Vertebrate animals display remarkable ability to tolerate high altitudes and compensate for the combined effects of cold and concomitant decreases in O₂ supply that potentially constrain aerobic metabolism [1]. The yak (Bos grunniens) is regarded as a typical seasonal grazing ruminant inhabiting in remote mountain regions of altitudes ranging from 3,000 to 5,500 m throughout the Hindu Kush-Himalayan and the Qinghai-Tibetan Plateau (QTP). More than 14 million domestic yaks provide the local Tibetan pastoralists with necessary resources and financial income, such as meat, milk, transportation, dung for fuel and hides for tented accommodation [2]. The alpine highlands where the yak is found characterised by severe climate at high altitudes and with grazing resources restricted by very short growing seasons. As the forage supply decreases sharply in winter, the herds generally suffer from malnutrition for almost 8 months of the year, and the resulting energy deficiency.

In mammals, the liver integrates nutrients uptake and circulating carbohydrates and lipids to peripheral tissues for the function of maintaining overall organism energy balance [3]. Low production, reduced fertility and immune related are known to be effected by energy deficiency in yaks [4]. During and prolonged starvation in the whole winter grazing seasons, an enhanced rate of gluconeogenesis that utilizes nonglycosidic intermediates such as lactate and pyruvate to produce glucose [5], in addition an increased hepatic fatty acid oxidation [6] are needed to provide the yak with insulation from cold as well as an energy reserve. Our previous study explored the effects of metabolites and environmental factors on lipid deposition and metabolism in liver and adipose tissues of yaks, provided the first example that the regulation of lipoprotein lipase involves some factors in addition to insulin and triglycerides for yak to better adapt to the harsh environment [7]. With the liver being the central metabolic organ in the body and is therefore assumed to be relatively vulnerable to hypoxemia [8]. Little is known, however, about the combined effects of cold exposure and nutrients stress on hepatic functional adaptations in this animal that native to high altitude. We hypothesized that hepatocytes maintain metabolic homeostasis by coordinating gene expression programs in response to dietary and systemic signals for the yaks.

It is well known that microRNAs (miRNAs) are a class of endogenous non-coding small RNAs of ~22 nucleotides in length found in most eukaryotes, which function as transcriptional and post-transcriptional regulators of gene expression [9]. They are potent modulators of diverse biological processes and pathologies comprising 1% to 5% of mammalian genes, including those liver-specific miRNAs involved in metabolic adaptation and energy homeostasis [10]. The function of miRNAs in energy metabolism was first studied in Drosophlia melanogaster, and revealed that miR-14 play a critical role in regulation of triacyglyceride metabolism on the whole-animal level [11]. miRNAs also safeguard insulin expression and secretion, thereby contributing to regulate adipocyte differentiation and maintenance of glucose homeostasis [12]. The liver-specific miRNA miR-122 is reported to be involved in the regulation of lipid metabolism and plays a prominent role in cholesterol and fatty acid metabolism [13]. Alterations of fatty acid metabolism-related mRNA levels for genes such as ACACA, ACACB, ACLY, FASN, LIPC, SCD1 were proposed as candidate biomarker for metabolic disease in mice even in cattle for anabolic steroid abuse screening [11]. Recently the importance of miRNA-directed gene regulation are coming into focus as more miRNAs and their targets were detected in the liver of dairy cow in negative energy balance [14]. In the case of beef cows, both liver specific and ubiquitously expressed miRNAs have been also reported [15]. In response to hypoxia, hypoxia inducible factor-1 alpha (HIF-1α) has recently been reported to induce transcription of miR-210 contributing to mitochondrial respiration and, thus, modulate a fundamental shift in cellular metabolism [16]. Together, these findings suggested a strong connection between miRNAs and energy metabolism. However, whether miRNAs play a role in the energy deficiency of yak at high altitude remains to be elucidated.

To address this question, we employed next-generation sequencing to characterize the potential roles of miRNAs in liver of yak under nutrition stress. The regulatory networks underlying interactions of miRNAs with their targeted genes and biological pathways were inferred to reveal their regulatory functions in energy metabolism.

Natural selection, imposed by hypoxia and cold, can have profound effects on energy generation and expenditure strategies in animals that native to high altitude. One such vertebrate species, Himalayan yak (Bos grunniens) separated from cattle (Bos taurus) approximately to 5 million years ago has successfully adapted by displaying a circannual rhythm to better adapt to the harsh environment of high altitude (~3,500 to 5,500 m) [19]. To date, hepatic miRNAs have been identified as essential mediators of energy metabolism through their function in modulation of glucose and lipid homeostasis, but a clear understanding of their role in the regulation of cellular metabolism in yak under harsh environment remains elusive. To dissect the role of hepatic miRNA in the regulation of energy deficiency in yak, the animals grazed in winter highlands selected as the model for exposed to the combined effects of nutrition stress and cold at high altitude. From using this model, we provide a comprehensive hepatic miRNA expression profiles via a deep sequencing approach. We found in total 369 known miRNAs (miRBase 19.0) and 387 pre-miRNAs producing 287 mature miRNAs. GO and KEGG pathway analyses show that those miRNAs may participate in many different metabolic-related processes. An analysis of the top 20 miRNAs showed that 11 of them may be involved in many regulatory aspects of the PI3K-Akt network. To the best of our knowledge, this is the first liver even yak miRNA-seq profiling study of moderate yak adaptation on the QTP.

In this study, objective preliminary analysis of the cDNA library has shown that 22-nt size class in liver of yak is the major type of sRNA, which is consistent with the majority of sRNA-lengths in bovine [20], and other ruminant species [21]. The mature miRNAs also showed a similar trend, which consistent with the typical size range of small RNA generated by Dicer cleavage. The total rRNA is regarded as a sign for the sample quality, and should be less than 60% in plant and nearly to 40% in animal tissues [21]. In the present study the total rRNA was 12.33%, indicating that the liver samples used were of a high quality.

The most dominant among the ten highly abundant miRNAs in our study, miR-122 is a liver-specific conserved miRNA. This dominance of expression by miR-122 is consistent with previous studies in dairy [19] and beef cows [22] where miR-122 was reported to be only expressed in liver, when compared with other tissues, and constituted more than 57% of all the miRNA reported in liver [22]. A recent study to indentify putative targets of differentially expressed miRNAs among differentially expressed hepatic genes in dairy cows in negative energy balance has revealed that tissue specific miR-122 and liver enriched miR-192 are two of the most abundant miRNAs [6]. MiR-122 reaches approximately 70% of the total miRNA population in the liver, and notably, its expression is sharply up-regulated in both mouse and human liver during embryonic development [23]. Historically, miR-122 is the first miRNA identified to regulate liver homeostasis and lipid metabolism and plays a prominent role in cholesterol accumulation and fatty acid metabolism [24]. Microarray analysis revealed reduced hepatic expression of a range of genes involved in the regulation of lipid biosynthesis such as acetyl-CoA carboxylase β (ACC2), stearoyl-CoA desaturase (SCD1), and adenosine triphosphate (ATP) citrate lyase (ACLY). Interestingly, most of the identified genes seem to be indirect targets of miR-122 because they lack seed sequences for miR-122, providing an explanation for their observed down-regulation in response to miRNA inhibition whereas the opposite would be expected for direct miRNA targets [25], suggests that miR-122 may negatively regulate a transcriptional repressor.

The next two most abundant circulating miRNA in our panel was let-7 family including let-7f and let-7a-5p. All let-7 family members are believed to exert similar functions because they share a common seed region (nucleotides 2 to 8), which mediates miRNA interaction with target mRNAs. The let-7 family members have been associated with hepatic development and disorders as well as glucose and insulin metabolism [26]. miR-192, which is also enriched in the liver in the present study and plays an important role in cellular responses to glucose stimulus, but is also expressed in kidney and gastrointestinal tract. miR-140, which is also among the highly expressed miRNAs in this study, has been reported to be abundantly expressed in human and mouse liver with implications in liver function and disorders [27]. Furthermore, miR-103 were previously predicted by bioinformatics to affect multiple mRNA targets in pathways that involve cellular acetyl-CoA and lipid levels [28], which were significantly upregulated in ob/ob mice, suggesting the potential role of in the pathogenesis of nonalcoholic fatty liver disease and confirm central importance to glucose homeostasis and insulin sensitivity, was only detected at a moderate level in yak in our study.

The hepatic expression of numerous genes including those involved in glycolysis, lipid metabolism, and carbohydrate metabolism may be altered in response to acute and chronic exposure to high altitude [29]. PI3-kinases were the first mammalian lipid kinases discovered and since then multiple mammalian PI3-kinases have been identified. They are responsible for coordinating a diverse range of cell functions including cell growth, proliferation, survival, metabolism and glucose homeostasis [30]. Recent studies suggest that PI3K is also regulated by hormones and by nutrients such as glucose and fatty acids. Furthermore, activation of the P13K-Akt signaling pathways may be one mechanism by which cells adapt and survive under conditions of hypoxia [31]. In the present study, some miRNAs were predicted to target genes such as AKT2, EIF4E2, JAK1, MAPK3, MTOR, PDPK1 involved in processes of the PI3K-Akt network, which play an important role in regulating glucose homoeostasis or lipid metabolism.

To better understand the functions of the identified miRNAs in lipid metabolism and adipogenesis, putative targets of the highly expressed top 20 miRNAs were use for PI3K-Akt network analysis. Glycogen synthase 1 (GYS1), as a predicted target gene of miR-122 regulation, which involved in cholesterol, fatty acid, and lipid metabolism [11]. Although its essential role as an energy reserve is common to all cells, there are differences in glycogen function between tissues. Liver glycogen contributes primarily to blood glucose homeostasis, being synthesized during periods of nutritional sufficiency, and subsequently converted to glucose, which is released into the bloodstream to counteract hypoglycaemia [32]. When oxygen becomes limiting, cells reduce mitochondrial respiration and increase ATP production through anaerobic fermentation of glucose. The HIFs play a key role in this metabolic shift by regulating the transcription of key enzymes of glucose metabolism. GYS1 gene induction correlated with a significant increase in glycogen synthase activity and glycogen accumulation in cells exposed to hypoxia [33]. Since fatty acid oxidation is limited and glycogen supplies glucose for anaerobic glycolysis, glucose assumes a more important role as a fuel for the heart in yaks. Significantly, knockdown of either HIF1a or GYS1 attenuated hypoxia-induced glycogen accumulation, while GYS1 overexpression was sufficient to mimic this effect, indicate that GYS1 regulation by HIF plays a central role in the hypoxic accumulation of glycogen [33].

The most of let-7 family was predict of target AKT2, primarily through glucose uptake in response to insulin through the translocation of glucose transporter type 4 (GLUT4) to the plasma membrane. Activation by AKT can also affect glucose by increasing the conversion of glucose to glucose-6-phosphate. This can either be stored through conversion back to glucose by glucogen synthase, or it can be catabolized to produce energy through glycolysis. The kinase AKT inhibits GSK3β from (in turn) inhibiting the glucogen synthase kinase activity, thereby stimulating the glucogen synthesis. However, AKT may also stimulate glycolysis through HIF-α. The lipid metabolism is also regulated by AKT through the inhibition of GSK3β which thereby promote the expression of genes involved in the cholesterol and fatty acid biosynthesis [34]. Thrombospondin1 (THBS1) was also the predicted target of let-7 family and it is noteworthy of differentially-expressed genes related to fatty acids metabolism in liver, which was previously reported as associated with the profile of intramuscular fatty acid composition in a genome-wide association study (GWAS) and RNA-Seq in pigs [35]. Therefore, they can be considered as interesting candidate genes and this suggest their role in the fatty acid metabolism processes in liver [35]. PDK1 (3-phosphoinositide-dependent protein kinase-1), were identified as possible targets of three miRNAs of let-7 family in our study, which plays an important role in regulating glucose homoeostasis and controlling expression of insulin-regulated genes, and a deficiency of the PDK1 pathway in the liver could contribute to development of diabetes, as well as to liver failure. Let-7a, let-7c, miR-181b, miR-185, miR-378 and miR-423-5p were predicted to target the evolutionarily conserved mechanistic Target of mTOR (Rapamycin), is a member of the PI3K related kinase (PIKK) family, functions as a molecular sensor of metabolism and cellular homeostasis and integrates environmental signals by altering the cellular metabolic processes [36]. More recent work indicates that mTOR Complex 1 (mTORC1) plays a significant role in protein synthesi, lipid biosynthesis, and inhibition of triacylglycerol lipolysis [37]. Thus it may have a significant impact on maintaining metabolic homeostasis in the whole body. mTORC1 enhances the translation of HIF-1α, a transcription factor, that in turn regulates the transcription of genes encoding glycolytic enzymes and glucose transporters [38]. As a result, mTORC1 promotes glucose uptake and activation of glycolysis to generate energy. The ability to control the rates of metabolic processes in response to changes in the internal or external environment is indispensable for all living cells. Mechanisms that are essential for metabolic control and maintenance of homeostasis are complex and involve transcriptional, translational, posttranslational, and allosteric regulation. Our results indicate that some miRNAs add a new level of regulation and fine tuning for gene expression that is likely to be important for a wide range of cellular functions, including signaling and metabolic control. However, their regulatory mechanisms warrant furher study.

In conclusion, the present study revealed 369 known and 41 novel miRNA candidates, most of which were predicted to be involved in the regulation of lipid as well as glucose homeostasis via the PI3K-Akt interaction network for the yak under deficiency energy. These findings contribute to an increased understanding of the roles of miRNAs in liver and to building the foundation for understanding their physiological functions and regulatory mechanisms for yak to better adapt to the harsh environment.