Contribution of microRNA-203 to the isoflurane preconditioning-induced neuroprotection

Abstract

A prior exposure to isoflurane, a common volatile anesthetic, provides neuroprotection (isoflurane preconditioning). To determine the role of microRNAs in this protection, we performed microRNA array assay on cerebral cortex harvested from rats exposed to isoflurane or isoflurane-exposed rat B35 neuron-like cells. We showed that isoflurane significantly increased microRNA-203 expression in B35 neuron-like cells. The microRNA-203 expression in rat cerebral cortex also trended to increase after isoflurane exposure. Over-expression of microRNA-203 increased the tolerance of B35 cells to oxygen-glucose deprivation and the expression of phospho-Akt, a protein kinase that promotes cell survival. Isoflurane preconditioning also reduced the injury of these cells after oxygen-glucose deprivation. These results suggest that isoflurane preconditioning-induced neuroprotection may involve increased expression of microRNA-203. This finding provides the initial evidence that micoRNA-203 is a target for isoflurane in the brain.

Fig 1. Role of miRNA-203 in isoflurane preconditioning-induced neuroprotection

A: Differentiated B35 cells were exposed to or were not exposed to 2% isoflurane for 1 h. Cells were harvested 6 h later for quantification of miRNA-203 by real-time PCR. Results are mean ± S.D. (n = 11). * P < 0.05 compared with control. B: Differentiated B35 cells were exposed to or were not exposed to 2% isoflurane for 1 h. They were then subjected to a 90 min oxygen-glucose deprivation (OGD) 24 h later. Lactate dehydrogenase (LDH) release assay was performed 24 h after the OGD. Results are mean ± S.D. (n = 6). * P < 0.05 compared with control. ^ P < 0.05 compared OGD only. C: Differentiated B35 cells were transfected with miRNA-203 duplex or Stealth RNAi negative control duplex (miRNA-N). LDH release was performed 24 h after the transfection. Results are mean ± S.D. (n = 9). * P < 0.05 compared with miRNA-N. D: Differentiated B35 cells were transfected with miRNA-203 duplex or the negative control duplex. They were subjected to various lengths of OGD. LDH release was performed 24 h after the OGD. Results are mean ± S.D. (n = 9). * P < 0.05 compared with the corresponding values of cells without OGD. # P < 0.05 compared the corresponding values of cells transfected with miRNA-203 duplex.

Fig 2. Effect of miRNA-203 on phospho-Akt (pAkt) and Akt expression

Differentiated B35 cells were transfected with miRNA-203 duplex or Stealth RNAi negative control duplex (miRNA-N). Cells were harvested at 24 h after the transfection for Western blotting. A: pAkt results. A representative image is presented on the top panel and the pooled results of pAkt after being normalized by the corresponding value of glyceraldehydes 3-phosphate dehydrogenase (GAPDH) are presented in the bar graph. B: total Akt results. A representative image is presented on the top panel and the pooled results of pAkt after being normalized by the corresponding value of GAPDH are presented in the bar graph. Results are mean ± S.D. (n = 5). * P < 0.05 compared with miRNA-N.