Luteolin Induces microRNA-132 Expression and Modulates Neurite Outgrowth in PC12 Cells

Lian-Fang Lin,Pei-Yi Chen,Szu-Ping Chiu,Ming-Jiuan Wu,Jui-Hung Yen,Gregg Roman

doi:10.1371/journal.pone.0043304

Abstract

Luteolin (3′,4′,5,7-tetrahydroxyflavone), a food-derived flavonoid, has been reported to exert neurotrophic properties that are associated with its capacity to promote neuronal survival and neurite outgrowth. In this study, we report for the first time that luteolin induces the persistent expression of microRNA-132 (miR-132) in PC12 cells. The correlation between miR-132 knockdown and a decrease in luteolin-mediated neurite outgrowth may indicate a mechanistic link by which miR-132 functions as a mediator for neuritogenesis. Furthermore, we find that luteolin led to the phosphorylation and activation of cAMP response element binding protein (CREB), which is associated with the up-regulation of miR-132 and neurite outgrowth. Moreover, luteolin-induced CREB activation, miR-132 expression and neurite outgrowth were inhibited by adenylate cyclase, protein kinase A (PKA) and MAPK/ERK kinase 1/2 (MEK1/2) inhibitors but not by protein kinase C (PKC) or calcium/calmodulin-dependent protein kinase II (CaMK II) inhibitors. Consistently, we find that luteolin treatment increases ERK phosphorylation and PKA activity in PC12 cells. These results show that luteolin induces the up-regulation of miR-132, which serves as an important regulator for neurotrophic actions, mainly acting through the activation of cAMP/PKA- and ERK-dependent CREB signaling pathways in PC12 cells.

Highlights

MicroRNAs are small (19–25 nucleotides) noncoding RNAs that are involved in several biological processes, such as development, morphogenesis, cell proliferation, cell differentiation and apoptosis [1]
Mature miRNAs are single-stranded RNA molecules that are derived from a immature form of hairpin precursor, which are processed from the primary miRNA gene transcripts, and usually bind to the complementary sequence in the 39-untranslated region (39-UTR) of multiple target genes, which leads to the translational repression or degradation of a target mRNA [5]
Luteolin Promotes miR-132 Expression in PC12 Cells To evaluate the effects of luteolin on the expression of miR-132, PC12 cells were cultured in low-serum medium (1% horse serum and 0.5% fetal bovine serum (FBS)) and treated with vehicle (0.1% dimethyl sulfoxide (DMSO)), forskolin (10 mM; as a positive control) or luteolin (20 mM) for the indicated period

Summary

Introduction

MicroRNAs (miRNAs) are small (19–25 nucleotides) noncoding RNAs that are involved in several biological processes, such as development, morphogenesis, cell proliferation, cell differentiation and apoptosis [1]. Mature miRNAs are single-stranded RNA molecules that are derived from a immature form of hairpin precursor (pre-miRNA) (approximately 70–100 nucleotides), which are processed from the primary miRNA gene transcripts (pri-miRNA), and usually bind to the complementary sequence in the 39-untranslated region (39-UTR) of multiple target genes, which leads to the translational repression or degradation of a target mRNA [5]. It has been shown that miR-132, one such miRNA that is enriched in the mammalian brain, could be induced by neurotrophic factors and that this could represent a mechanism for fine-tuning protein expression following neurotrophic action [9]. MiR-132 is induced by cyclic AMP (cAMP) response element binding protein (CREB) and is involved in the modulation of dendritic morphology, neurite outgrowth, synaptic plasticity and neuroprotection [10,11]

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