细胞外囊泡衍生的 miR-146a 通过靶向 SMAD4 成为高脂诱导动脉粥样硬化的新型串联机制

Kefeng Zhai, Liangle Deng, Yuxuan Wu, Han Li, Jing Zhou, Ying Shi, Jianhu Jia, Wei Wang, Sihui Nian, Ghulam Jilany Khan, Hesham R El-Seedi, Hong Duan, Lili Li, Zhaojun Wei
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引用次数: 0

摘要

导言:外泌体-miR-146a在动脉粥样硬化(AS)患者中明显增加,但其对AS的作用机制尚未完全阐明:探索外泌体释放的变化规律和机制,以及外泌体-miR-146a在AS中的作用和分子机制:方法:用ox-LDL处理THP-1巨噬细胞后,从巨噬细胞中分离并鉴定外泌体。方法:我们用氧化-LDL处理THP-1巨噬细胞后,从巨噬细胞中分离并鉴定了外泌体,然后用共免疫沉淀和银染色鉴定了参与调控外泌体释放的蛋白质。用 PKH67 标记外泌体以确认细胞能吸收外泌体,然后与 HVSMCs 共同培养检测细胞增殖和迁移。通过生物信息学和荧光素酶活性检测筛选并确定了miR-146a的靶基因,并通过qRT-PCR和Western blot检测了miR-146a和相关蛋白在HUVECs中的表达。建立了高脂饮食诱导的LDLR-/-小鼠AS模型,以研究外泌体-miR-146a对AS的影响:结果表明,实验性AS泡沫细胞的miR-146a表达量较高。结果表明,强直性脊柱炎实验性泡沫细胞miR-146a表达较高,NMMHC IIA和HSP70相互作用调控外泌体的释放。而且,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,调节 p38 MAPK 信号通路,从而介导 HUVECs 损伤。此外,外泌体-miR-146a 还诱导了 HVSMCs 的异常增殖和迁移。在miR-146a模拟小鼠中,miR-146a的表达量明显减少,而在miR-146a抑制剂小鼠中,miR-146a的表达量则有所增加:我们的研究结果表明,miR-146a是治疗强直性脊柱炎的潜在靶点,然而,要深入了解调控体内外泌体-miR-146a释放的机制,并开发出涉及miR-146a的有效治疗策略,还需进一步探索。
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Extracellular vesicle-derived miR-146a as a novel crosstalk mechanism for high-fat induced atherosclerosis by targeting SMAD4.

Introduction: Exosome-miR-146a is significantly increased in patients with Atherosclerosis (AS), but its mechanism and effect on AS have not been fully elucidated.

Objectives: 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.

Conclusion: 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.

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