Figure 5.

miR-615-5p/cZNF609 interaction is involved in regulating endothelial cell function (A) HUVECs were transfected with scrambled mimic (Scr), miR-615-5p mimic, miR-615-5p mimic plus cZNF609 siRNA, or left untreated, and then treated with or without H₂O₂ (100 μm) for 48 h. Apoptotic cells were analyzed using PI staining and quantified (n=4, *P<0.05 versus Ctrl group). Scale bar: 20 μm. (B and C) HUVECs were transfected with miR-615-5p mimic, miR-615-5p mimic plus cZNF609 siRNA, or left untreated. Transwell assay and quantification analysis was conducted to detect endothelial cell migration (B, n=4, *P<0.05 versus Ctrl group). The tube-like structures were observed 24 h after HUVEC seeding. Average length of tube formation for each field was statistically analyzed (C, n=4, *P<0.05 versus Ctrl group). (D) Diabetic C57BL/6 mice (8-week old, male) were received an intravitreous injection of scrambled agomir (Scr), miR-615-5p agomir, or left untreated (Ctrl). These agomirs were injected once monthly for a total of 6 months. The mice were infused with Evans blue dye for 2 h. Fluorescence images of flat-mounted retinas and quantification of Evans blue leakage was conducted (n=5, *P<0.05 versus Ctrl group). (E) Retinal trypsin digestion was conducted to detect acellular capillaries. Acellular capillaries were quantified in 30 random fields per retina and averaged. Red arrows indicated acellular capillaries. Scale bar, 50 μm (n=5, *P<0.05 versus Ctrl group). (F) Retinal vaso-obliteration (VO) and neovascularization of miR-615 overexpression mice and matched control mice at P12 and P17 were shown by staining retinal vasculature using GS lectin-Alexa Flour 549. Avascular areas were highlighted using white lines. Scale bar: 100 μm. Quantification of VO area at P12 and P17 and angiogenic area at P17 was also conducted (n=5, *P<0.05 versus Scr group). “^#” indicated significant difference between the marked groups. All data were from at least three independent experiments.