Murine microRNAs implicated in liver functions and aging process

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Abstract

Small non-coding microRNAs (miRNAs) are involved in gene regulation in various cellular and developmental processes, including mechanisms of aging. Here, the mouse liver was used as a paradigm for the study of miRNAs implicated in the aging process in mammals. Expression profiling of 367 murine miRNAs (Sanger Version 8.2) was assessed in livers from 4 to 33 months old mice, and their predicted targets were compared with proteomic profiling data generated from the same animals. Gradual increases in the levels of miR-669c and miR-709 were observed from mid-age of 18–33 months, while miR-93 and miR-214 were significantly up-regulated only in extremely old liver. In contrast, we did not identify any miRNAs showing significant down-regulation with age. Interestingly, the up-regulated miRNAs’ targets are associated with detoxification activity and regeneration capacity functions known to decline in old liver. In particular, three up-regulated miRNAs may contribute to the aging-related decline in oxidative defense by targeting various classes of glutathione S-transferases. Other proteins in decline in old liver and targeted by the up-regulated miRNAs are involved in mitochondrial functions or maintenance. Taken together, we identified the up-regulation of key miRNAs that may participate in the decline of regeneration and oxidative defense mechanisms in aging liver.

Introduction

MicroRNAs (miRNAs) are small, non-coding RNAs between 18 and 25 nucleotides long, generally involved in the suppression of gene expression in many cellular and developmental processes (Ambros, 2004, Alvarez-Garcia and Miska, 2005). While the identification of microRNAs is not complete, close to 500 miRNAs have been identified and registered in both man and mouse (Griffiths-Jones et al., 2006). Individual miRNAs have the potential to target hundreds of transcripts, typically by imperfect or perfect base pairing in the 3′-untranslated region (UTR) of mRNAs. Despite the growing number of experimentally demonstrated suppressions, most targets are predicted by computerized algorithms (Maziere and Enright, 2007). The multiple mechanisms of suppression through which miRNA may act include translational inhibition, mRNA de-adenylation and degradation, mRNA sequestration (Nilsen, 2007) and cleavage (Yekta et al., 2004), ultimately leading to decreased levels of targeted proteins.

In Caenorhabditis elegans, the roles of several microRNAs have been associated with the organism's lifespan (Ibáñez-Ventoso et al., 2006). Specifically, over-expression of miRNA lin-4 increases longevity, and is believed to act through the insulin-like signaling pathway (Boehm and Slack, 2006), a common module in various species controlling the rate of aging, implicating antioxidant genes such as superoxide dismutase and glutathione S-transferases (GSTs) (Kenyon, 2005). The identification of microRNAs involved in the aging process is expected in higher organisms, and may correspond to progressive deregulations in their expression commencing at mid-life (Wang, 2007).

In mammals, the liver is a vital organ involved in several functions including detoxification; its maintenance during aging is central to the animal's life expectancy (Schmucker, 2005). While the liver is known to have high regeneration capacity after hepatectomy, it is in fact a quiescent organ, gradually declining with age due to reduced regenerative capacity (Gagliano et al., 2007, Iakova et al., 2003, Schmucker, 2005) and decreased gene expression (Serste and Bourgeois, 2006). The decline in volume, drug metabolism and detoxification activity in the liver may reflect systemic aging processes in the elderly. In fact, evidence of extended lifespan by caloric restriction in mouse has been associated with the restoration of liver functions to younger levels (Cao et al., 2001), and alteration in insulin/IGFI receptor signaling (Bauer et al., 2004, Spindler and Dhahbi, 2007).

In this study, alterations in microRNA and protein levels in liver were investigated during aging in mice. MicroRNA expression in very old (33 months) mice was compared to younger mice, and correlations were made with corresponding proteomic data and miRNA target predictions. Four up-regulated microRNAs correlate with predicted targeted proteins found to decline in old liver, and linked to glutathione metabolism and mitochondrial functions. Functional attributions to these miRNAs in liver maintenance, as well as in aging mechanisms, are proposed.

Section snippets

Mouse strain and samples

Normal wild-type male C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, Maine). Two mice were housed in each cage, equipped with microisolator filter units, and housed in rooms with controlled temperature (22 ± 1 °C) and photoperiod (12:12 h). They were fed a minimum of 23% protein, 6.5% fat and a maximum of 4% fiber (Lab Diet Formula 5008; PMI Feeds, Ralston/Purina, St. Louis, MO) and water. Euthanasia of animals was performed by cervical dislocation in order to avoid

MicroRNA expression patterns in aging mouse liver

The quality and yield of small RNA extracted from livers from mice at different ages (i.e. 4, 10, 12, 18 and 33 months) did not differ significantly (Fig. 1), with an average ratio of 0.09 ± 0.02 of small over total RNA (one-way ANOVA P-value = 0.42). For each age group in three biological repeats, 1.0 μg of small RNA was 3′ end-labeled with DIG, and the miRNAs immunodetected after hybridization on the murine MMChip (Fig. 1).

MicroRNA expression profiles were analyzed by pairwise comparisons (T

Discussion

The role of miRNA expression in aging mammals was explored in murine liver, as a paradigm for the study of aging-related processes. The mouse lifespan is generally 24 months; those that reach the age of 33 months are extremely old. We observed a significant age-related increase in miRNA expression in mouse liver. Although no change in miRNA expression was found in aging mouse lung (Williams et al., 2007), our results are in agreement with reports on the increase of miRNA expression associated

Conflict of interest

None.

Acknowledgments

The authors gratefully acknowledge the contributions of Leszek Wojakiewicz, and Alan N. Bloch for proofreading this paper. This work was supported by a Research-enhancement funding support from the School of Medicine of the University of Louisville to EW.

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