MicroRNA Let-7f Mediates Mitochondrial Respiratory Deficit Induced by Repeated Ethanol Exposure and Withdrawal in HT22 Cells

Abstract

Aims: The expression of gene and gene product is typically inhibited by a small non-coding RNA (microRNA) or DNA methylation. The aim of this study is to investigate mechanisms involving microRNA let-7f by which the repeated cycles of ethanol exposure and withdrawal provoke mitochondrial respiratory damage. Study Design: The rat or cell model of repeated withdrawal from a high dose of ethanol exposure was used to mimic human alcoholics who repeat the cycles of heavy drinking and unsuccessful attempts at abstaining.  Place and Duration of Study: Department of Pharmacology and Neuroscience University of North Texas Health Science Center at Fort Worth, between June 2011 and March 2014. Methodology: Male adult rats received an ethanol program, consisting of two cycles of ethanol exposure (4 weeks) and withdrawal (2 weeks). At the end of the ethanol program, one hemisphere of each rat was used to measure the level of let-7f using TaqMan let-7f primers and qPCR. The other hemisphere was used to measure the methylation of cytosine in let-7f gene using bisulfite conversion and pyrosequencing.  Separately, HT22 cells (mouse hippocampal cells) were exposed to an ethanol program, consisting of two cycles of ethanol exposure (20 hours) and withdrawal (4 hours).  During the entire ethanol program, the cells were treated with let-7f antagomir (inhibitor) or a methylation-inducing methyl-donor. The role of let-7f in mitochondria was assessed by quantifying a mitochondrial enzyme, cytochrome c oxidase-IV (COX subunit IV) and real-time mitochondrial respiration using an immunoblot method and XF respirometry, respectively. Results: The level of let-7f increased (2.4±0.5 fold increase), whereas the methylation of let-7f gene decreased in the brain of rats that underwent repeated ethanol exposure and withdrawal (called “repeated-ethanol/withdrawal”). The methyl-donor treatment completely abolished the increase in let-7f induced by repeated-ethanol/withdrawal. let-7f antagomir treatment also abolished the inhibiting effect of repeated-ethanol/withdrawal on COX-IV and mitochondrial respiration. Conclusion: These data suggest that repeated-ethanol/withdrawal provokes the dysregulation of let-7f, thereby damaging brain mitochondria. Mitochondria-associated microRNA may be a potential research and drug target to manage alcoholism.