miR-126 promotes M1 to M2 macrophage phenotype switching via VEGFA and KLF4

<strong>Background. </strong>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. <strong>Methods.</strong> 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. <strong>Results. </strong>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. <strong>Conclusion.</strong> 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.研究背景：巨噬细胞极化（Macrophage polarization）与微小RNA（microRNA）在动脉粥样硬化（atherosclerosis, AS）的发生发展中发挥关键作用。M1型巨噬细胞表型可促进斑块形成，而M2型巨噬细胞表型则能缓解炎症反应并促进组织修复。已有研究证实，miR-126可在动脉粥样硬化情境下调控巨噬细胞极化，但miR-126的确切作用机制仍需进一步探究。实验方法：本研究采用氧化型低密度脂蛋白（oxidized low-density lipoprotein, ox-LDL）刺激THP-1细胞，构建泡沫细胞模型。分别使用miR-126模拟物及其阴性对照转染泡沫细胞，转染过程采用riboFECT CP转染试剂盒完成。通过逆转录实时定量聚合酶链反应（reverse transcription-quantitative PCR, RT-qPCR）定量检测泡沫细胞中miR-126及M1/M2型相关基因的表达水平；采用流式细胞术检测泡沫细胞中CD86阳性（CD86⁺）与CD206阳性（CD206⁺）细胞的占比。分别采用蛋白质印迹法与RT-qPCR检测血管内皮生长因子A（vascular endothelial growth factor A, VEGFA）与转录调节因子Krüppel样因子4（transcriptional regulator Krüppel-like factor 4, KLF4）的蛋白与mRNA表达水平。此外，将内皮细胞与巨噬细胞共培养后，检测内皮细胞的迁移能力。使用MG-132间接激活VEGFA的表达，同时检测KLF4的表达水平。实验结果：经ox-LDL处理后，巨噬细胞的凋亡激活与泡沫细胞生成均显著增强。使用miR-126模拟物及其阴性对照转染泡沫细胞后发现，miR-126可显著抑制泡沫细胞形成与吞噬作用，并促使促炎型M1巨噬细胞向抗炎型M2表型转化，具体体现为抗炎基因表达上调、促炎基因表达下调。此外，miR-126可显著降低VEGFA与KLF4的表达水平。蛋白质相互作用网络分析显示，miR-126、VEGFA与KLF4三者间存在显著的高相关性。miR-126还可能通过激活巨噬细胞过氧化物酶体增殖物激活受体γ（PPAR γ）的表达、有效抑制巨噬细胞炎症反应，从而促进内皮细胞迁移。MG-132可间接激活VEGFA的表达，同时KLF4的表达水平也显著升高，提示VEGFA与KLF4之间存在直接或间接的调控关系。研究结论：本研究证实，miR-126可逆转ox-LDL介导的巨噬细胞吞噬作用与细胞凋亡。综上，miR-126的潜在作用可体现为调控巨噬细胞功能及促进血管内皮迁移。