It has been well known that lifestyle, such as caloric restriction, exercise and high-fat diet, has significant influence on health. Although there are many studies that have attempted to clarify the molecular processes, it has not been fully understand the underlying common or unique mechanisms. Therefore, identifying the underlying mechanism is crucial to determine new targets, personalize treatment methods and bring a promise of translatability to human health. In the present study, this is the first report that compares the microRNAs profile in livers from these three lifestyle modification mice models. In addition, we also predicted the potential functions of DE microRNAs by GO and KEGG analysis. With this knowledge, our findings provide us with an overall vision of microRNAs in the molecular impact of lifestyle on health as well as useful clues for future and thorough research of the role of microRNAs.
The different energy intake and consumption status of lifestyle modifications were presented in our prepared mice models as reported previously[19, 20]. CR and endurance exercise are known robust lifestyle modifications that delay the onset of type 2 diabetes and metabolic syndrome. While high-fat diet induces obesity that leads to these diseases. The three lifestyle modification models guaranteed the miRNAs profiling results.
Many researchers commonly used microarrays to screen DE microRNAs in various pathophysiological processes [21]. Although more and more studies applied next-generation sequencing (NGS) to perform a comprehensive analysis of microRNA expression profile, it has been demonstrated that NGS and microarray measurements give similar results [22]. In addition, in this study, 4 ~ 6 DE microRNAs identified by microarray in each lifestyle treatment were verified via RT-qPCR. These results confirmed the reliability of our data and provided a credible base for further study.
Some findings have indicated that microRNAs are involved in the cellular and molecular mechanisms of lifestyle modifications [14–16]. However, most of the microRNA profiling studies of exercise focus on circulating microRNAs or microRNAs in skeletal muscle and heart [9, 14, 16]. Although there are some studies on microRNA profiling in liver of HF or CR[23–27], however, there is little comprehensive knowledge regarding the similarities and differences of microRNAs profile in liver between these beneficial and detrimental lifestyles. Therefore, we examined the overall microRNAs expression in the liver of mice subjected to CR, EX and HF. In general, about half microRNAs were detectable in liver and the responses of microRNAs to these lifestyle modifications were relatively mild. On one hand, only a small portion were responded to lifestyle modifications; on the other hand, most of the DE microRNAs changed within a small range. Different from these results, in some diseases or physiological process, such as Parkinson's Disease [28], fetal development [29], hepatocellular carcinoma[30], ischemia/reperfusion-induced acute kidney injury[31] and hepatitis C virus infection [32],there are more than one hundred DE microRNAs or the ranges of DE microRNAs change can be up to tens or more than one hundred folds. Among the three lifestyles, CR had the mildest impact on microRNAs, DE microRNAs in EX changed to the biggest range. Based on this result, to get the beneficial effects to health, maybe CR is a gentler choice. On the other side, most of the changes by the beneficial lifestyles were up-regulation, while the number of down- and up-regulated microRNAs by the detrimental lifestyle HF were about equal. More down-regulated microRNAs imply more up-regulated mRNA. It’s a possible way that HF disturbs homeostasis. In addition, our results showed some common DE microRNAs between different lifestyle modifications. For example, we found that miR-34a-5p was activated by HF and inhibited by CR. It has been reported that miR-34a was aberrantly elevated by HF and functionally involved into hepatic lipid metabolism [25, 33, 34]. In the brain of CR mice, there is a decreased expression of mmu-miR-34a [35]. Another example is mmu-miR-200b-5p, which was up-regulated by both CR and EX in the present study. Consistent with our findings, mmu-miR-200b-5p was also elevated in salivary post-running [36]. These data are essential and provide the ground work for a more thorough and comprehensive analysis of potential microRNAs involved in the effects on health by lifestyles.
To predict the potential functions of the DE microRNAs identified in present study, GO and KEGG analyses were performed on the predicted targets. The ontology covers three distinct aspects of gene function: molecular function (the activity of a gene product at the molecular level), cellular component (the location of a gene product's activity relative to biological structures), and biological process (a larger biological program in which a gene's molecular function is utilized) [17, 37]. KEGG is for understanding high-level functions and utilities of the biological system from molecular-level information [38, 39]. The GO and KEGG analysis showed that targets of the DE microRNAs in these lifestyle modifications were enriched in some common main functions, biochemical and signal transduction pathways, such as oxidation-reduction process and oxidoreductase activity, metabolic process and metabolic pathways, fatty acid metabolic process and fatty acid degradation, PPAR signaling pathway, etc. These gene functions and pathways of the targets, were also shown in some previous studies about lifestyle modifications. For example, exercise exerted profound changes in metabolism-associated genes, genes encoding proteins involved in oxidation, fatty acid transporter and fatty acid synthase [40]. PPARδ has also been observed in response to exercise [41]. The changes of the pathways, such as lipid metabolism, fatty acid degradation and metabolic pathways, have also been reported in CR[42, 43]. Studies have reported that HF has an important impact on the lipid metabolism process in rat liver [44]. The intake of a high-fat diet forces the body to maintain physiological balance by inhibiting fatty acid synthesis, promoting fatty acid oxidation, and accelerating fatty acid degradation. PPAR signaling pathway is also change significantly in rat liver after HF [45]. Besides, we also presented that targets of DE microRNAs in different lifestyle modification were also enriched in some different functions or pathways. For an instance, we found that CR altered expression of microRNAs implicated in regulating fatty acid oxidation, which is consistent with previous reports [42, 46]. Interestingly, we validated the expression change of a predicted target of mmu-miR-802-5p and mmu-miR-96-5p, Elovl2, in CR. Elovl2 is related with fatty acyl-CoA biosynthesis[47],while GO-MF term fatty-acyl-CoA binding was enriched in CR functions. Lamp2, another predicted target of mmu-miR-802-5p and mmu-miR-96-5p, belongs to autophagy–lysosome system and also reduces in liver of CR mice. A similar down-regulation of Lamp2 was reported by Junya Yamamoto and colleagues in mice livers after fasting [48].
Although there are some limitations in our study, such as a relatively small sample size, we confirmed some of the DE microRNAs and predicted targets with RT-qPCR. Differences of identified DE microRNAs do exist between our study and previous studies [23–27]. One possible reason is the differences of treatment, such as duration, age, diet ingredients, etc.; another possibility is difference between detection methods. Besides, although we observed inverse correlations between several microRNAs and their targets, direct evidences of repression by microRNAs on their targets need to be provided, and these mechanisms would have to be investigated further to gain more insight into these potential miRNA-target relationships. Cell culture experiments with overexpression or knockdown of microRNAs would enable us to elucidate this.