PurposeDiabetic retinopathy (DR) is a significant cause of blindness. Most research around DR focus on late-stage developments rather than early changes such as early endothelial dysfunction. Endothelial-to-mesenchymal transition (EndMT), an epigenetically regulated process whereby endothelial cells lose endothelial characteristics and adopt mesenchymal-like phenotypes, contributes to early endothelial changes in DR. The epigenetic regulator microRNA 9 (miR-9) is suppressed in the eyes during DR. MiR-9 plays a role in various diseases and regulates EndMT-related processes in other organs. We investigated the role miR-9 plays in glucose-induced EndMT in DR.MethodsWe examined the effects of glucose on miR-9 and EndMT using human retinal endothelial cells (HRECs). We then used HRECs and an endothelial-specific miR-9 transgenic mouse line to investigate the effect of miR-9 on glucose-induced EndMT. Finally, we used HRECs to probe the mechanisms through which miR-9 may regulate EndMT.ResultsWe found that miR-9 inhibition was both necessary and sufficient for glucose-induced EndMT. Overexpression of miR-9 prevented glucose-induced EndMT, whereas suppressing miR-9 caused glucose-like EndMT changes. We also found that preventing EndMT with miR-9 overexpression improved retinal vascular leakage in DR. Finally, we showed that miR-9 regulates EndMT at an early stage by regulating EndMT-inducing signals such as proinflammatory and TGF-β pathways.ConclusionsWe have shown that miR-9 is an important regulator of EndMT in DR, potentially making it a good target for RNA-based therapy in early DR.