）刺激的巨噬细胞（MФ）外泌体与血管平滑肌细胞（VSMC）之间联系中的作用。用oxLDL刺激的MФ处理VSMC，同时添加或不添加GW4869。通过微阵列检测oxLDL刺激的MФ和未处理的MФ中的miRNA谱，然后将候选miRNA进行逆转录定量聚合酶链反应（RT-qPCR）及在VSMC中的功能验证。将miR-186-5p模拟物/抑制剂转染到oxLDL刺激的MФ中，然后将其外泌体用于处理VSMC。随后，在VSMC中单独或联合改变miR-186-5p、SHIP2和磷脂酰肌醇-3激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白（PI3K/AKT/mTOR）信号通路。检测VSMC的活力、侵袭能力和凋亡情况。oxLDL刺激的MФ可诱导VSMC活力、侵袭能力增强，但抑制凋亡（均P<0.01）；而用GW4869处理以去除外泌体后，其作用减弱（均P<0.05）。与未处理的MФ相比，在oxLDL刺激的MФ中总共鉴定出45种失调的miRNA。oxLDL刺激的MФ处理后，VSMC中上调最明显的三种miRNA（miR-186-5p、miR-21-5p、miR-320b）均升高（均P<0.001）；而只有miR-186-5p模拟物显著增强了VSMC的活力和侵袭能力（均P<0.01）。此外，过表达miR-186-5p的oxLDL刺激的MФ外泌体可促进VSMC活力、侵袭能力，抑制凋亡，而过表达miR-186-5p的oxLDL刺激的MФ外泌体则表现出相反的作用（均P<0.05）。miR-186-5p在VSMC中负向调节SHIP2，并通过荧光素酶报告基因检测证实其与SHIP2结合（均P<0.05）。SHIP2过表达降低了VSMC活力、侵袭能力、PI3K/AKT/mTOR信号通路活性，增加了凋亡，并减弱了过表达miR-186-5p对这些功能的影响（均P<0.05）。此外，PI3K激活剂（740 Y-P）减弱了SHIP2过表达对VSMC活力、侵袭能力和凋亡的影响（均P<0.05）。总之，oxLDL刺激的MФ外泌体传递miR-186-5p以失活SHIP2介导的PI3K/AKT/mTOR信号通路，进而促进VSMC的活力和侵袭能力，加速动脉粥样硬化。
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