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

Authors: Zhu Jiang,  Ren Mei,  Xu Zhiguo

DOI: 10.3760/cma.j.issn.2095-0160.2017.08.005
Published 2017-08-10
Cite as Chin J Exp Ophthalmol, 2017,35(8): 695-702.

Abstract

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.

Key words:

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

Contributor Information

Zhu Jiang
Shaanxi Ophthalmic Medical Center, Department of Ophthalmology, Xi’an No.4 Hospital, Affiliated Guangren Hospital to School of Medicine, Xi’an Jiaotong University, Xi’an 710004, China
Ren Mei
Xu Zhiguo
(Read 52 times, 1 visits today)
Updated: September 4, 2019 — 1:34 pm