MicroRNAs in the Evaluation and Potential Treatment of Liver Diseases

Acute and chronic liver disease continue to result in significant morbidity and mortality of patients, along with increasing burden on their families, society and the health care system. This in part is due to increased incidence of liver disease associated factors such as metabolic syndrome; improved survival of patients with chronic predisposing conditions such as HIV; as well as advances in the field of transplantation and associated care leading to improved survival. The fact that one disease can result in different manifestations and outcomes highlights the need for improved understanding of not just genetic phenomenon predisposing to a condition, but additionally the role of epigenetic and environmental factors leading to the phenotype of the disease. It is not surprising that providers continue to face daily challenges pertaining to diagnostic accuracy, prognostication of disease severity, progression, and response to therapies. A number of these challenges can be addressed by incorporating a personalized approach of management to the current paradigm of care. Recent advances in the fields of molecular biology and genetics have paved the way to more accurate, individualized and precise approach to caring for liver disease. The study of microRNAs and their role in both healthy and diseased livers is one example of such advances. As these small, non-coding RNAs work on fine-tuning of cellular activities and organ function in a dynamic and precise fashion, they provide us a golden opportunity to advance the field of hepatology. The study of microRNAs in liver disease promises tremendous improvement in hepatology and is likely to lay the foundation towards a personalized approach in liver disease.


The Liver in Precision Medicine
The liver's enormous regenerative capacity in the face of acute and chronic injury has been recognized and studied for decades, both in humans and animal models. It is the only organ capable of regeneration and healing in a complex fashion that allows it to return to baseline functional capacity including, regulation of metabolism, synthesis, storage and redistribution of nutrients, detoxification, as well as immune modulation [1]. Whether we are studying the liver's response to external insults, or the normal physiologic repopulation of hepatocytes, the liver provides a unique opportunity to It has been well documented that miRNA expression, and detectable levels vary significantly both in liver tissue and serum depending on the conditions of the liver. The relative abundance of miRNAs is influenced directly by disease stage, dietary changes, and genetic manipulations in experimental models [45,46]. Fortunately, circulating endogenous miRNAs are very stable relative to RNAs in general and, therefore detection of aberrant miRNA profiles are the basis for their It has been well documented that miRNA expression, and detectable levels vary significantly both in liver tissue and serum depending on the conditions of the liver. The relative abundance of miRNAs is influenced directly by disease stage, dietary changes, and genetic manipulations in experimental models [45,46]. Fortunately, circulating endogenous miRNAs are very stable relative to RNAs in general and, therefore detection of aberrant miRNA profiles are the basis for their potential utility as a biomarker reflecting accurate and timely cellular changes ( Figure 2).
It is now well established that miRNAs play critical roles in numerous functions involving both cellular and organs growth, proliferation, differentiating, apoptosis, and metabolism. In humans, best estimates suggest that as much as 60%-70% of all genes are either directly or indirectly regulated by miRNAs. It is no easy task to elucidate the precise mechanisms involved for any one particular disease state. However, significant progress has been made, especially in the areas of hepatic malignancy, and other common liver pathologies such as alcoholic liver disease, NAFLD and chronic viral hepatitis. Table 1 provides a summary of sample studies evaluating numerous miRNA changes with target validation. Prior to an elaborate discussion of specific disease states, it is best to review the role of miRNAs in normal liver function as well as observed changes during natural homeostasis.. miRNA-based biomarkers. The schematic diagram shows the biogenesis of miRNAs and their secretion into body fluids through various mechanisms, which include mirovesicles, protein complexes, lipoproteins, apoptotic bodies, and passive release. The secreted miRNAs can be extracted with small RNA extraction kits and analyzed for the presence of mature miRNAs with different quantitative approaches. The profile of miRNA populations is used to determine the diagnosis and/or prognosis of the patient's clinical disorder. On the basis of diagnostic and prognostic utilities, a clinical decision can be made for the targeted and personalized treatment of the disease. Reprinted with permission from "Circulating MicroRNAs as Biomarkers: A New Frontier in Diagnostics" (Liver Transplant. 2012). It is now well established that miRNAs play critical roles in numerous functions involving both cellular and organs growth, proliferation, differentiating, apoptosis, and metabolism. In humans, best estimates suggest that as much as 60%-70% of all genes are either directly or indirectly regulated by miRNAs. It is no easy task to elucidate the precise mechanisms involved for any one particular disease state. However, significant progress has been made, especially in the areas of hepatic malignancy, and other common liver pathologies such as alcoholic liver disease, NAFLD and chronic viral hepatitis. Table 1 provides a summary of sample studies evaluating numerous miRNA changes with target validation. Prior to an elaborate discussion of specific disease states, it is best to review the role of miRNAs in normal liver function as well as observed changes during natural homeostasis.

MicroRNAs in Normal Liver Development and Maturation
MicroRNAs are some of the key players involved in almost every aspect of liver development and maturation. In hepatic development, the endodermal epithelium of the embryonic foregut will eventually produce hepatoblasts, which are the hepatic progenitor cells. These cells differentiate to give rise to cholangiocytes and hepatocytes. This differentiating process continues from approximately week 6 in the human fetus until birth [18]. Data suggest that some of these progenitor cells will be present through adulthood and form the basis for liver cell replacements during natural liver maintenance activities [47].
Data exist showing specific changes in miRNA levels that correlate with the transition of embryonic stem cells to definitive endoderm, which eventually transition to hepatoblasts in the developing liver. The up-and down-regulation of miRNAs is a time dependent phenomenon and a single miRNA might be up-regulated at a specific stage of development but down-regulated later [48,49]. For example, miR-20b and miR-146a are down-regulated during progenitor cell differentiation into hepatocytes, whereas miR 30a, miR-122, miR-143, and miR-542-5p are increased in their expression levels. In addition, during embryogenesis it is not surprising that a number of miRNAs are increased including, let-7 family, miR-22, miR-99a, miR-122, miR-125b and miR-192, whereas miRNAs 19b, 23a, 92a, 106, 125a and 127 are reduced in levels of expression [49].
Additional studies have shed important light on the interaction of miRNAs and their potential targets as well as their tissue specificity. As an illustration, miR-122 is one of the most abundant and liver specific miRNAs that becomes detectable early in embryogenesis and continues to be highly expressed into adulthood. By using a variety of miR-122 knockdown, knockout, and overexpression mouse models, a number of gene targets for this single miRNA have been identified. As an example, Cutl1 gene is negatively influenced by miR-122 [50,51].
Multiple studies have clearly illustrated the crucial role miRNAs play in development whether by targeting the mature mRNA itself or some of the key players in miRNA maturation in a negative feedback mechanism [52]. Additional studies evaluated differentiation of both human embryonic stem cells and mesenchymal stem cells from umbilical cord into hepatocytes and determined that several miRNAs were either up-regulated (miRNAs 10a, miR-122, and miR-143) or down-regulated (miR 20b, miR-30a and miR-146a) for expression. As the results were not consistent in the studies, it is postulated that the origin of the progenitor cell certainly influences the miRNA profile [48,53].

Hepatic Mass Maintenance
As with any other organ, the liver requires basic mechanisms that allow for replenishment of lost hepatocytes under normal conditions. Furthermore, the liver is the only organ that is capable of regeneration after massive injury. It is becoming more obvious that there is not a single type of cell that is responsible for this process. Rather, a number of potential hepatic progenitor cells have been identified and studied to gain a better insight into the liver's regenerative process. From animal models, it was traditionally believed that oval stem cells contributed directly to liver regeneration, however recent evidence expands liver regeneration beyond these cells [54]. More recent studies suggest that under certain physiological conditions mature hepatocytes are able to trans-differentiate into cholangiocytes [55]. Other studies have highlighted that peri-central hepatocytes replicate at a faster rate than those in the peri-portal region. It addition, it appears that they are the only hepatocyte population expressing genes activated by the Wnt signaling pathway, suggesting an important role for this population of cells in contributing to maintenance of the hepatocyte population [56]. In addition, it has been shown that in response to different injury models, the liver can induce different hepatic micro-compartments to activate potential progenitor cells. These include, but are not limited to junction of the hepatocyte bile canaliculi and the proximal biliary ductules at the canals of Hering; within the intralobular bile ducts; peripheral to the bile ducts; and within the hepatic parenchyma. Each of these subpopulations of hepatic cells contributes towards maintenance of normal hepatic mass depending on the nature of injury and rate of cell death [57].
The general underlying principals of liver repair and regeneration are (i) it is a well orchestrated process with clear starting and terminating points; (ii) it involves a number of metabolic and epigenetics changes; and (iii) wound healing process may be defective and can therefore lead to diseased states such as increased fibrosis. In general, liver injury followed by repair is achieved in the form of regeneration. Injury may take on many forms including surgical resection, acute and stressful events such alcoholic hepatitis, acute viral hepatitis or ischemic injury; as well chronic injury such as chronic viral hepatitis, chronic heavy alcohol consumption and NAFLD. Regeneration is the physiologic response to these stressors and it is a tightly controlled process involving well-synchronized effectors such as cytokines, growths factors, metabolic changes, progenitor cells; as well as precise interaction between the different liver cells including, hepatocytes, Kupffer cells, endothelial cell, macrophages and stellate cells. There are observed metabolic, genetic and epigenetic changes, which are tightly controlled [58][59][60].
Depending on the degree and duration of injury, regeneration may take on the form of either compensatory hypertrophy (increase in size of cells) or regeneration/proliferation where hepatocytes divide and increase in number and volume, and eventually reaching the appropriate volume to weight ratio which somehow signals termination of regeneration [60]. It is important to note the distinction between compensatory hypertrophy and true regeneration. After PH, the liver does not undergo true regeneration, which typically means regrowth of lost lobes, but rather it undergoes compensatory hypertrophy, i.e., growth of the remaining hepatic tissue.
The liver's response to injury is healing and regeneration. Regeneration is a tightly controlled process that involves a number of signals and factors between genetic code and the environment. It has the distinct phases of priming where hepatocytes prepare for division by moving from the G0 phase to G1 phase of the cell cycle; proliferation in which hepatocytes either increase in size alone or increase in size followed by division; and termination where a balance between apoptosis and proliferation is established and regeneration is halted once the specific liver volume to body weight ratio is reached [61][62][63].
In animal models, PH has provided the most data regarding the changes observed of key regulatory players including tumor necrosis factors, IL-6, hepatocyte growth factors, epidermal growth factor and transforming growth factors. Each of these metabolic regulators has a distinct role at different stages in the process of hepatocyte proliferation. For example, activin A and TGF-β are noted to be key factors involved in termination of proliferation of the liver [64][65][66][67][68][69].

Role of microRNAs and Liver Proliferation
A number of studies have documented the importance of miRNA involvement in every step of hepatic proliferation. It has now been established that there is a biphasic pattern of miRNA regulation post-PH. Within the first 3-18 h after PH in the rat, miRNAs are up-regulated followed by a down-regulation of as many as 70% of miRNAs by 24 h. The data suggested a negative feed-back mechanism that is present between miRNAs and their genes that control this process [61,[70][71][72]. In general, miRNAs are noted to accelerate or inhibit liver proliferation.
Examples of miRNAs that are associated with accelerated hepatic proliferation and regeneration include miR-21, miR-221, miR-23b, miR-122, miR-203, and miR-221 Specifically, miR-21 has been consistently observed to change during the early stages of regeneration. miR-21 knockout animal models suggest that miR-21 is responsible for cyclin D1 induction and subsequent G1 phase transition to S phase of hepatocyte cell cycle. Of interest, ursodeoxycholic acid which is a strong inducer of miR-21 appears to up-regulate miR-21 post-PH which may then lead to improved regeneration of the liver [73][74][75][76]. miR-221 is noted to inhibit hepatocytes apoptosis in mouse models, as well as delaying fulminant liver failure. It has long been observed to be up-regulated in hepatocellular carcinoma [77]. Experimental models showed that mice overexpressing miR-221 had rapid entry into the S-phase of cell cycle and up-regulation of cyclins D1, E1, A2, B1. The identified target gene for miR-221 is aryl hydrocarbon receptor nuclear translocator (Arnt) which when inhibited allowed for the promotion of hepatocyte proliferation [78,79]. miR-203 is another notable example, which epigenetically down-regulates the suppressor of cytokine signaling 3 (SOCS3), that is associated with inhibition of IL-6/STAT3 (signal transducer and activator of transcription 3). This in turn leads to promotion of hepatocyte proliferation [80,81].
Examples of miRNAs associated with inhibition of liver proliferation include: miR-26a, miR-33, miR-34a, miR-127, miR-150, and miR-378. This is achieved through a number of mechanisms that have been identified in the delay or inhibition of liver proliferation by miRNAs [76]. miR-26a appears to target cyclin D2 and E2 genes leading to impairment of proper cell cycle propagation and therefore, repression of liver regeneration. A model using adenovirus 5 (Ad5)-anti-miR26a-LUC to down-regulate miR-26a in mouse liver led to enhanced proliferation of hepatocytes and was associated with reduced liver enzymes and bilirubin after PH. The opposite was observed by using Ad5-miR26a-LUC [82]. miR-33, which is viewed as a key transcriptional factor in cholesterol biosynthesis and transportation, overexpression led to G1 cell cycle arrest and reduction of cellular proliferation [83].
miR-34a is involved in the termination phase of liver proliferation, as it is strongly induced during this period, and appears to suppress hepatocytes proliferation via inhibitory effects on potential targets such as cyclin D1, E2, CDK4, and CDK6 [84,85]. miR-127 overexpression was initially observed to suppress the growth of multiple cancers including liver [86,87]. Further studies of mouse models of liver regeneration noted that lower levels of miR-127 were observed in the initial 24 h after PH and that higher levels caused reduction in cell growth rate and cell cycle arrest in G2-M phase [88]. Two identified likely target genes were B-cell lymphoma 6 protein (Bcl6) and SET domain-containing protein 8 (Setd8). Both of these genes play a key role in cell cycle regulation and proliferative potential [89].
miR-150 down-regulation was associated with increased levels in both mRNA and vascular endothelial growth factor A (VEGF) proteins, which are expected to be elevated in the early phase of LR [90]. In humans, it was observed that patients receiving auxiliary liver transplantation had decreased tissue levels of miR-150 obtained from a liver biopsy [91]. Another key player is miR-378 which is observed to have an inverse relationship with ornithine decarboxylase (Odc1), which encodes the enzyme polyamine-synthesizing enzymes required for DNA synthesis. This ultimately led to suppression of liver proliferation [92].
It is important to note that a number of studies highlighting the role of miRNAs in liver proliferation after PH are based on animal models, and that relative abundance does not directly correlate with an anticipated role. Further studies are required to confirm post-hepatectomy changes in miRNA profile in humans and better understand their expression profile(s).

Hepatocellular Carcinoma
It is estimated that hepatocellular carcinoma (HCC) is the 5th most common cancer, and the 3rd cause of cancer-related deaths worldwide, with approximately 80% of HCC occurring in patients with underlying cirrhosis [93]. Certainly, the underlying liver disease as well as the chronicity of the disease, both influence the (i) risk of HCC development; (ii) rate of progression; and (iii) aggressive features of the tumor. However, there is an increasing number of studies identifying a complex network of factors and signals that influence the development and behavior of HCC. This is noted in dysregulation of cellular mechanisms that influence cell survival, fate and genome maintenance; which is a reflection of epigenetic events and cellular environmental changes [94,95]. miRNAs are gaining significant attention for their potential association with HCC, not only for the ability to act as potential biomarkers, but also as predictors of tumor behavior, and response to therapeutic targets for this multi-stage pathologic process. In addition, recent studies are evaluating their predicative role for recurrence after liver transplantation compared to available tools such as Milan criteria, and is currently estimated at 25%.
A number of studies have identified key miRNAs involved in the pathogenesis of HCC, both via down-or up-regulation of miRNA levels. For example, aggressive tumor growth and metastatic potential were found to be associated with lower levels of miR-199/b-3p and miR-122 [96][97][98].
Other studies noted that up-regulation of miR-221 and miR-222 was linked to cancer progression by enhancing cellular proliferation and migration. A number of target genes affected by dysregulated miRNAs have been identified in experimental models and observation studies both in animals and humans including the targeting of β-catenin, fibroblast growth factor receptor-1 (FGF-1), mTOR, P27, PTEN and TIMP3 tumor suppressors [99][100][101][102]. The differential expression of a number of miRNAs has been observed to be independent predictors for HCC recurrence after liver transplantation. These include miR-19a, miR-886-5p, miR-126, miR-223, miR-24 and miR 247 [103].
A recent study evaluated the use of specific miRNAs in combination of the Milan Criteria to develop a score model that may increase the accuracy for prediction of HCC recurrence. The most significant miRNAs were miR-214 and miR-3187 which allowed a cohort of patients to be classified as low and high-risk groups for recurrence [104]. This novel approach incorporated biological features of the tumor into the existing radiologic-based Milan criteria, further improving our ability to predict and manage the care of liver cancer.

NAFLD/NASH
Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are complex conditions that highlight the intricate relationship between the genome, and the environment. There is a worldwide increase in the prevalence of fatty liver disease, which is expected to be the leading cause of end-stage liver disease and need for liver transplantation within the next 1-2 decades. In addition, we must also recognize the parallel increase in all associated complications such as HCC and death [105]. The clinical challenges in fatty liver disease include accurate identification of patients with simple steatosis and low risk of progression to advanced liver disease versus those with progressive features of the disease. In addition, there is an urgent need for effective therapeutics given limitations of current strategies of life style modifications such as weight loss and exercise.
It is well established that a number of changes in miRNA expression profile is seen with NAFLD. For example, miR-34a has been noted to be significantly up-regulated both in animals and humans with features of the metabolic syndrome [106,107]. One of the main targets of miR-34a is NAD-deacetylase Sirtuin-1 (SIRT1). It is developing into a critical player involved in energy homeostasis as an activator of transcription factors Liver-X-receptor and proliferator-activated receptor-α, and an inhibitor of sterol regulatory element-binding protein 1c (SREBP1c), farnesoid X receptor (FXR) and PPAR-γ coactivator-1α [108]. In experimental models where miR-34a was silenced, successful restoration of SIRT1 and PPARα was achieved [109]. A recent study using a mouse model showed that inhibition of the p53 tumor suppressor protein resulted in reduced steatosis and lipotoxicity via a reduced effect of SIRT1 [110]. Other examples include miR-122, which was noted to be significantly down-regulated in NASH, miR-33a and miR-33b which were observed to inhibit genes involved in insulin signaling and fatty acid metabolism [111,112].
Few studies have evaluated the use of miRNA signatures as a prognostic tool. A panel of miRNAs was discovered to be approximately twice as high in healthy individuals compared to those with fatty liver disease, and they include miR -122, miR-192, miR-19a, miR-19b, miR-125, and miR-375. In this proof of concept study, miR-122 performed better than cytokeratin-18 and ALT in predicting presence and degree of fibrosis [113].
Ultimately, gaining a better understanding for the role of miRNA role in fatty liver disease would allow better-targeted therapies, whether against specific miRNAs or their target genes and mRNAs. A few studies have used antisense oligonucleotides to target mature miRNAs to reduce their biologic effect, as well as the use of viral vectors to deliver miRNA erasers, which would bind miRNA targets competitively but without producing the effect [114][115][116]. Challenges in this area include the complexity of miRNA interactions with multiple genes and targets, the optimal delivery method, and establishing the duration of therapy that is required for an effect.

Chronic Viral Hepatitis
Both chronic hepatitis B and C infections (HBV, HCV) have been associated with specific changes in miRNA expression profiles [117]. For example, a handful of miRNAs were noted to be down-regulated in patients with either chronic HBV or HCV infection compared to healthy controls including, miR-26a, miR-29c, miR-219 and miR-320. Furthermore, miR-122 levels showed direct correlation with HCV replication and stability. In fact, HCV RNA was reduced when miR-122 was inactivated, and increased with elevated levels of miR-122 in patients with active infection compared to healthy individuals [118,119]. Interestingly, the opposite effect was observed for miR-122 and chronic HBV infection, where observational studies noted an inverse relationship between the level of miR-122 and HBV viral DNA level. The effect appears to be mediated by inhibiting p53 mediated suppression of HBV enhancer element [120].
Of note, HCV genotype 3 has been associated with hepatic fatty accumulation, which was demonstrated by the induction of miR-27a as it targets genes involved in lipid metabolism such as transcription factor RXRα and the lipid transporter ATP-binding cassette subfamily A member 1 (ABCA1) [121].

Acute Liver Failure
Acute liver failure (ALF) is typically associated with massive loss of tissue, and compromise in function without evidence of underlying chronic liver disease. Given the rarity of the condition and high level of mortality without liver transplantation, there are few mechanisms known to be involved in recovery from ALF and associated hepatic proliferation. However, a recent study investigated the differential expression of miRNAs associated with liver regeneration, specifically miR-122, miR-21 and mi-221 from both serum and hepatic tissue. It was shown that those patients who had spontaneous recovery from ALF had significantly higher serum levels of all three of the aforementioned miRNAs compared to those who did not recover and either died or required liver transplantation. However, the hepatic tissue analysis showed that only miR-122 was significantly elevated in those who recovered, but not miR-221, or miR-21 [122].

Autoimmune Hepatitis
A recent study evaluated the expression profile of miRNAs in autoimmune hepatitis (AIH). Patients with confirmed AIH underwent microarray analysis to determine levels of miRNAs compared to healthy controls. miR-21 and miR-122 were elevated by more than 1.7 fold in the serum of untreated AIH patients; and their levels were down regulated by treatment with corticosteroids. However, there was a reduction in their levels once cirrhosis was present, and the relationship was inversely correlated with advancement of fibrosis stage [123].

In-born Errors of Metabolism and MicroRNAs
Alpha-1-antitrypsin (α1-AT) is a protein produced only by hepatocytes; and mutation of the coding gene may result in different genotypic and phenotypic manifestations, specifically MM and ZZ genotypes. α1-AT deficiency is manifest by decreased levels of circulating α1 protein leading to varying degrees of hepatic and/or pulmonary disease. A recent study identified different expression profiles of miRNAs in α1-AT deficiency patients with pulmonary disease. Specifically, miR-199-15-p was the most up-regulated miRNA in the monocytes of asymptomatic patients with the genotype ZZ compared to those with the MM genotype. Symptomatic patients with both genotypes were noted to have hypermethylation of miR-199a-2 promoter region, resulting in the inhibition of miR-199a-5p expression [124]. This highlights the potential use of miRNAs as not just markers of the disease but prognostic tools of the expected clinical course.
Wilson's disease (WD) is a disorder of copper metabolism that is inherited in an autosomal recessive manner resulting from mutation of the ATP7B gene leading to cooper overload. Evidence from animal models support the role of miR-122 in predicting outcomes of severe liver injury related to copper overload. Specifically, elevated levels of miR-122 followed over time appeared to more accurately predict worsening prognosis of fulminant liver failure compared to liver enzymes, AST, ALT and bilirubin [125]. This again, highlights the importance of miRNA and their prognostic utility both in terms of sensitivity to overall, liver health, and early predictor of outcome.
Crigler-Najjar Syndrome Type I (CN-1) is the result of a single point mutation in the uridine 5'-diphosphate-glucuronosyltransferase 1A1 (UGT1A1) gene catalyzing liver glucuronidation of hydrophobic bilirubin into a hydrophilic isoform for excretion into bile. Mutation of the enzyme results in the accumulation of unconjugated bilirubin with catastrophic consequences including permanent mental and motor dysfunction from hyperbilirubinemia.
The animal model of CN-1 disease is the Gunn rat, which has been extensively studied for both metabolic pathways, and treatment protocols. Numerous types of gene therapy have been used to correct the hyperbilirubinemia in the rat model. Most recently, miRNAs have facilitated a better understanding and management of the condition. In one study, by including miR-142 target sequences in a lentiviral vector, the immune system response was significantly circumvented by reducing the vector expression in antigen-presenting cells. This led to normalization of hyperbilirubinemia in Gunn rats compared to those without the miR-142 sequence, highlighting the ability of miRNAs to facilitate personalized medicine [126].

Concluding Remarks
The recent emergence of microRNAs as key players influencing almost every aspect of liver biology represents a milestone in our understanding and appreciation of liver health and disease. Specific changes in the expression profile of miRNAs are a reflection of the complex and delicate interaction between the genome and the environment, both globally and at the cellular level. Therefore, they offer a unique insight into the liver's capacity and ability to face stressful events acutely and chronically. They are valuable tools as markers of disease presence, stage and response to therapy, to name just a few. Their signature profiles can be established at all stages of hepatic development from embryonic to full term; during differentiation of progenitor cells; maintenance under normal physiologic conditions; and repair/regeneration under pathologic conditions including chronic viral disease, NASH/NAFLD, and in primary liver cancers. miRNAs are dynamic regulators, differentially expressed during periods of up-and down-regulation, as well as responsive to positive and negative feedback mechanisms that provide a precise reflection of cellular activities ( Figure 3).
Certainly, the application of miRNAs to personalized medicine is not without some limitations at this time. As stated, there is still more to learn and discover about miRNAs themselves as well as their interaction with each other and their target genes. It is absolutely critical to establish standardized techniques for reproducible and accurate measurements from any source. Key to our ability to translate the field of miRNAs to clinical practice is the need for more human studies in additional to the development of more accurate animal models for specific diseases. miRNAs hold a key to the future of personalized medicine, which aims to achieve specific goals of improved assessment of disease stage and activity; accurate prediction of disease course; and better anticipation of complications. Eventually, this will lead to an individualized management plan with reduction in adverse effects, reduction in cost by avoiding unnecessary tests and therapies, and improved adherence and patient satisfaction. The future of miRNAs in the practice of medicine is both exciting and personalized.  . Partial illustration highlighting the complex network of interaction between microRNAs and their target genes including negative feedback mechanisms. Reprinted with permission from "Using Expression Profiling Data to Identify Human MicroRNA Targets" using GenMiR ++ data analysis algorithm (Nat. Methods 2007).

Conflicts of Interest:
The authors declare no conflicts of interest.