Fig. 7. miRNAs in young and aged sEVs affect mitochondrial metabolism by regulating PGC-1α expression.
a, Western blot analysis of PGC-1α protein levels in NE-4C and C2C12 cells transfected with the scrRNA, a combination of miR-29a-3p, miR-29c-3p and miR-34a-5p mimics (miR-29a/29c/34a) or a combination of miR-144-3p, miR-149-5p and miR-455-3p mimics (miR-144/149/455). Left, representative western blots. Right, densitometric analysis (n = 6). b, Western blot analysis of APP, PARP-2 and HIF1an protein levels in NE-4C and C2C12 cells transfected with the scrRNA or the corresponding miRNA mimic. Left, representative western blots. Right, densitometric analysis (n = 6). c, Western blot analysis of PGC-1α protein levels in the hippocampus and muscle of aged mice injected with 200 μl of PBS or young mouse sEVs seven times over 2 weeks. Left, representative western blots. Right, densitometric analysis (n = 8 for hippocampus; n = 6 for muscle). d, Quantitative RT–PCR analysis of PGC-1α mRNA levels in the hippocampus and muscle of aged mice injected with 200 μl of PBS or young mouse sEVs seven times over 2 weeks (n = 4). e, Western blot analysis of PGC-1α protein levels in NE-4C and C2C12 cells incubated with 100 μl of PBS or young mouse sEVs for 24 h. Left, representative western blots. Right, densitometric analysis (n = 6). f, ATP synthesis rates in NE-4C and C2C12 cells transfected with scrRNA, miR-29a/29c/34a or miR-144/149/455 (n = 6). g, Mitochondrial complex V activity in NE-4C and C2C12 cells transfected with scrRNA, miR-29a/29c/34a or miR-144/149/455 (n = 6). h, Relative mtDNA content (MT-CO1/β2-MG) in NE-4C and C2C12 cells transfected with scrRNA, miR-29a/29c/34a or miR-144/149/455 (n = 6). Significance was determined using two-sided Student’s t-test in b–e and one-way ANOVA followed by Dunnett’s multiple comparison test in a and f–h. *P < 0.05, **P < 0.01 and ***P < 0.005.