OBJECTIVES: To investigate how miR-30b regulates mitophagy independently of the PINK1/Parkin pathway by targeting ubiquitin specific peptidase 14 (USP14) in neuronal oxygen-glucose deprivation/reoxygenation (OGD/R) injury, and to provide new insights for the treatment of neonatal hypoxic-ischemic encephalopathy.
METHODS: Fetal rat cortical neurons were isolated and an OGD/R model was established. Experiments were conducted in two parts. In part 1, cells were randomized into control, OGD/R, OGD/R+microRNA (miR)-negative control (NC), OGD/R+miR-30b mimic, OGD/R+miR-30b mimic+oe-NC, and OGD/R+miR-30b mimic+oe-USP14 groups. In part 2, cells were randomized into control, OGD/R, OGD/R+miR-NC, OGD/R+miR-30b mimic, OGD/R+miR-30b mimic+si-NC, OGD/R+miR-30b mimic+si-Parkin, and OGD/R+miR-30b mimic+si-Drp1 (dynamin-related protein 1) groups. miR-30b and USP14 mRNA levels were measured by real-time quantitative PCR. Cell viability was assessed using the CCK-8 assay and cell death by propidium iodide staining. Protein levels of USP14, microtubule-associated protein 1 light chain 3 (LC3) Ⅱ/LC3 Ⅰ, translocase of outer mitochondrial membrane 20 (TOMM20), PINK1, Parkin, and Drp1 were determined by Western blot. MitoTracker Green staining was used to evaluate mitochondrial morphology, and mitochondrial reactive oxygen species (ROS) were measured using the MitoSOX Red probe. AGO-RNA immunoprecipitation and dual-luciferase reporter assays were performed to validate the targeting relationship between miR-30b and USP14. 2
RESULTS: Compared with the OGD/R group, the OGD/R+miR-30b mimic group showed higher cell viability, miR-30b expression, LC3 Ⅱ/LC3 Ⅰ ratio, and Drp1 protein expression (<0.05), and lower PI positivity, mitochondrial ROS, USP14 mRNA and protein expression, TOMM20 protein expression, mitochondrial fragmentation index, and mitochondrial volume (<0.05). PINK1 and Parkin protein levels did not differ significantly between these two groups (>0.05). Compared with the OGD/R+miR-30b mimic+oe-NC group, the OGD/R+miR-30b mimic+oe-USP14 group exhibited reduced cell viability, miR-30b expression, and LC3 Ⅱ/LC3 Ⅰ ratio (<0.05), and increased PI positivity, mitochondrial ROS, USP14 mRNA and protein expression, TOMM20 protein expression, mitochondrial fragmentation index, and mitochondrial volume (<0.05). miR-30b was confirmed to target USP14. In addition, no significant differences were observed between the OGD/R+miR-30b mimic+si-NC and OGD/R+miR-30b mimic+si-Parkin groups in LC3 Ⅱ/LC3 Ⅰ ratio, fragmentation index, or average mitochondrial volume (>0.05). Compared with the OGD/R+miR-30b mimic+si-NC group, the OGD/R+miR-30b mimic+si-Drp1 group showed a decreased LC3 Ⅱ/LC3 Ⅰ ratio and increased fragmentation index and average mitochondrial volume (<0.05). P P P P P P P
CONCLUSIONS: miR-30b regulates mitophagy by targeting USP14 independently of the PINK1/Parkin pathway, and confers protection against neuronal OGD/R injury.
