MicroRNA-376b-3p Suppresses Choroidal Neovascularization by Regulating Glutaminolysis in Endothelial Cells.

Purpose: Choroidal neovascularization (CNV) is a common pathological change of various ocular diseases that causes serious damage to central vision. Accumulated evidence shows that microRNAs (miRNAs) are closely related with the regulation of endothelial metabolism, which plays crucial roles in angiogenesis. Here, we investigate the molecular mechanism underlying the regulation of endothelial glutamine metabolism by miR-376b-3p in the progression of CNV.

Methods: Human retinal microvascular endothelial cells (HRMECs) were transfected with control or miR-376b-3p mimics, and the expression of glutaminase 1 (GLS1), a rate-limiting enzyme in glutaminolysis, was detected by real-time PCR or Western blotting. The biological function and glutamine metabolism of transfected HRMECs were measured by related kits. Luciferase reporter assays were used to validate the CCAAT/enhancer-binding protein beta (CEBPB) was a target of miR-376b-3p. Chromatin immunoprecipitation and RNA immunoprecipitation assays were performed to verify the binding of CEBPB on the promoter region of GLS1. Fundus fluorescein angiography and immunofluorescence detected the effect of miR-376b-3p agomir on rat laser-induced CNV.

Results: The expression of miR-376b-3p was decreased, whereas GLS1 expression was increased in the retinal pigment epithelial-choroidal complexes of rats with CNV. HRMECs transfected with miR-376b-3p mimic showed inhibition of CEBPB, resulting in the inactivation of GLS1 transcription and glutaminolysis. Moreover, the miR-376b-3p mimic inhibited proliferation, migration and tube formation but promoted apoptosis in HRMECs, whereas these effects counteracted by α-ketoglutarate supplementation or transfection with CEBPB overexpression plasmid. Finally, the intravitreal administration of the miR-376b-3p agomir restrained CNV formation.

Conclusions: Collectively, miR-376b-3p is a suppressor of glutamine metabolism in endothelial cells that could be expected to become a therapeutic target for the treatment of CNV-related diseases.