Figure 4. miR-325-3p, which suppressed Nupr1 translation, was downregulated by sevoflurane in neuronal cells. (A) RT-qPCR for 10 Nupr1-targeting miRNAs in HCN-2 cells exposed to fresh gas (21% O2, 5% CO2, remainder N2) alone or with the addition of 3.4% sevoflurane for 6 h. (B, C) Bioinformatics analysis showing predicted binding of miR-325-3p to the 3'-UTR of rat (B) and human (C) Nupr1 mRNA. (D) Wild-type Nupr1 mRNA (Nupr1 3'-UTR) and Nupr1 mRNA with a mutation in the 3'-UTR miR-325-3p-binding site (Nupr1 3'-UTR mut) were prepared. A dual-luciferase reporter assay was performed using all combinations of miR-325-3p-modifying and Nupr1 3'-UTR plasmids. (E) MiR-325-3p was overexpressed using an miR-325-3p mimic in HCN-2 neuronal cells. Controls were transfected with NC mimic. miR-325-3p levels in these cells were measured using RT-qPCR. (F, G) Western blot (F) and RT-qPCR (G) for Nupr1 levels in miR-325-3p-modified HCN-2 cells. **p<0.05. N=6.