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    METTL3调控miR-301a促进缺氧环境下内皮集落形成细胞血管生成

    METTL3 regulates miR-301a to promote angiogenesis of endothelial colony-forming cells under hypoxic environment

      摘要
    • 摘要: 目的: 探讨缺氧环境对内皮集落形成细胞（ECFCs）血管生成能力的影响及甲基转移酶样3（METTL3）的调控作用。方法: 分离、培养犬外周血 ECFCs。实验分为正常对照组与缺氧实验组。缺氧实验组分别使用含 50 μmol/L、100 μmol/L 和200 μmol/L氯化钴（CoCl2）的培养基处理细胞24 h，对照组CoCl2浓度为0。CCK-8检测不同浓度CoCl2对ECFCs增殖的影响，实时荧光定量PCR（RT-qPCR）检测缺氧诱导因子-1α（HIF-1α）的表达，筛选适宜浓度的CoCl2用于缺氧环境构建。Transwell实验和管形成实验分别检测ECFCs迁移和血管生成能力。RT-qPCR和western blotting检测血管内皮生长因子（VEGF）、CD31、METTL3、miR-301a 的表达情况。构建沉默 METTL3 慢病毒转染 ECFCs，将 ECFCs 分为 NC-shRNA 组和 METTL3-shRNA组。RT-qPCR和western blotting检测ECFCs细胞中VEGF、CD31、METTL3、miR-301a表达水平的变化。结果: 与正常对照组比较，50 μmol/L和100 μmol/L的CoCl2培养ECFCs 24 h后可促进细胞增殖（均P＜0.000 1），HIF-1α随着CoCl2浓度升高而高表达（P＜0.01）。100 μmol/L CoCl2组中 ECFCs 迁移能力和血管生成能力提高（均 P＜0.01），VEGF、CD31、METTL3、miR-301amRNA水平表达升高（均P＜0.01），VEGF、CD31、METTL3蛋白表达升高（均P＜0.01）。与NC-shRNA组比较，METTL3-shR-NA 组中 VEGF、CD31、METTL3、miR-301a mRNA 表达降低（均 P＜0.05），VEGF、CD31、METTL3 蛋白表达降低（均 P＜0.05）。结论: 适宜的缺氧干预可提高ECFCs增殖、迁移和血管生成能力，METTL3/miR-301a轴可能参与调控血管生成。

       

      Abstract: Objective: To investigate the effect of hypoxic environment on the angiogenic capacity of endothelial colony forming cells (ECFCs) and the regulatory role of methyltransferase-like 3 (METTL3). Methods: Canine peripheral blood ECFCs were isolated and cultured. The experiment was divided into normal control group and hypoxia experimental group. Cells were treated with medium containing 50 μmol/L, 100 μmol/L and 200 μmol/L cobalt chloride (CoCl2) for 24 h in the hypoxia experimental group, and the concentration of CoCl2 in the control group was 0. CCK-8 was used to detect the effects of different concentrations of CoCl2 on the proliferation of ECFCs, and reverse transcription-quantitative PCR (RT-qPCR) was used to detect hypoxia-inducible factor-1α (HIF-1α) expression, and the appropriate concentrations of CoCl2 were screened for the subsequent construction of hypoxic environment. Transwell assay and tube formation assay were performed to detect the migration and angiogenic capacity of ECFCs. RT-qPCR and western blotting were performed to detect the expression of vascular endothelial growth factor (VEGF), CD31, METTL3, and miR-301a. Silencing METTL3 lentivirus was constructed to transfect ECFCs, and ECFCs were divided into NC-shRNA group and METTL3-shRNA group. RT-qPCR and western blotting were used to detect the changes in the expression levels of VEGF, CD31, METTL3, and miR-301a in ECFCs cells. Results: Compared with the normal control group, incubation of ECFCs with 50 μmol/ L and 100 μmol/L CoCl2 for 24 h promoted cell proliferation (both P＜0.0001), and HIF-1α was highly expressed in accordance with the elevated concentration of CoCl2 (P＜0.01). The migratory and angiogenic capacities of ECFCs were increased in the 100 μmol/L CoCl2 group (both P＜0.01), the expression of VEGF, CD31, METTL3, and miR-301a at the mRNA level was elevated (all P＜0.01), and the protein expression of VEGF, CD31, and METTL3 was elevated (all P＜0.01). Compared with the NC-shRNA group, VEGF, CD31, METTL3, and miR-301a mRNA expression was decreased in the METTL3-shRNA group (all P＜0.05), and VEGF, CD31, and METTL3 protein expression was decreased (all P＜0.05). Conclusion: Appropriate hypoxia intervention can improve the proliferation, migration and angiogenesis of ECFCs, and the METTL3/miR-301a axis may be involved in the regulation of angiogenesis.

       

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