Upregulation of MIR-132-5P via nicotine enhances cell survival in PC12 cells by targeting the anti-apoptotic protein BCL-2

Abstract

Objective Activation of nicotinic acetylcholine receptors (nAChRs) results in neuroprotection via a poorly understood molecular mechanism. Herein, we aimed to investigate the effect of nAChR stimulation with nicotine on the regulation of miRNA expression and identify the molecular pathway involved in neuroprotection. Materials and methods miRNA expression profiling using microarray was conducted to identify nicotine regulated miRNAs. The expression of miR-132-5p was validated by RT-qPCR analysis. Cell viability was assessed after treatment of cells with nicotine, miR-132-5p mimic or its inhibitor. The protein expression of CREB, Bcl-2 and Bax was determined by western blotting analysis. Results Using miRNA microarray we identified 37 miRNAs regulated by nicotine. The microarray and the RT-qPCR results showed a 1.67-fold and a 1.5-fold increase in miR-132-5p, respectively, upon nicotine treatment. Immunoblotting revealed >2-fold increase in the phosphorylation of CREB with nicotine, peaking at 4 h. Cells treated with nicotine showed increased viability from 35% to 54%, and the Bcl-2 immunoreactivity increased by 1.4-fold. Overexpression of miR-132-5p increased cell viability from 38% to 70% and increased the expression level of Bcl-2 by 3.9-fold. Inhibition of miR-132-5p decreased cell viability to 25%, whereas no change was seen in Bcl-2. Bax expression remained unchanged following treatment with miR-132-5p mimic or inhibitor. Conclusions Our results suggest that nAChR activation facilitates cell survival by upregulating miR-132-5p, which upregulates the anti-apoptotic protein, Bcl-2. These results present miR-132-5p as a potential novel therapeutic target to achieve neuroprotection via stimulation of nAChR.