Research Paper Volume 12, Issue 9 pp 8321—8338

Mitochondria-related miR-574 reduces sperm ATP by targeting ND5 in aging males

Figure 5. MiR-574 regulated mitochondrial function by directly targeting mt-ND5. (A) MiR-574 and positive control genes were detected in the mitochondrial fraction and cytoplasmic fraction in GC2 cells. (B) The putative site at which miR-574 binds to mt-ND5 in multiple species, the mutant vector was constructed by mutating miR-574 binding sites in mt-ND5. (C, D) mRNA levels of mt-ND5 in GC2 cells transfected with miR-574 mimic/inhibitor or their respective controls. ***p < 0.001, *p < 0.05. (E) Protein levels of mt-ND5 in GC2 cells transfected with miR-574 mimic/inhibitor or their respective controls. β-Actin was used to normalize the WB results. (F) Luciferase activity in GC2 cells co-transfected with miR-574 mimic/inhibitor or their respective controls and the WT/Mut luciferase reporter. (G) Anti-Ago2 RIP was performed in GC2 cells transiently overexpressing miR-574 or negative control, followed by qRT-PCR to detect mt-ND5 associated with Ago2.