高糖条件下miR-615-5p对人视网膜内皮细胞的影响研究

Effect of miR-615-5p on human retinal endothelial cells under high glucose conditions

目的探讨高糖条件下miR-615-5p对人视网膜内皮细胞(hRECs)迁移能力、成管能力、愈合能力的影响及其机制。方法取对数生长期的hRECs,将细胞分为低糖(LG)组、高糖(HG)组、miR-615-5p模拟物(mimic)组、miR-615-5p模拟物阴性对照(mi-nc)组、miR-615-5p抑制剂(inhibitor)组、miR-615-5p抑制剂阴性对照(in-nc)组。LG组hRECs用含5.5 mmol·L^(-1)葡萄糖的培养基培养,其余各组hRECs均用含25.0 mmol·L^(-1)葡萄糖的培养基培养。qRT-PCR分析LG组与HG组hRECs中miR-615-5p的相对表达情况;Transwell实验、成管实验、划痕愈合实验分析各组hRECs的细胞迁移能力、成管能力和愈合能力;免疫荧光实验分析miR-615-5p对hRECs中血管内皮生长因子A(VEGFA)表达量的影响。结果与LG组(1.00±0.28)相比,HG组hRECs中miR-615-5p的相对表达水平(6.36±1.31)上升(t=12.69,P<0.01)。Transwell实验中,HG组、mimic组、mi-nc组、inhibitor组和in-nc组的hRECs相对细胞迁移数分别为0.99±0.12、0.96±0.08、0.50±0.07、0.72±0.13和0.96±0.10,与mi-nc组相比,mimic组中hRECs的迁移能力显著增强(t=6.10,P<0.05);与in-nc组相比,inhibitor组中hRECs的迁移能力显著降低(t=7.21,P<0.05)。成管实验结果显示,HG组、mimic组、mi-nc组、inhibitor组和in-nc组hRECs的相对成管管长分别为1.00±0.15、1.24±0.13、0.81±0.13、0.40±0.05和0.86±0.04;与mi-nc组相比,mimic组hRECs的成管能力显著增强(t=5.52,P<0.01);相较于in-nc组,inhibitor组hRECs的成管能力显著降低(t=17.80,P<0.01)。划痕愈合实验显示,HG组、mimic组、mi-nc组、inhibitor组和in-nc组hRECs的创面愈合率分别为0.71±0.01、1.00±0.01、0.64±0.02、0.56±0.05和0.54±0.04,相较于mi-nc组,mimic组中hRECs的愈合能力显著增强(t=25.30,P<0.01)。免疫荧光实验显示,HG组、mimic组、mi-nc组、inhibitor组和in-nc组的VEGFA相对荧光强度分别为1.00±0.09、1.35±0.10、1.02±0.04、0.77±0.06和0.84±0.06,相较于mi-nc组,mimic组hRECs中VEGFA的表达水平增加(t=3.78,P<0.05)。结论高糖环境促进了hRECs中miR-615-5p的表达,进而促进了hRECs的成管能力、迁移能力和愈合能力,同时miR-615-5p的下游靶点可能是VEGFA。

Objective To investigate the effect and mechanism of miR-615-5p on migration,tube formation,and wound healing of human retinal endothelial cells(hRECs)under high glucose conditions.Methods hRECs in the logarithmic growth phase were divided into the low glucose(LG)group,high glucose(HG)group,miR-615-5p mimic(mimic)group,miR-615-5p mimic negative control(mi-nc)group,miR-615-5p inhibitor(inhibitor)group,and miR-615-5p inhibitor negative control(in-nc)group.Cells in the LG group were cultured in the medium containing 5.5 mmol·L^(-1) glucose,and cells in the other groups were cultured in the medium containing 25.0 mmol·L^(-1) glucose.The relative expression levels of miR-615-5p in hRECs in the LG and HG groups were measured by the quantitative real-time polymerase chain reaction.Transwell assay,Matrigel assay,and Scratch assay were used to analyze the migration,tube formation,and wound healing of hRECs.The immunofluorescence assay was used to analyze the effect of miR-615-5p on the expression of vascular endothelial growth factor A(VEGFA).Results Compared with the LG group(1.00±0.28),the relative expression level of miR-615-5p in hRECs in the HG group(6.36±1.31)was higher(t=12.69,P<0.01).Transwell assay results showed that the relative numbers of cells migrated in the HG,mimic,mi-nc,inhibitor,and in-nc groups were 0.99±0.12,0.96±0.08,0.50±0.07,0.72±0.13,and 0.96±0.10,respectively.Compared with the mi-nc group,the migration of hRECs in the mimic group was significantly stronger(t=6.10,P<0.05),while compared with the in-nc group,the migration of hRECs in the inhibitor group was significantly weaker(t=7.21,P<0.05).Matrigel assay results showed that the relative tube lengths of VEGFA in the HG,mimic,mi-nc,inhibitor,and in-nc groups were 1.00±0.15,1.24±0.13,0.81±0.13,0.40±0.05,and 0.86±0.04,respectively.Compared with the mi-nc group,the tube formation of hRECs in the mimic group was significantly increased(t=5.52,P<0.01),while compared with the in-nc group,the tube formation of hRECs in the inhibitor group was significantly reduced(t=17.80,P<0.01).Scratch assay results showed that the wound healing rates of hRECs in the HG,mimic,mi-nc,inhibitor,and in-nc groups were 0.71±0.01,1.00±0.01,0.64±0.02,0.56±0.05,and 0.54±0.04,respectively.Compared with the mi-nc group,the wound healing rate of hRECs in the mimic group was significantly higher(t=25.30,P<0.01).The immunofluorescence assay results showed that the relative fluorescence intensities of hRECs in the HG,mimic,mi-nc,inhibitor,and in-nc groups were 1.00±0.09,1.35±0.10,1.02±0.04,0.77±0.06,and 0.84±0.06,respectively.Compared with the mi-nc group,the expression level of VEGFA in hRECs in the mimic group was higher(t=3.78,P<0.05).Conclusion High glucose promotes the expression of miR-615-5p in hRECs,thus increasing the migration,tube formation,and wound healing of hRECs.VEGFA may be the downstream target of miR-615-5p.