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
10.3760/cma.j.cn511434-20240108-00007
- VernacularTitle:人脐带间充质干细胞外泌体靶向miR-126调节高糖诱导的人视网膜血管内皮细胞中血管内皮生长因子-A的表达
- Author:
Yingxue MA
1
;
Guanghui HE
;
Xiang GAO
;
Yan FU
;
Bin WU
Author Information
1. 天津市第一中心医院 天津市眼科医院 天津市眼科学与视觉科学重点实验室 天津市眼科研究所天津医科大学眼科临床学院，天津 300020
- Keywords:
miR-126;
Mammalian target of rapamycin;
Hypoxia-induced factor 1 α;
Vascular endothelial growth factor-A;
Human umbilical cord mesenchymal stem cells exos
- From:
Chinese Journal of Ocular Fundus Diseases
2024;40(5):372-378
- CountryChina
- Language:Chinese
-
Abstract:
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.
