HIF-1α protects PC12 cells from OGD/R-induced cell injury by regulating autophagy flux through the miR-20a-5p/KIF5A axis

Hypoxia inducible factor 1 alpha (HIF-1 alpha) has been reported to play a key role in protecting neurons from ischaemic injury. However, the exact molecular mechanisms remain largely unclear. PC12 cells were exposed to oxygen glucose deprivation/reoxygenation (OGD/R) conditions to mimic ischaemic injury in vitro. The expression of the HIF-1 alpha mRNA, miR-20a-5p, and kinesin family member 5A (KIF5A) mRNA was tested using qRT-PCR. Levels of the HIF-1 alpha, LC3I/II, P62, LAMP2, cathepsin B (CTSB) and KIF5A proteins were determined using western blotting. The CCK-8 assay was conducted to assess PC12 cell viability. DQ-Red-BSA and LysoSensor Green DND-189 dyes were employed to measure the proteolytic activity and pH of lysosomes, respectively. The interaction between miR-20a-5p and HIF-1 alpha or KIF5A was verified by performing chromatin immunoprecipitation (ChIP) and/or dual-luciferase reporter assays. TUNEL staining was adopted to assess PC12 cell death. GFP-LC3 and RFP-GFP-LC3 probes were used to examine the autophagy status and autophagy flux of PC12 cells. A rat middle cerebral artery occlusion-reperfusion (MCAO/R) model was established to investigate the role of the HIF-1 alpha/miR-20 alpha-5p/KIF5A axis in ischaemic stroke in vivo. OGD/R exposure initiated PC12 cell autophagy and injury. HIF-1 alpha expression was substantially increased in PC12 cells after OGD/R exposure. Overexpression of HIF-1 alpha reversed the effects of OGD/R on reducing cell viability, blocking autophagy flux and inducing lysosome dysfunction. These rescue effects of HIF-1 alpha depended on KIF5A. HIF-1 alpha negatively regulated miR-20a-5p expression by targeting its promoter region, and miR-20a-5p directly targeted and negatively regulated the KIF5A mRNA. Overexpression of miR-20a-5p abolished the effects of HIF-1 alpha on rescuing OGD/R-induced injury in PC12 cells. The effects of the HIF-1 alpha/miR-20a-5p/KIF5A axis were verified in MCAO/R rats. HIF-1 alpha protects PC12 cells from OGD/R-induced cell injury by regulating autophagy flux through the miR-20a-5p/KIF5A axis.

Automated summary

HIF-1 alpha protects PC12 cells from OGD/R-induced cell injury by regulating autophagy flux through the miR-20a-5p/KIF5A axis.