Key Laboratory of Transplant Engineering and Immunology of Health Ministry of China, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, Province, PR China.
J Biomed Sci. 2013 Dec 23;20(1):100. doi: 10.1186/1423-0127-20-100.
Hypoxia-inducible factor-1 alpha (HIF-1α) is one of the key regulators of hypoxia/ischemia. MicroRNA-494 (miR-494) had cardioprotective effects against ischemia/reperfusion (I/R)-induced injury, but its functional relationship with HIF-1α was unknown. This study was undertaken to determine if miR-494 was involved in the induction of HIF-1α.
Quantitative RT-PCR showed that miR-494 was up-regulated to peak after 4 hours of hypoxia in human liver cell line L02. To investigate the role of miR-494, cells were transfected with miR-494 mimic or miR-negative control, followed by incubation under normoxia or hypoxia. Our results indicated that overexpression of miR-494 significantly induced the expression of p-Akt, HIF-1α and HO-1 determined by qRT-PCR and western blot under normoxia and hypoxia, compared to negative control (p < 0.05). While LY294002 treatment markedly abolished miR-494-inducing Akt activation, HIF-1α and HO-1 increase under both normoxic and hypoxic conditions (p < 0.05). Moreover, apoptosis detection using Annexin V indicated that overexpression of miR-494 significantly decreased hypoxia-induced apoptosis in L02 cells, compared to control (p < 0.05). MiR-494 overexpression also decreased caspase-3/7 activity by 1.27-fold under hypoxia in L02 cells.
Overexpression of miR-494 upregulated HIF-1α expression through activating PI3K/Akt pathway under both normoxia and hypoxia, and had protective effects against hypoxia-induced apoptosis in L02 cells. Thus, these findings suggested that miR-494 might be a target of therapy for hepatic hypoxia/ischemia injury.
缺氧诱导因子-1α（HIF-1α）是缺氧/缺血的关键调节因子之一。微小 RNA-494（miR-494）对缺血/再灌注（I/R）诱导的损伤具有心脏保护作用，但它与 HIF-1α的功能关系尚不清楚。本研究旨在确定 miR-494 是否参与 HIF-1α的诱导。
定量 RT-PCR 显示，miR-494 在人肝细胞系 L02 缺氧 4 小时后上调至峰值。为了研究 miR-494 的作用，将细胞转染 miR-494 模拟物或 miR-阴性对照，然后在常氧或缺氧条件下孵育。结果表明，与阴性对照相比，miR-494 的过表达在常氧和缺氧条件下均显著诱导 p-Akt、HIF-1α 和 HO-1 的表达，qRT-PCR 和 Western blot 检测（p<0.05）。而 LY294002 处理明显消除了 miR-494 诱导的 Akt 激活，以及在常氧和缺氧条件下 HIF-1α 和 HO-1 的增加（p<0.05）。此外，用 Annexin V 检测凋亡表明，与对照相比，miR-494 的过表达显著降低了 L02 细胞缺氧诱导的凋亡（p<0.05）。miR-494 过表达还使 L02 细胞缺氧时 caspase-3/7 活性降低 1.27 倍。
miR-494 的过表达通过激活 PI3K/Akt 通路在常氧和缺氧条件下上调 HIF-1α 的表达，并对 L02 细胞缺氧诱导的凋亡具有保护作用。因此，这些发现表明 miR-494 可能是肝缺氧/缺血损伤治疗的靶点。
