Protective Effect of miR-374a on Chemical Hypoxia-Induced Damage of PC12 Cells In Vitro via the GADD45α/JNK Signaling Pathway.

Abstract

To explore the effect of microRNA-374a (miR-374a) on chemical hypoxia-induced pheochromocytoma (PC12) cell damage by mediating growth arrest and the DNA damage-45 alpha (GADD45α)/c-Jun N-terminal kinase (JNK) signaling pathway. PC12 cells were divided into a Control group (no treatment), Model group (treated with CoCl2 for 24h), negative control (NC) group (transfected with miR-374a negative control sequence and treated with CoCl2 for 24h), and miR-374a mimic group (transfected with miR-374a mimics and treated with CoCl2 for 24h). The viability and apoptosis of PC12 cells were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry, while the mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) content were assessed by Rh123 and dichloro-dihydro-fluorescein diacetate (DCFH-DA) methods. The expression of miR-374a and GADD45α/JNK proteins was detected using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. A significant decrease was found in the survival rate, MMP and miR-374a expression, while an increase was shown in the ROS content and GADD45α and p-JNK expression in hypoxic PC12 cells (all P < 0.05). A luciferase reporter gene assay demonstrated that GADD45α is the target gene of miR-374a. When transfected with miR-374a mimics, hypoxic PC12 cells showed an obvious elevation in survival rate and MMP but a great reduction in cell apoptosis and ROS content, as well as in the expression of GADD45α and p-JNK proteins (all P < 0.05). MiR-374a can protect PC12 cells against hypoxia-induced injury by inhibiting the GADD45α/JNK pathway, enhancing cell viability, suppressing oxidative stress, and inhibiting cell apoptosis, thereby becoming a potential therapeutic target for hypoxic damage.