Human umbilical cord mesenchymal stem cell exosomes target miR-126 regulate the expression of vascular endothelial growth factor-A in high glucose-induced human retinal vascular endothelial cells
Objective To explore the involvement of miR-126 and the role of mammalian target of rapamycin(mTOR)/hypoxia-induced factor 1 α(HIF-1 α)pathway in regulating human umbilical cord mesenchymal stem cells(hUCMSCs)exosomes(Exo)on vascular endothelial growth factor(VEGF)-A levels in high glucose-induced human retinal vascular endothelial cells(HRECs).Methods The hREC was cultured in EGM-2-MV endothelial cell culture medium with 30 mmol/L glucose and placed in hypoxic cell incubator by 1％oxygen concentration.The cell model of high glucose and low oxygen was established.After modeling,divided HRECs into Exo group,phosphate buffer saline(PBS)group,PBS+anti-miR126 group,Exo+anti-miR126 group,PBS+anti-mTOR group,and PBS+anti-HIF-1 α group.High-glucose and hypoxia-induced hREC in the PBS and Exo groups were respectively co-cultured with PBS and 100 μg/ml hUCMSC Exo.PBS+anti-mTOR group,PBS+anti-HIF-1 α group:500 nmol/L mTOR inhibitor ADZ2014,25 μmol/L HIF-1 α inhibitor YC-1 pretreatment for hREC 12 h,and then co-culture with PBS after High-glucose and hypoxia-induced.PBS+anti-miR126 group,Exo+anti-miR126 group:miR-126 LNA power inhibitor probe was transfected with high glucose,and co-cultured with PBS and hUCMSC Exo 6 h after transfection.Real-time polymerase chain reaction(qPCR)measured miRNA-126 expression levels in PBS,and Exo groups for 0,8,16 and 24 h.After 24 h of co-culture,the levels of mTOR and HIF-1 α in the cells of PBS and Exo groups were detected by immunofluorescence,Western blot and qPCR,respectively.Western blot,qPCR detection of VEGF-A expression levels in cells of the PBS+anti-mTOR and PBS+anti-HIF-1 α groups.The expression of VE GF-A,mTOR,and HIF-1 α mRNA was measured in cells of PBS+anti-miR126 group and Exo+anti-miR126 group by qPCR.Comparison between two groups was performed by t-test;one-way ANOVA was used for comparison between multiple groups.Results At 0,8,16 and 24 h,the relative mRNA expression of miR-126 gradually increased in the Exo group(F=95.900,P＜0.05).Compared with the PBS group,The mTOR,HIF-1 α protein(t=3.466,6.804),mRNA in HRECs in the Exo group,VEGF-A mRNA expression(t=8.642,7.897,6.099)were all downregulated,the difference was statistically significant(P＜0.05).The relative expression level of VEGF-A protein(t=3.337,7.380)and mRNA(t=8.515,10.400)was decreased in HRECs of the anti-mTOR+PBS group and anti-HIF-1 α+PBS group,differences were statistically significant(P＜0.05).The relative expression of VEGF-A,mTOR,and HIF-1 α mRNA was significantly increased in the cells of the Exo+anti-miR126 group,the differences were all statistically significant(t=4.664,6.136,6.247;P＜0.05).Conclusions miR-126 plays a role in regulating the effect of hUCMSCs exosomes on VEGF-A levels in high glucose-induced HRECs via mTOR-HIF-1 α pathway.
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维普
