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

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50μmol/L、100μmol/L和200μmol/L氯化钴(CoCl_(2))的培养基处理细胞24 h,对照组CoCl_(2)浓度为0。CCK-8检测不同浓度CoCl_(2)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CoCl_(2)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,50μmol/L和100μmol/L的CoCl_(2)培养ECFCs 24 h后可促进细胞增殖(均P<0.0001),HIF-1α随着CoCl_(2)浓度升高而高表达(P<0.01)。100μmol/L CoCl_(2)组中ECFCs迁移能力和血管生成能力提高(均P<0.01),VEGF、CD31、METTL3、miR-301a mRNA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-shRNA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。

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 50μmol/L,100μmol/L and 200μmol/L cobalt chloride(CoCl_(2))for 24 h in the hypoxia experimental group,and the concentration of CoCl_(2)in the control group was 0.CCK-8 was used to detect the effects of different concentrations of CoCl_(2)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 CoCl_(2)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 50μmol/L and 100μmol/L CoCl_(2)for 24 h promoted cell proliferation(both P<0.0001),and HIF-1αwas highly expressed in accordance with the elevated concentration of CoCl_(2)(P<0.01).The migratory and angiogenic capacities of ECFCs were increased in the 100μmol/L CoCl_(2)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.