OBJECTIVE: To explore the change rule and mechanism of exosomes release, and the role and molecular mechanism of exosome-miR-146a in AS.
METHODS: We isolated and identified exosomes from THP-1 macrophages after treating them with ox-LDL. Then used co-immunoprecipitation and silver staining to identify the proteins involved in regulating exosome release. PKH67 was used to label exosomes to confirm that cells can absorb them, and then co-culture with HVSMCs for cell proliferation and migration detection. The target genes of miR-146a were screened and identified through bioinformatics and luciferase activity assay, and the expression of miR-146a and related proteins was detected through qRT-PCR and Western blot in HUVECs. An AS model in LDLR-/- mice induced by a high-fat diet was developed to investigate the impact of exosome-miR-146a on AS.
RESULTS: The results showed that experimental foam cells from AS showed higher expression of miR-146a. It was observed that NMMHC IIA and HSP70 interacted to regulate the release of exosomes. And HUVECs can absorb exosomes derived from macrophages. In addition, we also found that miR-146a directly targeted the SMAD4 gene to modulate the p38 MAPK signaling pathway, thereby mediating HUVECs damage. Furthermore, exosome-miR-146a induced abnormal proliferation and migration of HVSMCs. The expression of miR-146a was significantly reduced in miR-146a-mimics mice and increased in miR-146a inhibitor mice whereas the inhibition of miR-146a effectively reduced while increasing miR-146a worsened AS in mice.
CONCLUSIONS: Our findings expressed the potential of miR-146a as a favorable therapeutic target for AS, however, further exploration is suggestive for deep understanding of the mechanisms regulating exosome-miR-146a release in vivo and to develop effective therapeutic strategies involving miR-146a.
目的:探讨外泌体释放的变化规律和机制,外泌体-miR-146a在AS中的作用及分子机制。
方法:我们用ox-LDL处理后,从THP-1巨噬细胞中分离并鉴定了外泌体。然后使用共免疫沉淀和银染色来鉴定参与调节外泌体释放的蛋白质。PKH67用于标记外泌体,以确认细胞可以吸收它们,然后与HVSMCs共培养用于细胞增殖和迁移检测。通过生物信息学和荧光素酶活性测定对miR-146a的靶基因进行筛选和鉴定,qRT-PCR和Westernblot检测HUVECs中miR-146a及相关蛋白的表达。在高脂饮食诱导的LDLR-/-小鼠中建立AS模型以研究外泌体-miR-146a对AS的影响。
结果:结果显示,来自AS的实验泡沫细胞显示出更高的miR-146a表达。观察到NMMHIIIA和HSP70相互作用以调节外来体的释放。HUVECs可以吸收来自巨噬细胞的外泌体。此外,我们还发现miR-146a直接靶向SMAD4基因来调节p38MAPK信号通路,从而调解HUVECs损伤。此外,外泌体-miR-146a诱导HVSMC的异常增殖和迁移。miR-146a的表达在miR-146a模拟小鼠中显着降低,在miR-146a抑制剂小鼠中增加,而miR-146a的抑制有效降低,而miR-146a的增加使小鼠的AS恶化。
结论:我们的发现表达了miR-146a作为AS的有利治疗靶点的潜力,然而,进一步的探索有助于深入了解体内外泌体-miR-146a释放的调节机制,并有助于开发涉及miR-146a的有效治疗策略.