Inhibitory effects of microRNA-375 on biological behaviour of human retinal capillary endothelial cells induced by hypoxia

Background

Studies showed that microRNA (miR)-375 suppresses the growth, apoptosis, migration and adhesion of tumor cells, and it plays a regulation to the changes of vascular endothelial growth factor (VEGF) in tumor tissue to arrest neovascularization.However, whether miR-375 intervenes the formation of new blood vessel in eyes is unelucidated.

Objective

This study was to explore the effects of miR-375 on human retinal capillary endothelial cells (HRCECs) function induced by hypoxia.

Methods

HRCECs were cultured using IMDM and divided into normal control group, CoCl₂ model group, CoCl₂+ miR-375 mimic group, CoCl₂+ miR-375 mimic control group, CoCl₂+ miR-375 inhibitor group and CoCl₂+ miR-375 inhibitor control group, and hypoxia cell models were created by adding 200 μmol/L CoCl₂.MiR-375 and frizzled 4 (FZD4) small interfering RNA (siRNA) were transfected into the cells by 50 nmol/L miRNA lipidosome for 48 hours.The proliferation of the cells was detected by MTT assay; migrated number of the cells was examined by Transwell chamber assay; ELISA was employed to detect the concentrations of VEGF and VE-cadherin in the medium; the expression of β-catenin, cyclinD1, matrix metalloproteinase-2 (MMP2) and VEGF proteins were analyzed by Western blot; tube length of vessel formation was evaluated by Matrigel assay.Cultured cells were divided into normal control group, CoCl₂ model group, CoCl₂+ mock group and CoCl₂+ FZD4 siRNA group, the relative expression of FZD4, a miR-375 targeted gene, was detected by luciferase reporter.

Results

The relative expression of miR-375 mRNA was significantly increased in the CoCl₂+ miR-375 mimic group compared with the CoCl₂+ miR-375 mimic control group and reduced in the CoCo₂+ miR-375 inhibitor group compared with the CoCo₂+ miR-375 inhibitor control group (t=-19.237, 8.764, both at P<0.01), with a higher transfected efficacy for miR-375.The cell proliferative fold, migrated cell number, VEGF and VE-Cadherin contents in the medium and the tube length were significantly different among the CoCl₂ model group, CoCl₂+ miR-375 mimic group, CoCl₂+ miR-375 mimic control group, CoCl₂+ miR-375 inhibitor group and CoCl₂+ miR-375 inhibitor control group (F=24.324, 26.776, 14.113, 19.225, 15.040, all at P<0.001), and those in the CoCl₂+ miR-375 mimic group were evidently reduced in the CoCl₂+ miR-375 mimic group compared with the CoCl₂+ miR-375 mimic control group, while those in the CoCl₂+ miR-375 inhibitor group were considerably elevated in comparison with the CoCl₂+ miR-375 inhibitor control group (all at P<0.01). The expressions of β-catenin, cyclinD1, MMP2 and VEGF protein were significantly different among the normal control group, CoCl₂ model group, CoCl₂+ miR-375 mimic group, CoCl₂+ miR-375 mimic control group, CoCl₂+ miR-375 inhibitor group and CoCl₂+ miR-375 inhibitor control group (F=11.753, 13.283, 16.770, 10.334, all at P<0.001). In addition, the cell proliferative fold, migrated cell number and the tube length were significantly increased in the CoCl₂ model group and CoCl₂+ mock group, and those in the CoCl₂+ FZD4 siRNA group were decreased in comparison with the CoCl₂+ mock group (all at P<0.05).

Conclusions

MiR-375 inhibits the growth, migration and tube formation ability of HRCECs in hypoxic status probably by regulating the activation of Wnt pathway via directly targeting FZD4.

MicroRNAs/physiology; Retina; Endothelium, vascular; Neovascularization, pathologic; Signal transduction/physiology; Wnt proteins/metabolism; Cell proliferation /drug effects; Gene expression regulation