| 摘要: |
| 目的 探讨装载miRNA-132的间充质干细胞外泌体在缺氧环境中对人脐静脉内皮细胞（HUVEC）的保护作用及其机制。方法 通过电转法分别将miRNA-132阴性对照（NC）和miRNA-132 mimics转入间充质干细胞来源的外泌体中，即为对照外泌体（Exo）和装载miRNA-132的外泌体（miRNA-132 Exo）。在低氧培养箱中，分别用等体积的PBS、Exo和miRNA-132 Exo与HUVEC共培养48h。利用CCK-8法检测各组细胞增殖能力，采用管样形成实验检测细胞成管能力，利用Transwell迁移实验检测细胞迁移能力。通过TargetScan数据库预测miRNA-132的下游靶基因，并通过双萤光素酶报告基因实验、qPCR和蛋白质印迹法进行验证。结果 电转法成功将miRNA-132 mimics转入外泌体中。在缺氧条件下培养48 h后，Exo组与miRNA-132 Exo组HUVEC的增殖能力、成管能力和迁移能力均优于PBS组（P均＜0.05），并且miRNA-132 Exo组HUVEC的增殖能力、成管能力和迁移能力均优于Exo组（P均＜0.01）。经TargetScan数据库筛选发现Ras p21蛋白活化子1（RASA1）可能为miRNA-132的靶点并与血管形成过程有关。双萤光素酶报告基因实验证实RASA1是miRNA-132的靶点，qPCR和蛋白质印迹法结果显示缺氧条件下加miRNA-132 Exo处理能抑制HUVEC中RASA1的表达。结论 无论是否装载miRNA-132，间充质干细胞来源的外泌体均可在缺氧环境中保护HUVEC的增殖能力、成管能力和迁移能力，miRNA-132可能通过抑制RASA1的表达增强了这种保护作用。 |
| 关键词: 微RNA-132 外泌体 人脐静脉内皮细胞 间充质干细胞 缺氧 细胞增殖 细胞迁移 新生血管形成 |
| DOI：10.16781/j.CN31-2187/R.20200656 |
| 投稿时间：2020-05-04修订日期：2021-09-02 |
| 基金项目: |
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| Effects of microRNA-132-loaded mesenchymal stem cell-derived exosomes on human umbilical vein endothelial cells under hypoxia condition |
| WANG Tian-bao,DU Jian-feng,XU Li-qing* |
(Department of Cardiovasology, the 4th People's Hospital of Shenyang, Shenyang 110000, Liaoning, China
*Corresponding author) |
| Abstract: |
| Objective To investigate the protective effects and mechanism of microRNA-132 (miRNA-132)- loaded exosomes derived from mesenchymal stem cells on human umbilical vein endothelial cells (HUVECs) under hypoxic condition.Methods Electroporation method was applied to transfect miRNA-132 negative control (NC) and miRNA-132 mimics into mesenchymal stem cell-derived exosomes as control exosomes (Exo) and miRNA-132 loaded exosomes (miRNA-132 Exo), respectively. Equal amount of phosphate buffered saline (PBS), Exo and miRNA-132 Exo were co-cultured with HUVECs for 48 h in a hypoxia incubator. Cell counting kit 8 (CCK-8) was used to detect the cell proliferative ability, tube formation assay was performed to measure the angiogenic ability, and Transwell migration assay was employed to investigate the cell migration ability. In addition, the downstream target genes of miRNA-132 were predicted by TargetScan database, and confirmed by dual-luciferase reporter gene assay, quantitative polymerase chain reaction (qPCR) and Western blotting.Results miRNA-132 mimics were successfully transfected into exosomes by electroporation method. After cultured under hypoxia for 48 h, the proliferative ability, angiogenic ability and migratory ability of HUVECs in the Exo and miRNA-132 Exo groups were significantly higher than those in the PBS group (all P＜0.05), and those in the miRNA-132 Exo group were signficantly higher than those in the Exo group (all P＜0.01). TargetScan database analysis result showed that Ras p21 protein activator 1 (RASA1) may be a target of miRNA-132, which was related to the process of angiogenesis. Dual-luciferase reporter gene assay confirmed that RASA1 was the downstream target of miRNA-132. The results of qPCR and Western blotting showed that treatment with miRNA-132 Exo under hypoxia could inhibit the expression of RASA1 in HUVECs.Conclusion Mesenchymal stem cell-derived exosomes with or without miRNA-132 load could both preserve the proliferative ability, angiogenic ability and migratory ability of HUVECs under hypoxia. miRNA-132 may enhance this protective effect by inhibiting the expression of RASA1. |
| Key words: microRNA-132 exosome human umbilical vein endothelial cell mesenchymal stem cell hypoxia cell proliferation cell migration neovascularization |
