Anhydrous glucose

Primary human glomerular endothelial cells (HGECs, ScienCell Company, Beijing Yuhengfeng Agency, China) were cultured in Endothelial Cell Medium containing 10% fetal bovine serum (Endothelial Cell Medium and serum were from ScienCell Company, Beijing Yuhengfeng Agency, China). The cells were used within six passages. Cells in the high insulin concentration group were treated with medium containing different concentrations of insulin: 7.14 ul, 14.28ul, 35.7ul, 71.4ul of Novolin insulin solution (Novo Nordisk, Copenhagen, Denmark) was added to 2 ml ECM medium separately(The ECM medium did not contain any insulin) to form 5 ng/ml, 10ng/ml, 25ng/ml, 50ng/ml high concentration insulin medium. Cells in the high glucose concentration group were treated with medium containing anhydrous glucose (Solarbio, Beijing, China) at 8.3, 11.1, 16.7, or 33.3 mmol/L. D-Mannitol (Solarbio, Beijing, China) was used as a control for osmolarity. HGECs were transfected with miR-21 mimics or miR-21 inhibitor (GenePharma, Shanghai, China) using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). The transfection was performed under strictly aseptic conditions according to the manufacturer’s instructions. The effectiveness of miR-21 mimics and inhibitor was verified by PCR after transfection (Figures 4A, E).

Human retinal endothelial cells (HRECs) (CSC; Kirkland, WA, USA) were cultured in endothelial Complete Classic Medium (CSC; Kirkland, WA, USA) containing 10% fetal bovine serum (Gibco, USA). The culture medium was changed every 2 days. When the cells were 85–90% confluent, it was passaged at a ratio of 1:3. The in vitro diabetic model was induced by culturing HRECs with high-glucose medium containing 30 mM anhydrous glucose (Solarbio, Beijing, China) for 48 h, and the cells cultured in normal medium containing 5 mM glucose were used as control under the same conditions. In order to ensure the accuracy and stability of the results, 8–10 generations of cells were selected for follow-up study.