HIF­1α protects PC12 cells from OGD/R­induced cell injury by regulating autophagy flux through the miR­20a­5p/KIF5A axis.

Hypoxia inducible factor 1α (HIF­1α) 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 HIF­1α mRNA, miR­20a­5p, and kinesin family member 5A (KIF5A) mRNA was tested using qRT-PCR. Levels of the HIF­1α, LC3I/II, P62, LAMP2, cathepsin B (CTSB) and KIF5A proteins were determined using western blotting. The CCK­8 assay was conducted to assess PC12 cell viability. DQ­Red­BSA and LysoSensor Green DND­189 dyes were employed to measure the proteolytic activity and pH of lysosomes, respectively. The interaction between miR­20a­5p and HIF­1α or KIF5A was verified by performing chromatin immunoprecipitation (ChIP) and/or dual­luciferase reporter assays. TUNEL staining was adopted to assess PC12 cell death. GFP­LC3 and RFP­GFP­LC3 probes were used to examine the autophagy status and autophagy flux of PC12 cells. A rat middle cerebral artery occlusion­reperfusion (MCAO/R) model was established to investigate the role of the HIF­1α/miR­20a­5p/KIF5A axis in ischaemic stroke in vivo. OGD/R exposure initiated PC12 cell autophagy and injury. HIF­1α expression was substantially increased in PC12 cells after OGD/R exposure. Overexpression of HIF­1α reversed the effects of OGD/R on reducing cell viability, blocking autophagy flux and inducing lysosome dysfunction. These rescue effects of HIF­1α depended on KIF5A. HIF­1α negatively regulated miR­20a­5p expression by targeting its promoter region, and miR­20a­5p directly targeted and negatively regulated the KIF5A mRNA. Overexpression of miR­20a­5p abolished the effects of HIF­1α on rescuing OGD/R­induced injury in PC12 cells. The effects of the HIF­1α/miR­20a­5p/KIF5A axis were verified in MCAO/R rats. HIF­1α protects PC12 cells from OGD/R­induced cell injury by regulating autophagy flux through the miR­20a­5p/KIF5A axis.

LncRNA RMRP accelerates autophagy-mediated neurons apoptosis through miR-3142/TRIB3 signaling axis in alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a major neurodegenerative disorder. The functions of lncRNA RMRP have been characterized mainly in various human cancers. However, the functional network of RMRP in AD progression remains unknown. METHODS: Human serum samples, AD transgenic (Tg) mice as well as SH-SY5Y cells were used in this study. The RNA expression patterns of RMRP, miR-3142 and TRIB3 were assessed by quantitative real-time PCR (qRT-PCR). Levels of apoptosis- or autophagy-associated biomarkers and TRIB3 level were evaluated using immunohistochemistry (IHC), western blotting or immunofluorescence assays, respectively. Bioinformatics methods and luciferase assays were used to predict and validate the interactions among RMRP, miR-3142, and TRIB3. Flow cytometry, TUNEL staining and EdU assays were used to examine the apoptosis and proliferation of neurons, respectively. RESULTS: The elevated RMRP and TRIB3 expressions and activation of autophagy were observed in AD. Knockdown of RMRP restrained neuronal apoptosis and autophagy activation in vitro and in vivo. Interestingly, TRIB3 overexpression reversed the biological effects of RMRP silencing on Aß1-42-induced cell apoptosis and autophagy. Further mechanistic analysis showed RMRP acted as a sponge of miR-3142 to elevate TRIB3 level. CONCLUSION: These data illustrated that knockdown of RMRP inhibited autophagy and apoptosis via regulating miR-3142/TRIB3 axis in AD, suggesting that inhibition of RMRP maybe a therapeutic strategy for AD.