Circular RNA circPHKA2 Relieves OGD-Induced Human Brain Microvascular Endothelial Cell Injuries through Competitively Binding miR-574-5p to Modulate SOD2

Figure 6

circPHKA2 mediated the regulation of SOD2 via serving as sponge for miR-574-5p. (a) Venn diagram showed the computational miRNAs that could target circPHKA2 and SOD2 according to CircInteractome, circBank, and starBase databases. (b) RIP and qPCR detected relative RNA enrichment of circPHKA2, SOD2, and miR-574-5p by anti-Ago2 in HBMEC, normalized to anti-IgG-mediated RIP. (c) Biotin-labelled miRNA capture examined relative RNA enrichment of circPHKA2 and SOD2 by probes of bio-miR-NC, WT-bio-miR-74-5p, and MUT-bio-miR-574-5p. (d) Schematic drawing showed the predicted binding sites between miR-574-5p and WT-circPHKA2 or MUT-circPHKA2 according to two positions of circPHKA2. (e) qPCR detected relative miR-574-5p expression in HBMEC administrated with commercial miR-574-5p or miR-NC. (f, g) Dual-luciferase reporter assay measured relative luciferase activity of WT-circPHKA2 vectors and MUT-circPHKA2 vectors in HBMEC administrated with commercial miR-574-5p or miR-NC. (h) Schematic drawing showed the predicted binding sites between miR-574-5p and WT-SOD2 3UTR or MUT-SOD2 3UTR. (i) Dual-luciferase reporter assay measured relative luciferase activity of WT-SOD2 3UTR vector and MUT-SOD2 3UTR vector in HBMEC administrated with commercial miR-574-5p or miR-NC. qPCR and western blotting examined relative SOD2 mRNA and protein expression in HBMEC administrated with (j, k) commercial miR-574-5p or miR-NC and (l, m) oe-circPHKA2 along with commercial miR-574-5p or miR-NC and NC along with miR-NC. .

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