miRNAs 29b and 181a Down-Regulate Expression of the Norepinephrine Transporter in PC12 Cells

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miRNAs 29b and 181a down-regulate expression of the norepinephrine transporter in PC12 cells. M.X. Deng, G. A. Ordway and M.-Y. Zhu. Dept. of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, Johnson City, TN, USA MicroRNAs are short non-coding RNAs that provide global regulation of gene expression at the post-transcriptional level. Such regulation has been found to play a role in stress-induced epigenetic responses in the brain. The noradrenaline transporter (NET) is a noradrenergic marker and regulates neurotransmitter signaling by rapidly clearing released norepinephrine from synapses. Our previous studies demonstrated that rat NET mRNA and protein levels are regulated by chronic stress and by administration of corticosterone. Whether miRNAs are intermediaries in the regulation of NET expression remains to be elucidated. The present study was undertaken to determine possible regulatory effects of miRNAs on NET expression in PC12 cells, a cell model for noradrenergic neurons. Using computational target prediction, we identified several miRNAs potentially related to regulation of NET expression. Mimics of these miRNAs were transfected into PC12 cells. NET protein expression was assayed by Western blotting 48 hours after transfection. miR29b- and miR181a-transfected cells showed significantly reduced NET protein levels. To identify the exact target loci, the 3’-UTR of NET mRNA was amplified by PCR from PC12 genomic DNA and cloned downstream of the red firefly gene of the pmirGlo vector. The NET 3’-UTR-bearing pmirGlo and miR29b or miR181a were co-transfected into PC12 cells and luciferase signals were measured 48 hours after transfection. Consistent with Western blots, co-transfection of these miRNAs with rat NET3’-UTR-containing plasmids resulted in reduced levels of luciferase activity in PC12 cells. We conclude that miR29b and miR181a can function as negative regulators of NET translation in vitro. Further studies to determine whether these miRNAs contribute to the regulation of NET expression induced by antidepressants are under way.

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