低氧诱导因子-1α 通过 miR-20a-5p/KIF5A 轴调控自噬流保护 PC12 细胞免受 OGD/R 诱导的细胞损伤。
HIF‑1α protects PC12 cells from OGD/R‑induced cell injury by regulating autophagy flux through the miR‑20a‑5p/KIF5A axis.
机构信息
Internal Medicine‑Neurology, The First Affiliated Hospital of Harbin Medical University, Harbin, P.R. China.
Department of Trauma Surgery, Harbin First Hospital, Harbin, P.R. China.
出版信息
Acta Neurobiol Exp (Wars). 2022;82(3):358-372. doi: 10.55782/ane-2022-034.
Hypoxia inducible factor 1α (HIF‑1α) 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‑1α mRNA, miR‑20a‑5p, and kinesin family member 5A (KIF5A) mRNA was tested using qRT-PCR. Levels of the HIF‑1α, 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‑1α 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‑1α/miR‑20a‑5p/KIF5A axis in ischaemic stroke in vivo. OGD/R exposure initiated PC12 cell autophagy and injury. HIF‑1α expression was substantially increased in PC12 cells after OGD/R exposure. Overexpression of HIF‑1α reversed the effects of OGD/R on reducing cell viability, blocking autophagy flux and inducing lysosome dysfunction. These rescue effects of HIF‑1α depended on KIF5A. HIF‑1α 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‑1α on rescuing OGD/R‑induced injury in PC12 cells. The effects of the HIF‑1α/miR‑20a‑5p/KIF5A axis were verified in MCAO/R rats. HIF‑1α protects PC12 cells from OGD/R‑induced cell injury by regulating autophagy flux through the miR‑20a‑5p/KIF5A axis.
缺氧诱导因子 1α（HIF-1α）已被报道在保护神经元免受缺血性损伤中发挥关键作用。然而，确切的分子机制在很大程度上仍不清楚。用氧葡萄糖剥夺/复氧（OGD/R）条件体外模拟缺血性损伤，检测 HIF-1α mRNA、miR-20a-5p 和驱动蛋白家族成员 5A（KIF5A）mRNA 的表达。用 Western blot 法检测 HIF-1α、LC3I/II、P62、LAMP2、组织蛋白酶 B（CTSB）和 KIF5A 蛋白水平。用 CCK-8 法检测 PC12 细胞活力。分别用 DQ-Red-BSA 和 LysoSensor Green DND-189 染料测定溶酶体的蛋白水解活性和 pH 值。通过染色质免疫沉淀（ChIP）和/或双荧光素酶报告基因检测验证 miR-20a-5p 与 HIF-1α 或 KIF5A 之间的相互作用。用 TUNEL 染色法评估 PC12 细胞死亡。用 GFP-LC3 和 RFP-GFP-LC3 探针检测 PC12 细胞自噬状态和自噬流。建立大鼠大脑中动脉闭塞再灌注（MCAO/R）模型，研究 HIF-1α/miR-20a-5p/KIF5A 轴在体内缺血性中风中的作用。OGD/R 暴露引发 PC12 细胞自噬和损伤。OGD/R 暴露后 PC12 细胞中 HIF-1α 表达显著增加。过表达 HIF-1α 逆转了 OGD/R 降低细胞活力、阻断自噬流和诱导溶酶体功能障碍的作用。HIF-1α 的这些挽救作用依赖于 KIF5A。HIF-1α 通过靶向其启动子区域负调控 miR-20a-5p 的表达，miR-20a-5p 直接靶向并负调控 KIF5A mRNA。过表达 miR-20a-5p 消除了 HIF-1α 对挽救 OGD/R 诱导的 PC12 细胞损伤的作用。在 MCAO/R 大鼠中验证了 HIF-1α/miR-20a-5p/KIF5A 轴的作用。HIF-1α 通过 miR-20a-5p/KIF5A 轴调节自噬流来保护 PC12 细胞免受 OGD/R 诱导的细胞损伤。
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