Figure 5.

circFoxO1 acts as a miRNA sponge in choroidal ECs

(A) The expression of nucleus Ctrl transcript (U6), cytoplasmic Ctrl transcript (GAPDH), FoxO1 mRNA, and circFoxO1 was detected by qRT-PCRs in the nucleus and cytoplasmic fractions of RF/6A cells (n = 4). (B) The entire sequence of circFoxO1 was cloned into the pRL-TK luciferase reporter to construct the LUC-circFoxO1 vector. RF/6A cells were co-transfected LUC-circFoxO1 with different miRNA mimics. Luciferase activity was detected using a dual-luciferase assay 48 h after transfection (n = 4; ∗p < 0.05 versus Ctrl group, one-way ANOVA, Bonferroni test). (C) RF/6A cells were co-transfected LUC-circFoxO1-Mut (without miR-145 binding site) with miR-145 mimic, Scr mimics, or were left untreated (Ctrl). Luciferase activity was detected using a dual- luciferase assay at 48 h post-transfection (n = 4; ∗p < 0.05 versus Ctrl group, one-way ANOVA, Bonferroni test). (D) The putative binding sites of miR-145 on circFoxO1 transcript are shown. (E) qRT-PCR assays were conducted to detect the expression level of miR-145 in the choroidal tissues of C57BL/6 mice after 8-week FD induction (n = 6; ∗p < 0.05 versus normal choroid, Mann-Whitney U test, Bonferroni test). (F) RF/6A cells were incubated with normal culture medium, H₂O₂ (200 μM, oxidative stress), or CoCl₂ (100 μM, hypoxic stress) for 24 h. qRT-PCR assays were conducted to detect miR-145 expression (n = 4; ∗p < 0.05 versus Ctrl group, one-way ANOVA, Bonferroni test). (G) RF/6A cells were transfected with pcDNA3.1 (vector), pcDNA3.1-circFoxO1, or were left untreated (Ctrl) for 24 h. qRT-PCR assays were conducted to detect miR-145 expression levels (n = 4; ∗p < 0.05 versus Ctrl group, one-way ANOVA, Bonferroni test). (H) RF/6A cells were transfected with Scr mimic, miR-145 mimic, or were left untreated (Ctrl) for 24 h. qRT-PCR assays were conducted to detect VEGFA and ANGPT2 expression (n = 4; ∗p < 0.05 versus Ctrl group, one-way ANOVA, Bonferroni test). (I) RF/6A cells were co-transfected VEGFA-Luc, VEGFA-Luc mutant (Mut), ANGPT2-Luc, or ANGPT2-Luc Mut with Scr mimic, miR-145 mimic, or were left untreated (Ctrl) for 24 h. Luciferase activity was detected at 24 h post-transfection (n = 4; ∗p < 0.05 versus Ctrl group, one-way ANOVA, Bonferroni test).