Figures & data
Table 1. Sequences of primers used for reverse transcription-quantitative PCR.
Figure 1. SOX17 knockdown inhibits angiogenesis and upregulates protein levels of tight junctions in Ang-II-treated HRMECs. (a-b) After transfection of sh-NC or sh-SOX17, SOX17 mRNA and protein levels in HRMECs with or without Ang-II treatment were detected by RT-qPCR and western blotting, respectively. (c) After transfection of sh-NC or sh-SOX17, angiogenesis of HRMECs with or without Ang-II treatment was examined by tube formation assays. (D) the protein levels of tight junctions (ZO-1, Occludin and Claudin-5) in HRMECs transfected with sh-NC/sh-SOX17 and treated with or without Ang-II were tested by western blotting. ** p <0.01, *** p <0.001.
Figure 2. SOX17 is targeted by miR-194-5p. (a) Four upstream miRnas (miR-141-3p, miR-200a-3p, miR-194-5p and miR-26-5p) of SOX17 were predicted using Targetscan. (b) the expression of candidate miRnas in HRMECs treated with or without Ang-II was detected by RT-qPCR. (c) MiR-194-5p and SOX17 levels in HRMECs transfected with miR-194-5p mimics or NC mimics were tested by RT-qPCR. (d) a binding site between miR-194-5p and SOX17 was predicted using Targetscan. (e) the binding relationship between miR-194-5p and SOX17 was confirmed by luciferase reporter assay. (f) SOX17 protein level in HRMECs transfected with NC mimics/mir-194-5p mimics and treated with or without Ang-II was quantified by western blotting. ** p <0.01, *** p <0.001.
Figure 3. SOX17 overexpression reverses the effects of miR-194-5p upregulation on angiogenesis and protein levels of tight junctions in Ang-II-treated HRMECs. (a-b) the mRNA and protein levels of SOX17 in Ang-II-stimulated HRMECs after overexpressing SOX17 were detected by RT-qPCR and western blotting, respectively. (c) Angiogenesis of Ang-II-treated HRMECs after upregulating miR-194-5p or SOX17 was examined by tube formation assays. (b) the protein levels of tight junctions (ZO-1, Occludin and Claudin-5) in HRMECs upon Ang-II treatment after upregulating miR-194-5p or SOX17 were tested by western blotting. *p <0.05, ** p <0.01, *** p <0.001.
Figure 4. MiR-194-5p inactivates VEGF pathway by downregulating SOX17. (a) Protein levels of genes (VEGFA, VEGFB and VEGFR) involved in the VEGF signaling pathway were quantified by western blotting in Ang-II-stimulated HRNECs overexpressing miR-194-5p or SOX17. *** p <0.001.
Figure 5. Overexpressing miR-194-5p mitigates retinal thickness and elevates tight junction proteins of Ang-II-treated mice. (a) the effect of Ang-II on the b-wave ratio in electroretinograms (ERGs) was determined. (b-e) H&E staining was used to assess the central retinal thickness, IPL thickness and INL thickness in sham-operated mice and Ang-II-treated mice after injection with AAV-NC or AAV-miR-194-5p. (f) Protein levels of tight junctions in retinal tissues of sham-operated mice and Ang-II-treated mice after injection with AAV-NC or AAV-miR-194-5p were quantified by western blotting. *p <0.05, ** p <0.01, *** p <0.001.
Figure 6. MiR-194-5p upregulation inhibits VEGF pathway in Ang-II-treated mice. (a) Protein levels of genes (VEGFA, VEGFB and VEGFR) involved in the VEGF signaling pathway were quantified by western blotting in retinal tissues of sham-operated mice and Ang-II-treated mice after injection with AAV-NC or AAV-miR-194-5p was examined by western blotting. n = 10 mice/group. *p <0.05, ** p <0.01, *** p <0.001.
Figure 7. MiR-194-5p negatively regulates SOX17 in Ang-II-treated mice. (a-b) the expression of miR-194-5p and SOX17 in retinal tissues of sham-operated mice (n = 10 mice/group) and Ang-II-treated mice (n = 10 mice/group) after injection with AAV-NC or AAV-miR-194-5p was detected by RT-qPCR. (c) Correlation between miR-194-5p expression and SOX17 expression in retinal tissues of Ang-II-treated mice (n = 20) was analyzed by Pearson correlation analysis. (d) Fluorescence in situ hybridization (FISH) was performed to determine the in situ expression of SOX17 in retinal tissues of mice with or without Ang-II after injection of AAV-NC or AAV-miR-194-5p. *p <0.05, ** p <0.01, *** p <0.001.
