Fig. 6.

miRNA binding sites are not critical for circFndc3b function: a CircFndc3b sequence was cloned into the 3′UTR of the pGL3 Luciferase reporter to construct LUC-circFndc3b. MCECs were co-transfected with LUC-circFndc3b and different miRNA mimics (30 nM). Luciferase activity was detected using the dual luciferase assay at 48 h post transfection. Data are Mean ± SEM of 3 independent experiments, **P < 0.01 ***p < 0.001 vs scrambled control group (one-way ANOVA); b–d RT-qPCR analysis of circFndc3b target miRNAs (miR-93-3p, miR-298-5p and miR-412-3p) in 3 and 7 days post-MI hearts compared to sham, data normalized to U6 snRNA; Data are Mean ± SEM of n = 3–5/group. *p < 0.05 vs sham hearts (one-way ANOVA); e–g Representative images of H9c2 cells (Scale bar 20 μm) treated with control plasmid or WT-circFndc3b or Mut-circFndc3b plasmids and subjected to hypoxic stress (1% O₂, 48 h). TUNEL assay was performed; h Quantification of TUNEL + cells presented as the % TUNEL + positive cells and DAPI-stained nuclei. Data are Mean ± SEM of 6 independent experiments. ***p < 0.001 vs control plasmid (one-way ANOVA); i, j Representative images and quantification of apoptotic cardiomyocytes in the myocardium at 10 days after MI in AAV9 control or AAV9 WT-circFndc3b or Mut-circFndc3b administered mice. TUNEL staining for detecting apoptosis (red) of cardiomyocytes (α-SA, green florescence) and DAPI (blue) for nuclear staining. Arrows indicate TUNEL and α-SA + cells (Scale bar 100 μm). n = 3/each group. ***p < 0.001 vs AAV9 control (two-sided unpaired students t-test)