Protective effect of microRNA‑340‑5p against oxygen‑glucose deprivation/reperfusion in PC12 cells through targeting neuronal differentiation4.

Abstract

The expression levels of microRNA (miR)-340-5p are reportedly decreased in the peripheral blood during acute ischemic stroke; however, the direct effect and mechanism of action of miR-340-5p in ischemic stroke remains largely unknown. The present study aimed to investigate the effects of miR-340-5p, and its mechanism of action, on PC12 cells following oxygen-glucose deprivation/reperfusion (OGD/R) induction. OGD/R-induced PC12 cells served as the cellular model and subsequently, mRNA expression levels of miR-340-5p and neuronal differentiation 4 (Neurod4) were analyzed using reverse transcription-quantitative PCR. Tumor necrosis factor-α, interleukin (IL)-1β and IL-6 expression levels were detected using ELISA kits, and flow cytometry was used to determine the rate of cellular apoptosis. In addition, a nitric oxide (NO) synthase activity assay kit was used to detect NO levels and a NADPH assay kit was used to measure NADPH levels. Western blotting was also performed to analyze protein expression levels of bax, bcl-2, cleaved caspase 3 and phosphorylated endothelial NOS (eNOS), and the target gene of miR-340-5p was predicted using TargetScan software and verified using a dual-luciferase reporter assay. The expression levels of miR-340-5p were decreased in PC12 cells following OGD/R induction and Neurod4 was identified as a target gene of miR-340-5p. In addition, miR-340-5p overexpression reduced inflammation, apoptotic rate, NO production and NADPH levels, in addition to increasing eNOS expression in PC12 cells following OGD/R induction. Notably, the overexpression of Neurod4 reversed the aforementioned effects of miR-340-5p on PC12 cells following OGD/R induction. In conclusion, the findings of the present study suggested that miR-340-5p may protect PC12 cells against OGD/R through targeting Neurod4, which could provide important implications for the treatment of ischemia-reperfusion injury based on miR-340-5p expression levels in vivo.