Abstract
ABSTRACT MicroRNAs have been implicated as critical regulatory molecules in many cerebrovascular diseases. Recent studies demonstrated miR-22 might provide a potential neuroprotective effect. However, the neuroprotective effect of miR-22 in ischemia/reperfusion (I/R) injury has not been thoroughly elucidated. In this study, the PC12 cells were subjected to 4 h oxygen and glucose deprivation (I) and 24 h reoxygenation (R). The PC12 cells were pre-transfected with miR-22 or anti-miR-22 or siRNA-mediated downregulation of p53-upregulated-modulator-of-apoptosis (PUMA)(PUMA siRNA) or their controls at 24 h prior to exposure to I/R. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot were employed to analyze mRNA and protein expression. PI and Annexin V assays and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay were used to quantify the rate of apoptosis. We found that miR-22 expression was significantly downregulated in the PC12 cells subjected to I/R. Loss of function of miR-22 increased PC12 apoptosis after I/R, and overexpression of miR-22 decreases PC12 apoptosis after I/R. PUMA protein was upregulated in the I/R group as compared with the sham group. The increased PUMA protein expression and apoptosis induced by I/R was reversed by transfection with PUMA siRNA. We concluded that I/R enhanced apoptosis and PUMA expression in PC12 cells via downregulation of miR-22. Enhanced miR-22 expression reversed both PUMA expression and apoptosis induced by I/R in PC12 cells. miR-22/PUMA axis has important implications for their clinical applications.
Highlights
Cerebral ischemia is a serious condition associated with vascular disease, affecting patients worldwide
PUMA was upregulated in PC12 cells subjected to I/R As shown in Figure 1(b,c), under normal condition, no significant change of PUMA expression was found in the PC12 cells during 0, 12, 24 and 48 h
A significant increase in PUMA protein expression was observed at the I/R-12 h compared to the normal condition group
Summary
Cerebral ischemia is a serious condition associated with vascular disease, affecting patients worldwide. Despite hundreds of promising preclinical trials demonstrating efficacy of neurontargeted therapies in animal models of stroke, the only clinical treatment remains early restoration of blood flow with thrombolysis [1]. The failure to translate neuron-targeted approaches to useful clinical therapy suggests that alternative cellular targets in the brain may more effectively coordinate the complex intra- and intercellular signaling cascades that contribute to neuronal injury. The apoptosis is a prominent cellular injury mechanism, understanding the mechanisms underlying cerebral neuron apoptosis is still the key prerequisite for the treatment of brain ischemia/reperfusion (I/R) injuries effectively [2,3].
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