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

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.