Microvascular dysfunction is a key pathological mechanism of diabetic retinopathy (DR). In recent years, it has been found that the phenomenon of "metabolic memory" is prevalent in diabetic patients, and diabetic microangiopaplasia cannot be avoided even if patients’ blood glucose is well controlled. Therefore, it is necessary to explore DR from a genetic perspective. miR-126 is the unique microRNA specifically expressed in vascular endothelial cells, which is closely related to the formation of neovascularization and can affect the stability of DR microvessels as well as the germination and migration of endothelial cells, and its gene level is significantly negatively correlated with the expression of vascular endothelial cell growth factor. The potential value of intracellular and circulating miR-126 in the regulation of DR microvascular homeostasis, early diagnosis and treatment, and monitoring of disease course has attracted great attention. However, studies in this area are mostly hypothesis-driven and still have some limitations. It is believed that with the rapid development of genomics, the miRNA spectrum and its molecular mechanism in eye development and eye diseases will gradually become clear, which may lead to a breakthrough in the intervention of individual refractory retinal diseases and establish a new miRNA diagnosis and treatment method in the future.
ObjectiveTo 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). MethodsThe 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 hof 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. ResultsAt 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-Aprotein (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). ConclusionsmiR-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.