https://orcid.org/0000-0001-8788-9583
Figures & data
Figure 1. The expression levels of circ-UBAP1 in human vitreous humor and hRMECs. (a) Vitreous humor samples were collected from DR patients (n = 30) and non-DR people (n = 30) to analyze the expression of circ-UBAP1 using RT-qPCR. (b) circ-UBAP2 expression was measured after hRMECs were treated with HG or NG. Each experiment was performed in triplicate. ***P < 0.001. DR, diabetic retinopathy; hRMECs, human retinal microvascular endothelial cells; HG, high glucose; NG, normal glucose
Figure 2. Knockdown of circ-UBAP2 attenuated oxidative stress triggered by HG in hRMECs. (a) The transfection efficiency of circ-UBAP2. (b) MDA contents, (c) SOD, and (d) GSH-PX activities in hRMECs were detected using the corresponding kits after the indicated treatments. (e) Western blotting for the proteins of Nrf2, HO-1, and SOD-1. GAPDH was used to normalize the protein levels. Each experiment was performed in triplicate. *P < 0.05; ***P < 0.001. MDA, malondialdehyde; SOD, superoxide dismutase; GSH-PX, glutathione peroxidase; hRMECs, human retinal microvascular endothelial cells
Figure 3. Knockdown of circ-UBAP2 inhibited viability, migration, and tube formation of the HG-treated hRMECs. (a) MTT assay was performed to determine the cell viability. (b) The migrated cells were fixed and stained after the transwell migration assay. (c) Quantification of B. (d) Images of the tube-like structures were observed under an inverted microscope after 24 h incubation. (e) Quantification of D. Each experiment was performed in triplicate. *P < 0.05; **P< 0.01; ***P < 0.001. HG, high glucose; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; hRMECs, human retinal microvascular endothelial cells
Figure 4. miR-589-5p was sponged by circ-UBAP2. (a) The binding sequences of miR-589-5p and circ-UBAP2. (b) Luciferase activities of hRMECs. (c) The expression level of miR-589-5p when the hRMECs were transfected with si-circ-UBAP2. (d) The expression levels of miR-589-5p in the vitreous humor samples from the DR patients (n = 21) and the non-DR people (n = 21). (e) The expression levels of miR-589-5p in hRMECs treated with NG or HG. (f) FISH was performed to detect the co-location between circ-UBAP2 and miR-589-5p. Each experiment was performed in triplicate. ***P < 0.001. WT, wild type; MUT, mutant type; DR, diabetic retinopathy; si-, small interference RNA; hRMECs, human retinal microvascular endothelial cells; HG, high glucose; NG, normal glucose
Figure 5. Inhibition of miR-589-5p abrogated the effects of si-circ-UBAP2 on oxidative stress and dysfunctions of HG-treated hRMECs. (a) The transfection efficiency of miR-589-5p. (b) MDA contents, (c) SOD, and (d) GSH-PX activities in the hRMECs were detected using the corresponding kits after the indicated treatments. (e) Western blotting was used to determine the protein expressions of Nrf2, HO-1, and SOD-1. GAPDH was used to normalize the protein expressions. (f) MTT assay was performed to determine the cell viability. (g) The migrated cells were fixed and determined using a transwell migration assay. (h) Quantification of G. (i) Images of tube-like structures were observed under an inverted microscope. (j) Quantification of I. Each experiment was performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001. HG, high glucose; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; hRMECs, human retinal microvascular endothelial cells; MDA, malondialdehyde; SOD, superoxide dismutase; GSH-PX, glutathione peroxidase
Figure 6. EGR1 was the target gene of miR-589-5p. (a) The binding sequences of miR-589-5p and EGR1. (b) Luciferase activities of hRMECs. (c) The transfection efficiency of EGR1. (d) The expressions of EGR1 in the vitreous humor samples from the DR patients (n = 21) and the non-DR people (n = 21). (e) The expressions of EGR1 in hRMECs treated with NG or HG. Each experiment was performed in triplicate. ***P < 0.001. WT, wild type; MUT, mutant type; DR, diabetic retinopathy; EGR1, Early Growth Response Protein 1; hRMECs, human retinal microvascular endothelial cells; HG, high glucose; NG, normal glucose
Figure 7. EGR1 exacerbated oxidative stress and dysfunctions of HG-treated hRMECs. (a) The expression level of EGR1 after the cells were transfected with EGR1 overexpression plasmid. (b) MDA contents, (c) SOD, and (d) GSH-PX activities in hRMECs were detected using the corresponding kits after the indicated treatments. (e) Western blotting for the proteins of Nrf2, HO-1, and SOD-1. GAPDH was used to normalize the protein levels. (f) MTT assay was performed to determine the cell viability. (g) The migrated cells were fixed and stained after the transwell migration assay. (h) Quantification of G. (i) Images of the tube-like structures were observed under an inverted microscope. (j) Quantification of I. Each experiment was performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001. HG, high glucose; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; hRMECs, human retinal microvascular endothelial cells; MDA, malondialdehyde; SOD, superoxide dismutase; GSH-PX, glutathione peroxidase
Data availability statement
All the data are available from the corresponding author due to reasonable request