miRs, while currently being exploited to understand disease pathologies, have been less appreciated in the role of functional gene repression during cellular growth and liver development. miRs, specifically let 7 and lin4, control larval development transition in Caenorhabditis elegans, as well as apoptotic pathways and growth in Drosophila[5]. Recent studies have elegantly demonstrated that miRs regulate differentiation of human cells and development of whole organ systems, which shows tissue-specific expression. miR 122 accounts for approximately 70% of the total liver miR population; however, specific roles for this miR outside of disease-associated functions [e.g. hepatitis C virus (HCV) and hepatocellular carcinoma (HCC)] are less clear, especially with regard to normal liver development. Xu et al[6] have shown that miR 122 is strongly upregulated during liver embryonic development with expression correlative to liver enriched transcription factors (LETFs) C/EBPα, HNF1α and HNF3 isoforms, which are all necessary for proper hepatocyte development and function. Additionally, the transcriptional repressor CUTL1, which is known to promote proliferation and suppress differentiation, is gradually repressed by miR 122 during development. In vitro miR 122 knockdown experiments have shown restoration of CUTL1 expression and of downstream target genes, which emphasizes that, during development, miR 122, as an effector molecule of LETFs, regulates a delicate balance between differentiation and proliferation[6]. Quantitative analysis of miRs in fetal vs adult livers has demonstrated that miR expression levels are higher in the fetal period and expression levels during this time are dynamic[5]. Additionally, recent studies have demonstrated that 162 miRs exhibit tissue-specific distribution in mice[7]. Endoderm-derived liver, jejunum and pancreas display differential miR enrichment, with miRs 122, 21, 101b, 107, 192 and 221 among those with the highest reads per million concentrations. Additionally, mapping of transcriptional start sites has demonstrated that divergent sites are used by miRs in endoderm-derived liver tissue as opposed to stem cells. Maternal influences on miR expression have also been identified and may be affected by diet. For example, studies by Zhang et al[8] have shown that, as a result of maternal high-fat diet, expression of miR let 7c is decreased in offspring, along with 23 other miRs including 122 and 194. let 7c was identified by Tzur et al[9] as being highly expressed in adult compared to embryonic liver, and that profibrotic transforming growth factor-β receptor 1 is a target of this miR. The miR 30 family has recently been shown to be crucial for liver development, because it is upregulated during later stages that influence known regulators of hepatic function, including epidermal growth factor receptor. GW182, a crucial component in miR-mediated gene repression, is also a target of this miR family, which indicates a more global role for miRs in earlier stages of epigenetic silencing via the RNA-induced silencing complex[10]. Functional analyses in zebrafish larvae have demonstrated that biliary morphogenesis is under the regulation of miR 30a, as knockdown studies have resulted in defective bile duct excretion[11]. The liver, as a vital multifunctional organ, must maintain proper epigenetic programming to ensure xenobiotic and bile metabolism, and vitamin and glucose storage among a plethora of other tasks, which emphasizes the importance of proper cellular differentiation and development.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic fat accumulation without significant alcohol consumption or other underlying etiology[12]. NAFLD incidence is rising as a result of the increased population of obese individuals worldwide, with severity of the disease ranging from steatosis/steatohepatitis (NASH) to cirrhosis. Although most patients with NAFLD are asymptomatic, clinical manifestations of the disease are well described and include hyperechoic appearance of the liver on ultrasound, dyslipidemia, systemic arterial hypertension, and insulin resistance. Although disease pathogenesis is not completely understood, recently, a role for miRs in NAFLD has been established[13]. Lipid droplet accumulation in hepatocytes as a marker of steatosis is associated with various liver diseases including NAFLD. Recent high-content screening studies have shown that 11 miRs are able to alter lipid droplet formation/retention[14]. Specifically, miR 181d decreased droplets by approximately 60%, which subsequently reduced cellular triglycerides and cholesterol. Peroxisome proliferator-activated receptor (PPAR)α, a major contributor to NAFLD pathogenesis, has recently been identified as a target of miR 10b, which has been shown to regulate steatosis level in L02 cells (steatotic hepatocyte model)[15]. Additionally, post-transcriptional regulation of PPARα by miR 10b is maintained by a single binding site, as demonstrated by mutation analyses. NASH can progress to end-stage cirrhosis in approximately 15% of patients, with the mechanism that underlies disease progression being undefined. Recent studies in humans have demonstrated that miR expression profiles are altered in NASH. Unsurprisingly, miR 122 is significantly underexpressed (63%) in NASH subjects compared to those without the metabolic syndrome[16]. Overexpression of miR 122 in HepG2 cells results in a significant decrease in sterol regulatory element binding proteins (SREBP1-c, SREBP2), FAS and 3-hydroxy-3-methyl-glutaryl-CoA reductase; all of which are key lipogenic genes in human NASH. Lesser known miRs 34a and 146b are significantly overexpressed (99 and 80%, respectively) in NASH. In a smaller sample study (n = 12/group), seven miRs have been found to be differentially expressed, including 132, 150, 197 and 99; the latter two are significantly associated with pericellular fibrosis[17]. It is of particular importance to note that previous studies have shown that miR signatures change in accordance with disease stage and source of hepatic injury (ethanol or high fat diet)[18,19]. These studies emphasize the importance of avoiding generalization when examining miRs as potential biomarkers, because miR expression patterns of end-stage disease may be similar but expression profiles that lead to organ dysfunction appear to be divergent.

Along with NASH, HCV is one of the most common global causes of hepatic fibrosis and cirrhosis. According to the World Health Organization, over 180 million people are infected with the single-stranded RNA virus, with > 70% of that population at risk for developing cirrhosis[20]. Pegylated interferons in conjunction with ribavirin have improved treatment of chronic hepatitis C; however, a large percentage of the infected population remains unresponsive to available regimens, which emphasizes the need to better understand the mechanisms of cellular infection, for development of novel drug therapies. Numerous studies have emphasized a role for miR 122 in several aspects of hepatic function and disease, including HCV replication[21]. Plasma levels of miR 122 are elevated in hepatitis B virus (HBV)- and HCV-infected patients, as well as in models of alcohol and drug-induced liver damage reinforcing a role for miRs as biomarkers[22]. However, recent studies by Ura et al[23] have shown that miR expression patterns are divergent in HBV- and HCV-infected livers. Additionally, elegant studies by Hou et al[24] have demonstrated that miR 196 directly acts on the 3’UTR of Bach1, the transcriptional repressor of heme oxygenase 1, a critical cytoprotective enzyme. This miR also inhibits HCV expression (both genotype 1b and 2a) in human hepatocytes in vitro, which shows clinical promise. Accordingly, parallel miR and mRNA expression profiling studies in genotype-1b-expressing cells have shown numerous anti-correlated miR/mRNA pairs affected by the presence HCV[25]. Among the noted anti-correlated pairs that have been detected in a human cell line that expresses the HCV Con1 replicon, miR 130a/b is correlated with decreased PPARγ expression, which is a known regulator of the quiescent hepatic stellate cell (HSC) profile. Additional miR/mRNA profiling in HCV-infected hepatoma cells has shown a total of 108 differentially expressed miRs, and among this pool, miRs 24, 149, 638 and 1182 are predicted to control HCV entry, replication and viral propagation[26]. Scagnolari et al[27] have examined miR expression in peripheral blood mononuclear cells from healthy and chronic-HCV-infected patients and have found that miRs 1, 30, 128, 196 and 296 are differentially expressed and that treatment with interferon (IFN)α induces all aforementioned miRs. Although limited by small sample size, baseline levels of miRs do not appear to differ significantly between responders and non-responders; however, expression of miRs 128 and 196 appears higher in the responsive population. Additionally, Pedersen et al[28] have demonstrated this same set of miRs, which possess sequence-predicted targets within HCV genomic RNA, and are induced by IFNβ stimulation. The tumor suppressor gene DLC-1 (deleted in liver cancer-1) is also a target of miR repression[29]. Levels of DLC-1 are decreased in HCV-infected cells, which are correlated with the increase in miR 141. Depletion of this miR inhibits viral replication, while addition of exogenous miR 141 increases replication rates, which indicates that miR regulation of tumor suppressor genes can influence HCV infection and play a role in HCC development (for a comprehensive review of miRs in HCC see[30,31]).

Alcoholic liver disease (ALD) is a major healthcare burden worldwide and a leading cause of liver-related mortality. It is well known that ethanol consumption alters methylation status and other epigenetic factors, including histone tail signatures, signal transduction pathways and transcription factor expression. Ethanol metabolism affects chromatin condensation, which results from direct DNA methylation or histone protein alterations (acetylation, methylation or phosphorylation)[32]. Additionally, DNA methylation is connected to direct interference by small RNAs[33]. Therefore, it is plausible that, as a result of epigenetic remodeling, ethanol exposure can also alter miR expression. Ethanol has been shown to repress miRs 9, 21, 153 and 335 in neural stem cells and neural progenitor cells that alter cellular development and maturation[34]. Additionally, miR 21 influences PTEN/PI3K signaling, which ultimately contributes to the fibrotic response, and both miRs 9 and 335 are predicted (by TargetScan) to bind to targets that influence the progression of ALD[35]. Previous studies have highlighted the importance of miRs in the gut-liver axis and have shown that ethanol exposure alters miR profiles in liver cells[36]. Specifically, endotoxemia and direct ethanol exposure modulate hepatic miRs 182, 183, 705, 1224 and 199a-3p[37]. Symptomatic of ALD, hepatic microcirculation is disrupted when increased portal pressure arises as a result of altered endothelin-1 (ET-1) expression, with subsequent increases in NADPH oxidase and hypoxia inducible factor-1α[38]. Recent studies have shown that miR 199 is involved in ET-1 expression in rat liver sinusoidal endothelial cells and human endothelial cells, by functioning as a negative regulator of ET-1 transcription and thus vascular tone in ALD[39]. For still unknown reasons, only 20%-30% of chronic heavy alcohol users develop hepatic fibrosis. Future studies of miRs involved in ALD may allow us to uncover additional factors that contribute to the disparity that exists in disease development.

Commonalities of miR expression can also be seen among fibrotic disorders of different organ systems, including cardiac and renal fibrosis, with an overarching effect of the miR 29 family in regulation of effector cellular differentiation or in translation of extracellular matrix (ECM) components[35]. Additionally, miRs involved with translation of profibrotic transforming growth factor (TGF)β/SMAD signaling are shared amongst pathologies. Roderburg et al[40] have examined miR expression profiles in a CCl₄ rodent model of hepatic fibrosis, and microarray analyses have revealed 31 differentially expressed miRs; 10 of which are overexpressed in fibrotic tissue, including miRs 125-p, 199b, 221 and 302c. Marked downregulation has been observed in a pool of 21 miRs; specifically, miR 29 family members were significantly reduced. Additional experiments in an alternate model of fibrosis (bile duct ligation; BDL) have confirmed that miRs 29b/c are significantly reduced compared to sham controls. Downregulation of miRs 29a/b/c could also be observed in explanted liver samples with patients denoted by a Desmet fibrosis score of 2-4. Low plasma levels of miR 29a correlate with advanced stage liver fibrosis in human patients. The idea that low miR levels in the tissue correlate with low miR in the circulating plasma is in opposition to recent hypotheses that an inverse correlation between tissue and plasma miR concentrations exists as a byproduct of vesicles that release miRs[41]. Hepatic fibrosis is influenced by several epigenetic factors that control the wound-healing response. Recent studies by Mann et al[42] have shown that miR 132 is significantly decreased in fibrotic livers as demonstrated in two different models (BDL, CCl₄), and this downregulation influences HSC activation. Collectively, these studies have emphasized that, during development of fibrosis, there are important changes in miR expression that regulate wound-healing transcripts; however, effects exerted by miRs appear to act in concert with other epigenetic factors to direct disease progression.