METTL3 regulates miR-301a to promote angiogenesis of endothelial colony-forming cells un-der 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 an-giogenic 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 construct-ed 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 MET-TL3 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.
hypoxiaendothelial colony-forming cellsangiogenesismethyltransferase like 3
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