Sex and age differences in the expression of liver microRNAs during the life span of F344 rats

Joshua C. Kwekel, Vikrant Vijay, Tao Han, Carrie L. Moland, Varsha G. Desai, James C. Fuscoe

Physiological factors such as age and sex have been shown to be risk factors for adverse effects in the liver, including liver diseases and drug-induced liver injury. Previously, we have reported age- and sex-related significant differences in hepatic basal gene expression in rats during the life span that may be related to susceptibility to such adverse effects. However, the underlying mechanisms of the gene expression changes were not fully understood. In recent years, increasing evidence for epigenetic mechanisms of gene regulation has fueled interest in the role of microRNAs (miRNAs) in toxicogenomics and biomarker discovery. We therefore proposed that significant age and sex differences exist in baseline liver miRNA expression, and that comprehensive profiling of miRNAs will provide insights into the epigenetic regulation of gene expression in rat liver. To address this, liver tissues from male and female F344 rats were examined at 2, 5, 6, 8, 15, 21, 52, 78, and 104 weeks of age for the expression of 677 unique miRNAs. Following data processing, predictive pathway analysis was performed on selected miRNAs that exhibited prominent age and/or sex differences in expression. Of the 314 miRNAs found to be expressed, 214 were differentially expressed; 65 and 212 miRNAs showed significant (false discovery rate (FDR) <5% and ≥1.5-fold change) sex- and age-related differences in expression, respectively. Thirty-eight miRNAs showed 2-week-specific expression, of which 31 miRNAs were found to be encoded within the Dlk1-Dio3 cluster located on chromosome 6. This cluster has been associated with tissue proliferation and differentiation, and liver energy homeostasis in postnatal development. Predictive pathway analysis linked sex-biased miRNA expression with sexually dimorphic molecular functions and toxicological functions that may reflect sex differences in hepatic physiology and disease. The expression of miRNAs (miR-18a, miR-99a, and miR-203, miR-451) was also found to associate with specific sexually dimorphic hepatic histopathology. The expression of miRNAs involved in regulating cell death, cell proliferation, and cell cycle was found to change as the rats matured from adult to old age. Overall, significant age- and sex-related differences in liver miRNA expression were identified and linked to histopathological findings and predicted functional pathways that may underlie susceptibilities to liver toxicity and disease.