Zhao Jin, Ou Shu-Lin, Wang Wei-You, Yan Chang, Chi Lu-Xiang
Center of Translational Medicine, College of Pharmacy, Third Military Medical UniversityChongqing 400038, China.
Department of Cardiology, Southwest Hospital, Third Military Medical UniversityChongqing 400038, China.
Am J Transl Res. 2017 Jul 15;9(7):3433-3442. eCollection 2017.
Injury and endothelial cell apoptosis are hall marks of atherosclerosis (AS). However, the mechanisms underlying its pathogenesis remain ill-defined. Recent evidence of a role for microRNAs in AS-associated endothelial cell apoptosis encouraged us to address this question. Here, AS was developed in ApoE (-/-) mice supplied with a high-fat diet (HFD), compared to ApoE (-/-) mice supplied with a normal diet (ND). Mouse endothelial cells were isolated from the aortic arch using flow cytometry based on their expression of CD31. Human aortic endothelial cells (HAECs) were treated with oxidized low-density lipoprotein (ox-LDL) as an model for AS. Gene expression was quantified by RT-qPCR and protein levels were analyzed by Western blotting. Apoptosis was evaluated by FITC Annexin V Apoptosis assay and by TUNEL staining. Predicting binding patterns between miRNAs and the 3'-UTR of mRNA from the target gene was performed by bioinformatics analyses and confirmed by a dual luciferase reporter assay. We found that HFD mice, but not ND mice, developed AS in 12 weeks. A significant reduction in endothelial cells and a significant increase in mesenchymal cells were detected in the aortic arch of the HFD mice, compared to those of ND mice. Endothelial cell apoptosis was significantly higher in HFD mice, seemingly due to functional suppression of protein translation of anti-apoptotic Bcl-2 protein through upregulation of miR-1907, confirmed by analysis. Moreover, inhibition of miR-1907 abolished the effects of ox-LDL-induced apoptotic cell death on HAECs. Thus, AS-associated endothelial cell apoptosis may partially result from downregulation of Bcl-2, via upregulation of miR-1907 which binds and suppresses the translation of Bcl-2 mRNA.
损伤和内皮细胞凋亡是动脉粥样硬化（AS）的标志。然而，其发病机制仍不清楚。近期有证据表明微小RNA在AS相关的内皮细胞凋亡中发挥作用，这促使我们探讨这一问题。在此，将给予高脂饮食（HFD）的载脂蛋白E基因敲除（ApoE (-/-)）小鼠与给予正常饮食（ND）的ApoE (-/-)小鼠进行比较，以建立AS模型。基于CD31的表达，使用流式细胞术从主动脉弓分离小鼠内皮细胞。用人主动脉内皮细胞（HAECs）作为AS模型，用氧化型低密度脂蛋白（ox-LDL）进行处理。通过逆转录定量聚合酶链反应（RT-qPCR）对基因表达进行定量，通过蛋白质印迹法分析蛋白质水平。通过FITC膜联蛋白V凋亡检测法和TUNEL染色评估细胞凋亡。通过生物信息学分析预测微小RNA与靶基因mRNA的3'-非翻译区（3'-UTR）之间的结合模式，并通过双荧光素酶报告基因检测进行验证。我们发现，12周时HFD小鼠而非ND小鼠发生了AS。与ND小鼠相比，在HFD小鼠的主动脉弓中检测到内皮细胞显著减少，间充质细胞显著增加。HFD小鼠的内皮细胞凋亡显著更高，这似乎是由于通过上调miR-1907对抗凋亡Bcl-2蛋白的蛋白质翻译进行功能抑制所致，经分析得到证实。此外，抑制miR-1907消除了ox-LDL诱导的HAECs凋亡性细胞死亡的作用。因此，AS相关的内皮细胞凋亡可能部分是由于miR-1907上调，通过结合并抑制Bcl-2 mRNA的翻译导致Bcl-2下调所致。
