miR-424 up-regulation inhibiting RF/6A cells function under high glucose condition via CCND1

Backgrounds: Retinal vascular dysfunction is an important factor to the progression of diabetic retinopathy(DR). Multiple abnormal microRNAs (miRNAs) contributes to the pathogenesis of vascular dysfunction. However, the role and underlying mechanism of miR-424 in retinal vascular endothelial cells dysfunction under hyperglycemia stress remain obscure. Methods: Rhesus macaque choroid retinal endothelial cell line (RF/6A) cells were cultured under normal glucose (NG) and high glucose (HG) condition. qPCR was used to quantify the mRNA expression of miR-424 and Cyclin D1 (CCND1) and western blot was applied to detect the protein amount of CCND1. RF/6A cells were transfected with miR-424 mimics, miR-424 inhibitor, miR-424 inhibitor+ siRNA-CCND1 or with vehicle molecules. The cell proliferation, wound healing, tube formation ability and cell cycle were evaluated by MTT assay, scratch wound healing assay, tube formation assay and flow cytometric analysis, respectively. Interaction between miR-424 and CCND1 was predicted with bioinformatics and confirmed by Dual Luciferase Reporter Analysis.Results: Compared with NG, miR-424 was up-regulated and cell phenotype such as proliferation, wound healing and tube formation were inhibited in HG. The phenotypes can be reversed by inverting miR-424 expression under different conditions. CCND1 was confirmed as one of target genes of miR-424 and it can be modulated at transcriptional or translation level. Manipulation of silencing CCND1 can reverse the influences, such as promotion in proliferation, scratch wound tube formation and cell cycle, induced by transfecting miR-424 inhibitor into RF/6A cells under HG. Conclusions: Overexpression of miR-424 in RF/6A cells under HG stress significantly inhibited the cell function such as cell proliferation, wound healing, tube formation through suppressing CCND1 and blocking cell cycle.