Zhejiang Chinese Medical University, Hangzhou, China.
The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China.
PeerJ. 2023 Mar 31;11:e15180. doi: 10.7717/peerj.15180. eCollection 2023.
Macrophage polarization and microRNA play crucial roles in the development of atherosclerosis (AS). The M1 macrophage phenotype contributes to the formation of plaques, while the M2 macrophage phenotype resolves inflammation and promotes tissue repair. MiR-126 has been found to play a role in regulating macrophage polarization in the context of AS. However, the exact mechanism of miR-126 requires further research.
The foam cell model was established by stimulating THP-1 with oxidized low-density lipoprotein (ox-LDL). We transfected foam cells with miR-126 mimic and its negative control. The transfection of miR-126 was implemented by riboFECT CP transfection kit. The levels of miR-126 and M1/M2 associated genes in foam cells were quantified using reverse transcription-quantitative PCR (RT-qPCR). Additionally, the expressions of CD86 and CD206 cells in foam cells were determined by flow cytometry. Western blotting and RT-qPCR were used to determine the protein and mRNA levels of the vascular endothelial growth factor A (VEGFA) and the transcriptional regulator Krüppel-like factor 4 (KLF4), respectively. Additionally, we detected endothelial cell migration after co-culturing endothelial cells and macrophages. MG-132 was used to indirectly activate the expression of VEGFA, and the expression of KLF4 was also evaluated.
The activation of apoptosis and production of foam cells were boosted by the addition of ox-LDL. We transfected foam cells with miR-126 mimic and its negative control and observed that miR-126 greatly suppressed foam cell development and inhibited phagocytosis. Moreover, it caused pro-inflammatory M1 macrophages to switch to the anti-inflammatory M2 phenotype. This was reflected by the increase in anti-inflammatory gene expression and the decrease in pro-inflammatory gene expression. Additionally, miR-126 dramatically decreased the expressions of VEGFA and KLF4. The protein-protein interaction network analysis showed a significantly high correlation between miR-126, VEGFA, and KLF4. MiR-126 may also promote EC migration by activating macrophage PPAR γ expression and effectively suppressing macrophage inflammation. MG-132 indirectly activated the expression of VEGFA, and the expression of KLF4 also significantly increased, which indicates a direct or indirect relationship between VEGFA and KLF4.
Our study shows that miR-126 can reverse ox-LDL-mediated phagocytosis and apoptosis in macrophages. Consequently, the potential role of miR-126 was manifested in regulating macrophage function and promoting vascular endothelial migration.
巨噬细胞极化和 microRNA 在动脉粥样硬化（AS）的发展中起着至关重要的作用。M1 巨噬细胞表型有助于斑块的形成，而 M2 巨噬细胞表型则能缓解炎症并促进组织修复。已经发现 miR-126 在 AS 背景下调节巨噬细胞极化中发挥作用。然而，miR-126 的确切机制仍需要进一步研究。
用氧化低密度脂蛋白（ox-LDL）刺激 THP-1 建立泡沫细胞模型。我们用 miR-126 模拟物及其阴性对照转染泡沫细胞。通过 riboFECT CP 转染试剂盒转染 miR-126。采用逆转录定量 PCR（RT-qPCR）检测泡沫细胞中 miR-126 及 M1/M2 相关基因的水平。此外，通过流式细胞术检测泡沫细胞中 CD86 和 CD206 细胞的表达。采用 Western blot 和 RT-qPCR 分别检测血管内皮生长因子 A（VEGFA）和转录调节因子 Krüppel 样因子 4（KLF4）的蛋白和 mRNA 水平。此外，我们还检测了共培养内皮细胞和巨噬细胞后内皮细胞的迁移情况。用 MG-132 间接激活 VEGFA 的表达，并评估 KLF4 的表达。
ox-LDL 的加入促进了细胞凋亡和泡沫细胞的产生。我们用 miR-126 模拟物及其阴性对照转染泡沫细胞，发现 miR-126 能显著抑制泡沫细胞的发展并抑制吞噬作用。此外，它能使促炎 M1 巨噬细胞向抗炎 M2 表型转化。这反映在抗炎基因表达增加和促炎基因表达减少上。此外，miR-126 显著降低了 VEGFA 和 KLF4 的表达。蛋白质-蛋白质相互作用网络分析表明，miR-126、VEGFA 和 KLF4 之间存在显著的高相关性。miR-126 还可能通过激活巨噬细胞的过氧化物酶体增殖物激活受体 γ（PPAR γ）表达并有效抑制巨噬细胞炎症来促进 EC 迁移。MG-132 间接激活了 VEGFA 的表达，KLF4 的表达也显著增加，这表明 VEGFA 和 KLF4 之间存在直接或间接关系。
我们的研究表明，miR-126 可以逆转 ox-LDL 介导的巨噬细胞吞噬作用和凋亡。因此，miR-126 的潜在作用体现在调节巨噬细胞功能和促进血管内皮细胞迁移上。
