Fig. 5: ADEV-miRNA-382-5p downregulates OPA1 expression in neuronal mitochondria in the context of a mitochondrial imbalance.

a Overlap of potential targets of miRNA-382-5p predicted by the TargetScan, miRWalk, miRDB, RNAhybrid, and miRanda databases. b Venn diagram of putative target genes of miRNA-382-5p overlapping with mitochondria-related genes. c Wild-type and mutant OPA1 3′-UTR reporter constructs. d Luciferase reporter assay results for HEK293T cells cotransfected with the indicated wild-type or mutant 3’-UTR constructs and the miRNA-382-5p mimic (n = 6 per group; one-way ANOVA). e, f WB analysis and densitometric quantification of OPA1 levels in primary neurons transfected with or without the miRNA-382-5p mimic (n = 6 per group; Student’s t test). g MitoTracker was used to label mitochondria in primary neurons, mitochondrial morphology was examined via fluorescence microscopy, and mitochondrial morphological features were quantified (aspect ratios) using ImageJ software. h The boxed area next to each micrograph shows an magnified version of the white square (n = 6 per group, one-way ANOVA). i, j Representative images of MitoSOX fluorescence and quantitative comparison of mitochondria-derived ROS levels (n = 6 per group; one-way ANOVA). k, l Representative images of fluorescence staining and quantitative comparison of JC-1 aggregates (red fluorescence) and JC-1 monomers (green fluorescence) in cultured primary neurons from different experimental groups (n = 6 per group; one-way ANOVA). The data are presented as the means ± SDs; **p < 0.01, ***p < 0.001, and NS not significant.