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Circulating microRNAs: Emerging Biomarkers of Liver Disease Marco Arrese, MD1

Akiko Eguchi, PhD2

Ariel E. Feldstein, MD2

1 Department of Gastroenterology, Escuela de Medicina, Pontificia

Universidad Católica de Chile, Santiago, Chile 2 Department of Pediatric Gastroenterology, Rady Children’s Hospital, University of California San Diego, California

Address for correspondence Ariel E. Feldstein, MD, Department of Pediatrics, University of California San Diego, 9500 Gilman Drive, MC 0715, La Jolla, CA 92037-0715 (e-mail: [email protected]).

Abstract

Keywords

► ► ► ► ► ► ► ►

liver disease biomarkers microRNAs liver injury clinical applications extracellular vesicles exosomes microparticles

Development of reliable, noninvasive biomarkers that allow for diagnosis, risk stratification, and monitoring of disease changes over time or in response to specific therapies represents a key priority in the field of hepatology. Recent evidence has uncovered the role of microRNAs as potential ideal biomarkers of liver injury in various acute and chronic liver diseases. These small noncoding RNAs are released to the extracellular space in a stress-specific manner and are remarkably stable in most bodily fluids including blood, where they circulate in specific compartments including extracellular vesicles and protein complexes. In this review, the authors provide a concise overview of available information on the emerging role of quantitation of circulating microRNAs in different liver diseases and their use as biomarkers for both diagnosis and prognosis assessment. Additionally, several key issues that still need to be addressed for microRNAs to become useful tools in daily clinical practice are critically reviewed.

The recognition that microRNAs (miRNAs), naturally occurring small RNA molecules that play an important role in the regulation of gene expression, are released into the extracellular space and are remarkably stable in various bodily fluids has sparked interest for their potential as disease biomarkers.1–4 Indeed, miRNAs have several characteristics that make them a potentially ideal diagnostic tool, including tissue and stress-dependent expression and the availability of relatively simple and sensitive methods for detection, such as quantitative polymerase chain reaction (PCR).4 Due to the significant limitations of currently available noninvasive tools to diagnose and monitor liver damage in various forms of liver disease, and growing evidence for a prominent role of specific miRNA in liver pathobiology,5 miRNA profiling, particularly in blood, represents a rapidly growing area of research interest and holds promise for the development of reliable mechanistic biomarkers for monitoring liver damage.6,7 In this review, we focus on current evidence for the utility of circulating

Issue Theme miRNA in Liver Pathobiology, Diagnosis, and Therapy; Guest Editor, Gregory J. Gores, MD

miRNAs as biomarkers for various forms of acute and chronic liver disorders.

Circulating MicroRNAs as Biomarkers Physiology, Pathophysiology, and Tissue Specificity Various miRNAs have been identified to be important regulators of liver development during embryogenesis, as well as being involved in liver physiology and pathophysiology in the adult liver.5,8–10 In fact, most of the hepatic functions, including glucose, lipid, iron, and drug metabolism are under the tight regulatory influence of miRNAs.11,12 Release miRNAs into the extracellular space and then into circulation may serve physiological purposes (i.e., cell-to-cell communication) or may represent liver cell damage in the setting of liver injury or inflammation. In addition, circulating miRNAs may originate from any of the resident parenchymal or nonparenchymal cells as well as from incoming inflammatory cells. Also, the existence of different

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DOI http://dx.doi.org/ 10.1055/s-0034-1397348. ISSN 0272-8087.

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Semin Liver Dis 2015;35:43–54.

MicroRNAs as Biomarkers of Liver Disease

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export systems that selectively export miRNAs and protect them from degradation has been demonstrated.13 In fact, although cell-free RNAs would be predicted to be rapidly degraded in a ribonuclease- (RNase-) rich environment like blood,14 the stability of circulating miRNAs is remarkable, suggesting that these small RNA molecules are resistant to the action of RNase. The mechanisms underlying this resistance have been the focus of intense investigation and relates to the various forms in which miRNA circulates in blood.3,4 Indeed, miRNAs may circulate in blood encapsulated in extracellular vesicles (EVs) or in a nonmembrane-bound form associated with specific proteins such as argonaute 2 (Ago2) and lipoproteins, which shield miRNAs from degradation (►Fig. 1).15–17 Three types of EVs have been found in blood18:

Fig. 1 (A) Mechanisms responsible for the export and stability of miRNAs in circulation. Pre-miRNA (precursor microRNA) is processed by Drosha, a nuclear RNase III enzyme, and is exported to the cytoplasm. There the premiRNA is turned to miRNA by Dicer. miRNA is unwound to single RNA and loaded into the RISC complex. The RISC complex mainly binds to 3′UTR of target genes that can lead to translational repression. Different export systems exist that result in selective release of miRNAs and protect them from degradation. (B) miRNAs may circulate in blood encapsulated in extracellular vesicles (EVs), or in a non-membrane bound form associated with specific proteins such as Argonaute2 (Ago2), and lipoproteins, which shield miRNAs from degradation. Three types of EVs have been found in blood, including exosomes, microparticles (MPs), and apoptotic bodies. All three types of EVs can carry miRNAs in the circulation. On the other hand, non- EV-associated miRNAs circulate in a ribonucleoprotein complex, in particular with Ago2, or associated to lipoproteins enriched in apolipoprotein A-I, the main protein component of high-density lipoprotein (HDL). Seminars in Liver Disease

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(1) exosomes, which are small, 50 to 100 nm in diameter, and are released by exocytosis as a result of fusion of multivesicular bodies with the plasma membrane; (2) microparticles (MPs) that are between 100 to 1000 nm in size and are generated through cell membrane shedding in a process that involves a regulated sorting of membrane proteins into the shed MP and flipping of phosphatidylserine from the inner to the outer membrane during cellular activation or early apoptosis; and (3) apoptotic bodies that are larger than 1000 nm and are formed during the execution phase of the apoptotic process, when the cell’s cytoskeleton breaks up and causes the membrane to bulge outward, separating from the cell and taking a portion of cytoplasm with them.19,20 All three types of EVs can carry miRNAs in the circulation. On the other hand, non-EVassociated miRNAs circulate in a ribonucleoprotein complex, in particular with Ago2, the effector component of the miRNAinduced silencing complex that directly binds miRNA and mediates mRNA repression in cells,21 or associated to lipoproteins (8–12 nm) particles that are enriched in apolipoprotein A-I, the main protein component of high-density lipoprotein (HDL).22 Whereas in healthy individuals, miRNA appears to predominantly circulate in the nonmembranebound form associated with Ago2 protein,23 the proportion of the various forms of miRNAs in pathophysiologic states remains incompletely understood as the existence of EVassociated and Ago2-associated miRNAs may reflect different release mechanisms or be related to the cell type of origin, which may vary in health and disease. Recent data highlight the potential importance of different miRNA forms in disease states. Of note, in a study with direct implications to liver biomarkers, Pirola et al24 demonstrated that as opposed to healthy individuals in whom miR-122, a liver-specific miRNA (see below), is present in the circulation only in an Ago2 complex fraction, in patients with nonalcoholic fatty liver disease (NAFLD) the majority of serum miR-122 circulates in Ago2-free forms. The specific compartment was not further assessed in this study. In this context, using experimental models of NAFLD, we have found that circulating miR-122 is enriched in the vesicular fraction in plasma,25 the levels are dynamic, increasing over time and correlating with histological features of disease severity (unpublished data). Further support for the importance of compartmentation was provided by work from Bala et al.7 These authors elegantly showed that the enrichment of miR-122 as well as other miRNAs in one particular compartment in the circulation is dependent on the type of liver injury, being predominantly in exosomes in models of alcoholic liver disease (ALD) and in exosome-free fractions in acetaminophen- (APAP-) induced liver injury. Future studies addressing the importance of changes in both concentration and compartmentalization of circulating miRNAs and the potential occurrence of differential packaging of circulating miRNA in apoptotic bodies, shedding vesicles or exosomes in different forms of liver diseases may have significant impact on the development of miRNA as biomarkers. With regard to liver tissue specificity of circulating miRNAs, particular attention has been focused on miR-122, as it accounts for approximately 70% of the total liver miRNA population, and plays a critical role in liver homeostasis and

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MicroRNAs as Biomarkers of Liver Disease

Circulating MicroRNA: Current Challenges for Quantitation A relevant issue that poses potential problems for the use of miRNAs as biomarkers relates to the lack of standardization in the procedures involved in sample collection, storage, RNA isolation methods, and assessment of the quality and quantity of miRNA species in samples, which makes comparison of published studies difficult. Furthermore, as differential enrichment of miRNA in various compartments in circulation described above may become an important step in biomarker development, handling and collection procedures to reliably separate the various fractions such as miRNA-Ago or miRNA vesicles (exosomes or microparticles) will be needed. For instance, it is a common procedure to isolate exosomes via a commercially available kit using either size filtration or precipitation against exosome surface markers, such as CD9, CD63, and CD81. However, the exosome-free fraction contains a variety of other extracellular vesicles, such as microparticles and apoptotic bodies, as well as miRNA-Ago.7 Furthermore, depending on the RNase treatment, miRNA can be favorably obtained from either miRNAAgo or vesicle fraction. Treating with RNase inhibitor will support the stability of miRNA-Ago complex outside of vesicles, whereas treating samples with RNase A will favor miRNA-vesicle fraction because they are protected from the enzyme by the phospholipid layer.29,30 Currently, the most frequently used assays for quantitation of miRNA in bodily fluids include microarrays, quantitative real-time PCR, and next-generation sequencing.31 Unlike the analysis using cells or tissues, choosing the right setting of internal controls for normalization of circulating miRNA is challenging; it often relies on the identification of miRNAs that are minimally perturbed and most stable across all samples tested in one particular setting. However, miRNAs, especially encapsulated miRNA in the vesicular fractions, can be significantly altered in certain disease status. A proposed alternative is the use of synthetic miRNAs, such as Caenorhabditis elegans miRNA oligonucleotides, which are spiked into the samples coming from the same volume of plasma or serum when miRNA extraction is processed. In the future, the establishment of guidelines for miRNA isolation, handling, quantitation, and normalization procedures will be an important step toward the use of miRNA measurements in blood and other bodily fluids as disease biomarkers.

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Biomarkers of Liver Injury: Current Status and MicroRNA Advantages An ideal hepatic biomarker should be able to detect liver damage with high sensitivity and specificity as well as adequately assess the degree or severity of hepatic injury (i.e., correlation with histological damage) and the restoration of normal liver function.32,33 Ideally, a liver injury biomarker should also be able to predict the ultimate outcome of injury, that is to define whether a patient will recover or worsen, ultimately leading in some cases to liver failure. Serum levels of alanine aminotransferase (ALT) fulfill some of these criteria and are widely used as a sensitive biomarker for the detection of liver injury in both a clinical and preclinical setting, although limitations and gaps regarding their use have been increasingly recognized.34–36 Concerns regarding specificity exist, particularly due to problems in differentiating hepatic and extrahepatic causes (i.e., skeletal muscle disorders) of elevated ALT serum levels. In addition, serum levels of ALT do not always truly reflect liver injury and do not measure liver function. In the context of acute liver injury, changes in serum ALT are not able to forecast the occurrence of idiosyncratic (i.e., drug-induced) injury, and are a poor predictor of outcome.37,38 Similarly, it has become clear that serum ALT measurements have significant limitations in the diagnosis and staging of various chronic liver disorders. For example, in patients with NAFLD the entire spectrum of disease severity can be seen in individuals with normal ALT levels.35,36 Circulating miRNAs, particularly those liver-enriched (see below), exhibit great potential to address some of the gaps and limitations described for the more conventional markers of liver injury6 in some settings they can even outperform serum ALT levels as sensitive, specific, and predictive hepatic biomarkers. In fact, some circulating miRNAs fulfill many of the criteria required for a good hepatic biomarker (►Table 1). Although several issues need to be addressed before routinely using miRNAs as a reliable “liver test” able to provide increased sensitivity and enhanced specificity in early detection of liver injury,2,37 data assessing the potential utility of quantitation of miRNA in various bodily fluids and in particular blood as useful indicators of liver injury or even hepatocarcinogenesis are rapidly growing. In the sections below, we summarize the available information on the emerging role of measuring circulating miRNAs in different liver diseases (►Table 2) and the use of miRNAs as biomarkers for both diagnosis and prognosis assessment.

MicroRNA as Biomarkers for Specific Liver Diseases Drug-Induced Liver Injury Although drug-induced liver injury (DILI) is a relatively uncommon cause of acute liver injury, it remains a leading cause of acute liver failure (ALF) in the United States and many other parts of the world.39,40 Over the last two decades, significant advances have been made in the understanding of the mechanisms involved in DILI induced by intrinsic Seminars in Liver Disease

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pathophysiology as an important regulator of lipid metabolism, hepatic circadian regulation, hepatocyte differentiation, and hepatocarcinogenesis.26,27 An early study by Wang and colleagues suggested that this miRNA could be useful to assess liver injury by quantitation of its levels in blood.28 Using an experimental model of liver injury due to APAP in mice, they showed dose- and time-dependent changes in the blood levels of miRNA-122 as well as miRNA-192 (another liver-enriched miRNA) that paralleled the degree of liver damage. The changes in plasma levels were detected early and were more sensitive than measuring liver enzymes.28 Interestingly, the increase in plasma levels of miRNA-122 was accompanied by a decrease in the levels of this miRNA in liver tissue.

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Table 1 The attributes of circulating miRNAs as liver biomarkers Feature

Requirement

Data/promises and shortcomings

Specific

Tissue-specific

miR-122 and miR-192 expression are both highly abundant and specific to the liver.26,27

Sensitive

Can differentiate between pathological conditions Levels should change in an early and significant manner during different stages of injury

Preclinical studies suggest various miRNAs can be highly sensitive increasing at early stages before any changes in serum ALT levels.28,43

Predictive

Able to determine prognosis (i.e., disease progression, therapy response)

Serum levels of miR-122 and miR-1 are related to survival in patients with cirrhosis and hepatocellular carcinoma88,100 and correlates with histological disease severity in hepatitis C-infected patients.61

Robust

Easily identified and quantified using modern techniques. Stability in regular storage conditions Detectability in tissue samples (even in formalin-fixed, paraffin-embedded samples)

Technical issues related to sample collection, storage, RNA isolation methods, and assessment of the quality and quantity of miRNA in samples need to be addressed. Also, normalization of miRNA data using selected reference RNAs is a critical issue to assess clinical significance.29–31

Translatable

Data can be used to bridge pre-clinical and clinical gaps Detection should be feasible in a clinical setting

Advancing Estimating miRNA assays cost remain difficult since to date they have been used primarily for research purposes.37

Noninvasive

Present in circulation and/or other biofluids.

miRNAs are present and highly stable in most bodily fluids.3,4

Abbreviations: ALT, alanine aminotransferase; miRNA, microRNA.

hepatotoxins such as APAP, while the interplay between genetic and environmental factors as well as immunologic and metabolic factors on the development and severity of DILI in particular in the cases of idiosyncratic DILI remains incompletely understood.41,42 This is in part attributable to the lack of reliable biomarkers that aid in early detection and risk stratification of patients with DILI. Therefore, the development of accurate noninvasive biomarkers is still needed for dissecting pathophysiological mechanisms and for clinical decision making. Early studies in animal models identified miR-122 and miR-192, as well as miR-193, as potential biomarkers for APAP toxicity, showing that their levels increase early during APAP exposure and correlate with severity of liver damage showing better sensitivity than serum ALT measurement.28,43 In addition to miR-122, inflammatory miRNAs, such as miR-155, -146a, and -125b were also shown to be increased in circulation in an APAP mouse model.7 This study further demonstrated that during APAP toxicity, circulating miRNAs were enriched in exosome-free compartments in contrast to liver injury associated with ALD where the enrichment occurred mainly on the vesicular compartment. Thus, the results of this study suggested for the first time that analyzing the various miRNA compartments in circulation may aid in differentiation of the etiology of liver injury. Unfortunately, this study did not address the specific compartment that is enriched during APAP toxicity such as Ago2, Seminars in Liver Disease

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lipoproteins, or microparticles. Moreover, the role of compartmentation in other non-APAP forms of DILI remains unknown. Recent studies have examined the role of circulating miRNA determination as biomarkers of DILI in humans.38,44–46 In a small cohort of patients with APAPinduced acute liver injury, both miR-122 and miR-192 were found to be substantially elevated when compared with patients without liver disease.44 The study used U6 small nuclear RNA (snRNA), typically used as a cell/tissue miRNA normalizer, as an internal control for normalization of the circulating miRNA levels. Although a study of a large group of healthy subjects and individuals with various disease conditions showed that U6 may have significant variability in blood samples, this was particularly influenced by age—suggesting that this snRNA is not a reliable internal normalizer for serum miRNA determinations.47 The authors subsequently demonstrated that U6 performed similarly to a set of other internal controls, but suggested that the miRNA let-7d may be a more suitable normalizer in a subgroup of the patients and healthy subjects included in the original study, highlighting the important challenges that are still present for the development of miRNAs as reproducible, clinically useful biomarkers of disease (discussed in detailed in the section above). Both miRNA-122 and miRNA-192 were also elevated when compared with patients with non-APAP acute liver injury.44 In a follow-up study by the same group, measurement of miR-122, as well as other markers of cell

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Table 2 Selected reports in circulating miRNAs in different liver diseases miRNAs

Source

Fraction

Clinical correlation

Experimental

Human

Ref.

miR-122, -192, -193

Plasma

Not assessed

Increased

Yes

No

28 43

miR-122, -155, -146a, -125b

Plasma

Exosome-free

Increased

Yes

No

7

miR-122, -192

Serum/plasma

Not assessed

Increased

No

Yes (n ¼ 53)

44

miR-122

Plasma

Not assessed

Increased

No

Yes (n ¼ 129)

45

miR-122

Plasma

Not assessed

Increased

No

Yes (n ¼ 35)

46

miR-122

Serum

Not assessed

Increased

Yes

No

53

miR-122

Plasma

Microparticle

Increased

Yes

No

25

miR-122, -192

Serum

Ago-free

Increased

No

Yes (n ¼ 65)

24

miR-122, -21, -34a, -451

Serum

Not assessed

Increased

No

Yes (n ¼ 403)

54

miR-432–5p, -578, -155–5p

Serum

Not assessed

Increased (male NAFLD> female NAFLD)

No

Yes (n ¼ 5)

55

miR-432–5p, -301b

Serum

Not assessed

Increased (male NAFLD > male control)

No

Yes (n ¼ 5)

55

miR-122

Serum

Not assessed

Increased levels correlated with serum ALT activity and with necroinflammatory activity.

No

Yes (n ¼ 68)

61

miR-122

Serum

Not assessed

Increased, superior to ALT in discriminating chronic HCV-infected patients with a normal ALT from healthy controls

No

Yes (n ¼ 102)

62

miR-20a, -92a

Serum And plasma

Not assessed

Increased, miR-20a correlated with fibrosis

No

Yes (n ¼ 86)

63

miR-134, -320c, -483–5p

Serum

Not assessed

Increased

No

Yes (n ¼ 36)

60

miR-122, -194

Serum

Not assessed

Increased

No

Yes (n ¼ 63)

69

miR-122, -572, -575 and -638

Serum

Not assessed

Increased

No

Yes (n ¼ 10)

70

Not assessed

Increased

No

Yes (n ¼ 198)

71

NAFLD/NASH

HCV

HBV

miR-22, -122 miR-122

Serum

Not assessed

Increased

No

Yes (n ¼ 89)

72

miR-99a-5p, -122–5p, -122–3p, -125b-5p

Plasma

Not assessed

Increased plasma levels inversely correlated

No

Yes (n ¼ 42)

73

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DILI

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Table 2 (Continued) miRNAs

Source

Fraction

Clinical correlation

Experimental

Human

Ref.

with immunological control of HBV in children HCC miR-122

Serum

Not assessed

Increased but unable to discriminate between HBV infection with and without HCC

No

Yes (n ¼ 58, HBV-related HCC)

80

miR-16 and -199a

Serum

Not assessed

Decreased, positive HCC predictions of HCC in patients with negative results of conventional markers.

No

Yes (n ¼ 105)

81

miR-143, -215

Serum

Not assessed

Increased

No

Yes (n ¼ 95)

82

miR-15b, -130b

Serum

Not assessed

Increased, combined use provides high sensitivity and accuracy for HCC detection

No

Yes (n ¼ 57)

83

miR-25, -375, -let-7f

Serum

Not assessed

Increased, miR-375 alone had high sensitivity and specificity

No

Yes (n ¼ 120)

84

miR-122, -192, -21, -223 -26a, -27a, -801

Plasma

Not assessed

Increased

No

Yes (n ¼ 457)

85

miR-21

Serum

exosomes

Increased, exosomal miR-21 provided increased sensitivity of detection than serum miR-21

No

Yes (n ¼ 30)

87

microRNA-1 and microRNA-122

Serum

Not assessed

Higher miR-1 and miR122 serum levels showed longer overall survival

No

Yes (n ¼ 195)

88

miR-122

Serum

Not assessed

Increased

No

No

93

miR-190, -743b

Serum

Not assessed

Decreased

No

No

92

miR-505–3p, -197–3p

Serum

Not assessed

Decreased

No

Yes (n ¼ 10 PBC patients)

94

miR-299–5p

PMC cells

N/A

Increased

No

Yes (n ¼ 58 PBC patients)

96

miR-200b/429 cluster

Serum

Not assessed

Increased

Yes

Yes (n ¼ 24 biliary atresia patients)

97

Cholestasis

Abbreviations: DILI, drug-induced liver injury; HBV, hepatitis B virus; HCV, hepatitis C virus; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis.

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death, significantly outperformed ALT, international normalized ratio, and plasma APAP concentration for the prediction of APAP-induced liver injury.45 Finally, in a recent study, Ward et al found a set of 11 miRNAs that discriminate APAPrelated liver injury from ischemic hepatitis and interestingly were sensitive to n-acetyl-cysteine treatment.48 Future studies by independent groups to validate these results, as well as to assess the utility of miRNA determination in non-APAP forms of acute liver injury, are needed. Finally, further research is needed to assess whether determination of an individual miRNA versus a panel of miRNAs in isolation or combined with other clinical or laboratory data, as it has been shown in the context of hepatocellular carcinoma (HCC), is the best approach for their use as biomarkers

Nonalcohol-Related Fatty Liver Disease Nonalcohol-related fatty liver disease (NAFLD) has become the most common form of chronic liver disease in both children and adults affecting up to 30% of the American population. Nonalcohol-related fatty liver disease is tightly associated with obesity and encompasses a wide spectrum of conditions associated with the overaccumulation of fat in the liver, ranging from hepatic steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. Nonalcoholic steatohepatitis is a serious condition, with approximately 5% to 25% of patients progressing to fibrosis and cirrhosis with its associated complications of portal hypertension, liver failure, and HCC.49 It is now the most rapidly growing indicator for liver transplantation in patients with HCC.50 Liver biopsy, an invasive procedure associated with possibly significant complications, presently remains the only reliable method to differentiate hepatic steatosis from NASH and to establish the severity of liver injury and fibrosis.51 Thus, the development of reliable and accurate noninvasive tools to diagnose and monitor patients over time has become an area of intense investigation. Specifically, over the next 5 to 10 years there will be an exponential growth in the number of clinical trials to identify effective therapies targeted mainly to those patients with NASH, as well as those with NASH and fibrosis.52 The development of adequate biomarkers that can be used as main outcome measurements, as well as companion diagnostics, is crucial to the success of these programs. The potential role of various miRNAs in the pathogenesis of NAFLD/NASH and as biomarkers of disease severity in NAFLD/ NASH has been recently explored. Using a time course experiment with the methionine and choline deficient (MCD) dietary model of NASH, Clarke et al demonstrated that determination of serum levels of miRNA-122 is more sensitive than conventional measurements of serum liver enzymes for assessment of early changes of hepatic steatosis, but was not sufficient to differentiate the various stages of the disease with the levels reaching a plateau as early as 3 days after initiation of the diet.53 Studies from our group demonstrated that miR-122 is enriched in circulating microparticles in mice on the MCD diet25; however, while more recently using the choline-deficient amino aciddefined (CDAA) model of NASH, we found that as opposed to the findings using serum, the levels of miRNA-122 in the vesicular compartment are dynamic, increasing over time, and correlating

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with histological features of disease severity (unpublished data), suggesting that quantification of this miRNA content in EVs may be the method of choice in the context of NASH. Supporting this concept in its application to human NASH, Pirola et al recently demonstrated that in NAFLD patients miR-122 was enriched in the Ago-free fraction in circulation; quantification allowed for discrimination between those with hepatic steatosis and those with NASH.24 In addition to miR-122, other miRNAs that have been shown to be increased in circulation of NAFLD patients include miR-21, miR-34a, and miR-45154 however, in this study the diagnosis of NAFLD was based on liver ultrasound determination, thus the utility of these miRNAs to distinguish between hepatic steatosis and NASH could not be assessed. A more recent study uncovered a gender difference in blood levels of various miRNAs; the levels were higher in male versus female patients with NAFLD. Male NAFLD patients had significantly upregulated expressions of the miR-432–5p, miR-578, and miR-155–5p compared with female NAFLD patients. The miR-432–5p and miR-301b levels were significantly higher in NAFLD male patients compared with male controls. In contrast, female NAFLD patients did not have significantly different miRNA levels compared with female controls.55 These data, however, were based upon five patients with NAFLD. Although intriguing, these differences require exploration in larger, appropriately powered studies. In the future, larger, well-designed studies including the full spectrum of NAFLD, assessing the relevance of the specific compartments of miRNA circulating in blood, and comparing their performance with routinely used tools to noninvasively diagnose and stage NAFLD will provide the data still needed to support the use of miRNA determination as clinically useful biomarkers for NAFLD.56

Chronic Viral Hepatitis Hepatitis C virus (HCV) infection influences the expression of a significant number of hepatic miRNAs.57,58 Although the biological consequences of viral-related aberrant miRNA expression are not known in detail, current evidence suggests that this may influence the viral life cycle and regulate critical molecular pathways in the infected cell facilitating viral hijacking of host cellular machinery.58,59 Different studies have shown that HCV-associated changes in hepatic miRNA expression may have proviral and antiviral roles, with the latter being amenable as therapeutic targets. Studies assessing circulating miRNAs as biomarkers of disease severity or progression in HCV-infected patients are scarce.60 Viral infection in the liver implies the interaction of various cell types; hence, the precise cellular source of the different circulating miRNAs in HCV-infected sera has not been well delineated. The same holds true for the function of these extracellular circulating miRNAs, which may represent the mere release of miRNAs into circulation or active secretion from either resident or inflammatory cells in a cell-specific manner. Published studies have shown that the liver-enriched miR-122 can be a sensitive although not specific, marker of liver disease in HCV-infected patients.61 Of note, the expression of miR-122 in serum correlates with serum ALT levels and with the degree of histological necroinflammatory activity in patients with chronic HCV suggesting that this miRNA can Seminars in Liver Disease

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potentially outperform ALT as a serum biomarker of liver disease in HCV-infected patients.57,62 Another recent study found that circulating miR-20a and miR-92a are elevated specifically in HCV-related liver diseases and that miR-20a correlated with liver disease progression.63 Also, Swetha et al, using miRNA profiling, found that the expression of miR-134, miR-320c, and miR-483–5p are significantly upregulated in serum from HCV infection.60 Finally, there have been attempts to assess miRNA expression in the urine of HCVinfected patients64; however, the studies show limited diagnostic performance. In the case of hepatitis B virus (HBV), viral infection modulates the expression of several host cellular miRNAs that may contribute to a favorable environment for viral replication either by directly influencing viral gene expression or by contributing to viral immune evasion.65,66 Details on the role of miRNAs in these processes are beyond the scope of the present review and are summarized elsewhere.67 Although information on the role of circulating miRNAs as biomarkers of viral infection is limited because several reports show that differentially expressed miRNAs in HBV infection are associated with different disease stages including viral-related hepatocarcinogenesis,65 serum or plasma levels of miRNA species may indeed reflect the disease status and the ongoing liver injury in the setting of HBV infection. Moreover, it has been shown that circulating HBV surface antigen particles carry selective pools of miRNAs either originated from infected hepatocytes or from immunoregulatory cells.68 These data indeed provide the basis for using miRNAs in the diagnosis of HBV infection and HBV- HCC. In one of the first reports analyzing circulating miRNAs in the serum of HBV-infected patients, Ji et al69 found that the number of miRNA species detected increases along with the severity of the liver disease with miR-122 and miR-194 being consistently associated to chronic infection. Also, Zhang et al70 profiled the serum miRNA expression in patients with HBV infection, patients with NASH, and controls and found that miR-122, miR-572, miR-575, miR-638, and miR-744 serum levels were sensitive and specific to diagnose chronic hepatitis B. Arataki et al71 recently reported their results with198 patients and found that miRNA-22 and miR-122 were significantly elevated in HBV-infected patients and that miR-22 was higher than miR-122 and correlated better with ALT levels. In the West, Waidmann et al72 found that serum miR-122 levels correlated with the level of ALT, HBV viral load, and serum levels of HBV surface antigen, and that miR-122 serum concentration was able to discriminate between HBV carrier patients with high or low risk for disease progression. Also, Winther et al73 recently reported data on plasma levels of a miRNA panel in children with chronic hepatitis B in different immunological phases of the disease. Interestingly, miRNA plasma levels were found to be higher in immune-tolerant children than in immuneactive children. Because children were followed over several years, the authors found that plasma levels of selected miRNAs (miR-99a-5p, miR-122–5p, miR-122–3p, and miR125b-5p) decrease over time, although the significance of this decline is unknown. This report, although with some Seminars in Liver Disease

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limitations, also found that miR-122 may be a good and sensitive marker of liver injury in HBV infection. In summary, current data suggest that measuring circulating miRNA in serum or plasma of HBV-infected patients might be of help for the diagnosis and prognosis of HBV-related diseases. Therefore, miR-122 could be a promising biomarker miRNA to be used for both estimation of ongoing liver injury and patient risk stratification.

Hepatocellular Carcinoma The incidence of HCC is increasing: Now this cancer is the sixth most common malignancy worldwide.74 Unless successful curative therapy (ablation, surgery, or transplantation) is performed prognosis remains dismal with expected overall 5-year survival rate of 5% to 9% from the time of clinical diagnosis of HCC. A major challenge in the field of HCC is the fact that many tumors are detected in a late stage due to lack of adherence to surveillance protocols by clinicians as well as for the lack of appropriate biomarkers.75 In fact, despite remarkable advances in the field, no new biomarkers have been developed for clinical use; those available such as α-fetoprotein (AFP) or des-gammacarboxyprothrombin (DCP) are still of debatable usefulness.76 Because miRNAs have been shown to be involved in both pathogenesis and progression of HCC,77,78 they indeed may serve as potentially useful diagnostic tools allowing early diagnosis, as well as accurate staging and prognostication. A myriad of studies assessing miRNA expression in HCC has been performed. Initially, miRNA expression was examined mainly in neoplastic tissue and compared with adjacent nonneoplastic liver tissue. In these studies, more than 200 differentially expressed miRNAs have been identified as down- or upregulated in HCC in recent studies (reviewed in ref.78). However, considerable variation, including nonreproducible results, has been found among studies. This may be due to technical differences, heterogeneous samples (race, gender, different liver disease stages), and distinct disease background (cirrhosis associated to HBV or HCV infection, steatohepatitis, or others) that alters miRNAs expression. Among the miRNAs that are aberrantly upregulated, miR-21, miR-221/-222/-224, and miR181 are the more frequently reported, whereas miR-29, miR122, miR-145, miR-101, miR-200, miR-148b, and miR-199a/b are more commonly downregulated.79 Although this information is relevant to study disease pathogenesis and progression and may help to perform a molecular classification of HCC or to identify markers of HCC prognosis after resection or transplantation, its diagnostic usefulness is limited. This is due to the fact that the majority of HCC cases are diagnosed without a liver biopsy and therapeutic decisions are adopted mainly on radiological grounds.74 Thus, assessment of circulating serum miRNAs as biomarkers for early HCC diagnosis has become the focus of more recent studies because this strategy could have more impact in clinical practice. In recent years, several studies have been published studying different miRNAs in serum or plasma of HCC patients, but data exhibit considerable variability with regard to diagnostic accuracy. Although some studies have focused on specific candidate miRNAs, other groups have used array techniques aiming to identify “panels” of miRNAs suitable for clinical use in the setting of chronic liver

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performance as a screening test in a large cohort of the target population for HCC screening will prove test utility.

Cholestasis MicroRNA expression is dysregulated in cholestasis both in experimental models and in human cholestatic liver disease such as primary biliary cirrhosis (PBC).89 Moreover, altered miRNA expression may have a functional role in disease development because key cellular processes for the hepatocyte such as bile acid homeostasis as well as cellular events related to liver cell injury (i.e., apoptosis, endoplasmic reticulum stress, and fibrosis) are modulated by miRNAs in the liver.90,91 Reports on tissue expression of different miRNAs, either in rodents with cholestasis or patients with cholestatic liver disease, are increasingly appearing in the literature (for a recent summary see ref. 89) Nevertheless, available data on circulating miRNAs and cholestasis are scarce. As in other models of liver injury, the liverenriched miR-122 is increased in the serum of animals with bile duct ligation; serum miR-190 and miR-743b were specifically downregulated in both obstructive and hepatocellular cholestasis rat models.92,93 In a recent human study in PBC patients, analysis of circulating miRNAs by Illumina deep small-RNA sequencing showed that expression of miR-505–3p and 197–3p was reduced in PBC patients compared with healthy controls and patients with viral hepatitis.94 Additional studies have examined the miRNA expression profile in peripheral mononuclear cells from PBC patients. Quin et al identified 17 miRNAs to be differentially expressed in peripheral mononuclear cells from four PBC patients compared with healthy controls providing informative data on certain molecular pathways at play in PBC.95 However, the lack of controls with other liver diseases does not allow assigning PBC-specificity to the observed changes. In addition, Katsushima et al found that miR-299–5p is increased in peripheral mononuclear cells from patients in whom therapy with ursodeoxycholic acid has failed,96 suggesting that measuring miR-299–5p could be useful for predicting response to bile acid therapy. Unfortunately, the above-mentioned studies involve a relatively small number of patients, and overlap with autoimmune liver disease limits the interpretation of data. Thus, the potential usefulness of miRNAs as biomarkers for the diagnosis and/or monitoring of cholestatic liver diseases including prediction of response to ursodeoxycholic acid needs to be demonstrated in further prospective studies with appropriate controls. Lastly, serum microRNAs of the miR-200b/429 cluster have been used as a diagnostic tool to differentiate biliary atresia from other forms of neonatal hyperbilirubinemia with good sensitivity and specificity.97 Indeed, early detection of biliary atresia is relevant as early intervention would improve patient outcome. However, published data are scant and studies are ongoing.98

Other Conditions Circulating microRNAs have also been explored as markers in miscellaneous liver diseases such as acute hepatitis,99 acute liver failure,46 cirrhosis,100,101 and liver transplantation102 however, available data are limited and more studies are needed to assess their utility as diagnostic and prognostic tools for severe liver injury in these settings. Seminars in Liver Disease

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disease. Thus, Qi et al demonstrated that miR-122 is increased in the serum of HCC patients compared with healthy controls, but did not discriminate between HBV patients with HCC or those without HCC.80 On the other hand, Qu et al showed the usefulness of serum levels of 3 miRNAs (miR-16, miR-195, and miR-199a) either alone or in combination with conventional serum markers (AFP and DCP) to differentiate HCC from chronic liver disease.81 The latter authors found that miR-16 had the highest sensitivity for HCC. Additionally, the value of serum miR-143 and miR-215 for HCC was recently studied by Zhang et al82 in 95 patients and found acceptable diagnostic power with areas under the receiver operating characteristic (ROC) curve of 0.795 and 0.816, respectively. Serum miR-15b and miR-130b levels have been also found to be upregulated in HCC and the combined use of serum levels of both miRNAs provided high sensitivity and accuracy for HCC detection in one study.83 Interestingly, serum levels of these miRNAs were markedly reduced after surgery indicating their tumoral origin. In trying to find a miRNA diagnostic panel, Li et al found that 13 miRNAs were differentially expressed in serum of HBV-positive patients and showed that they allowed to discriminate HBV-associated HCC cases from controls and also differentiated HBV-positive HCC cases from non-HCC HBV cases.84 In this study, three miRNAs (miR-25, miR-375, and let-7f) were found to distinguish HCC cases from controls with miR-375 being the most powerful HCC predictor. In the same line, Zhou et al studied HBV-related HCC in plasma from 934 individuals and were able to identify a miRNA panel able to diagnose early-stage HBVassociated HCC that could also differentiate HCC from chronic HBV hepatitis, cirrhosis, and healthy individuals.85 Of note, the miRNAs found to be useful for HCC diagnosis in this study (miR122, miR-192, miR-21, miR-223, miR-26a, miR-27a, and miR801) were different to those found by Li et al,84 likely due to methodological issues. A recent comprehensive systematic review of published literature analyzing 30 studies including 1,314 patients with HCC and 1,407 controls found that measuring multiple miRNAs was more accurate than determining individual miRNAs.86 Results were similar for Asian and nonAsian individuals, and serum-based miRNA assays exhibited higher sensitivity compared with plasma-based miRNA assays. Finally, two recent studies deserve mention. In a report by Wang et al,87 the expression of miR-21 was assessed in serum exosomes isolated from sera obtained from HCC patients. miR21 was found to be enriched in this fraction, was higher in HCC patients compared with patients with chronic HBV or healthy volunteers, and correlated with tumor stage and cirrhosis. Lastly, Koberle et al88 found that serum miRNA-1 has prognostic value in patients with HCC as serum levels of this microRNA were independently associated with overall survival in a singlecenter case control study. Altogether, data (summarized above, see also ►Table 2) show that circulating miRNAs may indeed serve as biomarkers for HCC diagnosis, but further validation with appropriately powered studies is needed before clinical application occurs. In particular, appropriate controls (matched by age, sex, race, as well as by etiology and severity of the underlying liver disease) should be used when assessing the specificity of miRNAs for detecting HCC. Ultimately, prospective assessment of assay

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Conclusions Over the last decade we have witnessed significant progress in the field of hepatology with regards to molecular mechanisms of disease pathogenesis as well as novel therapies to treat various acute and chronic liver conditions. However, a liver biopsy, an invasive procedure prone to sampling errors and observer variability, has remained the gold standard for diagnosis, risk-stratification, and monitoring disease progression/regression over time. Development of reliable, clinically useful noninvasive biomarkers is a key priority for this field. Circulating miRNAs have emerged as attractive biomarker candidates because they fulfill many of the key characteristics of an ideal biomarker: They are very stable in various bodily fluids, the expression of some miRNAs is tissue- and diseasespecific, and their levels can be easily measured using relatively simple and sensitive assays such as quantitative PCR. Indeed, as we reviewed in the sections above, changes in blood levels of various miRNAs in patients with several liver diseases have been recently characterized, but definitive miRNA signatures for liver diseases remain to be defined. Although quantitation of circulating miRNAs is a promising approach, several key issues still need to be addressed, including the confirmation of the clinical associations of miRNA’s presence in larger and independent studies, a better understanding of the importance of the various miRNA circulating forms during liver injury of various etiologies, and the development of protocols for adequate sample preparation, quantitation, and normalization techniques. These studies may pave the way to the development of a new generation of reliable, mechanism-based disease biomarkers.

Abbreviations

Acknowledgments This work was partially funded by grants from the Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT 1110455 to M.A.) and the Comisión Nacional de Investigación Científica y Tecnológica (grant PFB 12/ 2007, Basal Centre for Excellence in Science and Technology, M.A.)—both from the Government of Chile—and the Gilead Research Scholar Award (AE) and R01 DK082451 (AEF).

References 1 De Guire V, Robitaille R, Tétreault N, et al. Circulating miRNAs as

2

3

4

5 6

7

8

AFP Ago ALD ALF ALT APAP CDAA DCP DILI EVs HBV HCC HCV HDL MCD miRNA and miR MPs NAFLD NASH PBC RNase ROC snRNA TLR

alpha-fetoprotein argonaute alcoholic liver disease acute liver failure alanine aminotransferase acetaminophen choline deficient amino acid-defined des-gamma-carboxyprothrombin drug-induced liver injury extracellular vesicles hepatitis B virus hepatocellular carcinoma hepatitis C virus high-density lipoprotein methionine and choline deficient microRNA microparticles nonalcoholic fatty liver disease nonalcoholic steatohepatitis primary biliary cirrhosis ribonuclease receiver operating characteristic small nuclear RNA Toll-like receptor

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9

10 11 12

13

14

15

16

17 18

sensitive and specific biomarkers for the diagnosis and monitoring of human diseases: promises and challenges. Clin Biochem 2013;46(10–11):846–860 Srivastava SK, Bhardwaj A, Leavesley SJ, Grizzle WE, Singh S, Singh AP. MicroRNAs as potential clinical biomarkers: emerging approaches for their detection. Biotech Histochem 2013;88(7): 373–387 Chen X, Ba Y, Ma L, et al. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res 2008;18(10):997–1006 Mitchell PS, Parkin RK, Kroh EM, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A 2008;105(30):10513–10518 Szabo G, Bala S. MicroRNAs in liver disease. Nat Rev Gastroenterol Hepatol 2013;10(9):542–552 Hornby RJ, Starkey Lewis P, Dear J, Goldring C, Park BK. MicroRNAs as potential circulating biomarkers of drug-induced liver injury: key current and future issues for translation to humans. Expert Rev Clin Pharmacol 2014;7(3):349–362 Bala S, Petrasek J, Mundkur S, et al. Circulating microRNAs in exosomes indicate hepatocyte injury and inflammation in alcoholic, drug-induced, and inflammatory liver diseases. Hepatology 2012;56(5):1946–1957 Lakner AM, Bonkovsky HL, Schrum LW. microRNAs: fad or future of liver disease. World J Gastroenterol 2011;17(20):2536–2542 Pogribny IP, Beland FA. Role of microRNAs in the regulation of drug metabolism and disposition genes in diabetes and liver disease. Expert Opin Drug Metab Toxicol 2013;9(6):713–724 Chen Y, Verfaillie CM. MicroRNAs: the fine modulators of liver development and function. Liver Int 2014;34(7):976–990 Hsu SH, Ghoshal K. MicroRNAs in liver health and disease. Curr Pathobiol Rep 2013;1(1):53–62 Takata A, Otsuka M, Yoshikawa T, Kishikawa T, Ohno M, Koike K. MicroRNAs and liver function. Minerva Gastroenterol Dietol 2013;59(2):187–203 Hulsmans M, Holvoet P. MicroRNA-containing microvesicles regulating inflammation in association with atherosclerotic disease. Cardiovasc Res 2013;100(1):7–18 Tsui NB, Ng EK, Lo YM. Stability of endogenous and added RNA in blood specimens, serum, and plasma. Clin Chem 2002;48(10): 1647–1653 Turchinovich A, Samatov TR, Tonevitsky AG, Burwinkel B. Circulating miRNAs: cell-cell communication function? Front Genet 2013;4:119 Chen X, Liang H, Zhang J, Zen K, Zhang CY. Horizontal transfer of microRNAs: molecular mechanisms and clinical applications. Protein Cell 2012;3(1):28–37 Rani S. MicroRNA profiling of exosomes isolated from biofluids and conditioned media. Methods Mol Biol 2014;1182:131–144 Lemoinne S, Thabut D, Housset C, et al. The emerging roles of microvesicles in liver diseases. Nat Rev Gastroenterol Hepatol 2014;11(6):350–361

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

52

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20 21 22

23

24

25

26

27

28

29

30

31 32

33

34

35

36

37

38

39

state-of-the-art: emerging role of extracellular vesicles. Cell Mol Life Sci 2011;68(16):2667–2688 Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 2013;200(4):373–383 Eguchi A, Dowdy SF. siRNA delivery using peptide transduction domains. Trends Pharmacol Sci 2009;30(7):341–345 Vickers KC, Palmisano BT, Shoucri BM, Shamburek RD, Remaley AT. MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol 2011; 13(4):423–433 Arroyo JD, Chevillet JR, Kroh EM, et al. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci U S A 2011; 108(12):5003–5008 Pirola CJ, Fernández Gianotti T, Castaño GO, et al. Circulating microRNA signature in non-alcoholic fatty liver disease: from serum non-coding RNAs to liver histology and disease pathogenesis. Gut 2014 Povero D, Eguchi A, Niesman IR, et al. Lipid-induced toxicity stimulates hepatocytes to release angiogenic microparticles that require Vanin-1 for uptake by endothelial cells. Sci Signal 2013; 6(296):ra88 Esau C, Davis S, Murray SF, et al. miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metab 2006;3(2):87–98 Tsai WC, Hsu SD, Hsu CS, et al. MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis. J Clin Invest 2012; 122(8):2884–2897 Wang K, Zhang S, Marzolf B, et al. Circulating microRNAs, potential biomarkers for drug-induced liver injury. Proc Natl Acad Sci U S A 2009;106(11):4402–4407 Köberle V, Pleli T, Schmithals C, et al. Differential stability of cellfree circulating microRNAs: implications for their utilization as biomarkers. PLoS ONE 2013;8(9):e75184 Cheng L, Sharples RA, Scicluna BJ, Hill AF. Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood. J Extracell Vesicles 2014;3: 10.3402/jev.v3.23743 Wang Z, Yang B. MicroRNA Expression Detection Methods. Heidelberg, Germany: Springer; 2010 Ramaiah SK. Preclinical safety assessment: current gaps, challenges, and approaches in identifying translatable biomarkers of drug-induced liver injury. Clin Lab Med 2011;31(1):161–172 Aithal GP, Guha N, Fallowfield J, Castera L, Jackson AP. Biomarkers in liver disease: emerging methods and potential applications. Int J Hepatol 2012;2012:437508 Kim WR, Flamm SL, Di Bisceglie AM, Bodenheimer HC; Public Policy Committee of the American Association for the Study of Liver Disease. Serum activity of alanine aminotransferase (ALT) as an indicator of health and disease. Hepatology 2008;47(4): 1363–1370 Mofrad P, Contos MJ, Haque M, et al. Clinical and histologic spectrum of nonalcoholic fatty liver disease associated with normal ALT values. Hepatology 2003;37(6):1286–1292 Molleston JP, Schwimmer JB, Yates KP, et al; NASH Clinical Research Network. Histological abnormalities in children with nonalcoholic fatty liver disease and normal or mildly elevated alanine aminotransferase levels. J Pediatr 2014;164(4):707–713.e3 Hong H, Tong W. Emerging efforts for discovering new biomarkers of liver disease and hepatotoxicity. Biomarkers Med 2014; 8(2):143–146 Starkey Lewis PJ, Merz M, Couttet P, et al. Serum microRNA biomarkers for drug-induced liver injury. Clin Pharmacol Ther 2012;92(3):291–293 Fontana RJ. Pathogenesis of idiosyncratic drug-induced liver injury and clinical perspectives. Gastroenterology 2014;146(4): 914–928

53

40 Björnsson ES. Epidemiology and risk factors for idiosyncratic

drug-induced liver injury. Semin Liver Dis 2014;34(2):115–122 41 Urban TJ, Daly AK, Aithal GP. Genetic basis of drug-induced liver

injury: present and future. Semin Liver Dis 2014;34(2):123–133 42 Stephens C, Andrade RJ, Lucena MI. Mechanisms of drug-induced

liver injury. Curr Opin Allergy Clin Immunol 2014;14(4):286–292 43 Su YW, Chen X, Jiang ZZ, et al. A panel of serum microRNAs as

44

45

46

47

48

49 50

51

52

53

54

55

56

57

58

59

60

61

specific biomarkers for diagnosis of compound- and herb-induced liver injury in rats. PLoS ONE 2012;7(5):e37395 Starkey Lewis PJ, Dear J, Platt V, et al. Circulating microRNAs as potential markers of human drug-induced liver injury. Hepatology 2011;54(5):1767–1776 Antoine DJ, Dear JW, Lewis PS, et al. Mechanistic biomarkers provide early and sensitive detection of acetaminophen-induced acute liver injury at first presentation to hospital. Hepatology 2013;58(2):777–787 Dubin PH, Yuan H, Devine RK, Hynan LS, Jain MK, Lee WM; Acute Liver Failure Study Group. Micro-RNA-122 levels in acute liver failure and chronic hepatitis C. J Med Virol 2014;86(9):1507–1514 Qi R, Weiland M, Gao XH, Zhou L, Mi QS. Identification of endogenous normalizers for serum microRNAs by microarray profiling: U6 small nuclear RNA is not a reliable normalizer. Hepatology 2012;55(5):1640–1642, author reply 1642– 1643 Ward J, Kanchagar C, Veksler-Lublinsky I, et al. Circulating microRNA profiles in human patients with acetaminophen hepatotoxicity or ischemic hepatitis. Proc Natl Acad Sci U S A 2014;111(33): 12169–12174 Angulo P. Nonalcoholic fatty liver disease. N Engl J Med 2002; 346(16):1221–1231 Wong RJ, Cheung R, Ahmed A. Nonalcoholic steatohepatitis is the most rapidly growing indication for liver transplantation in patients with hepatocellular carcinoma in the U.S. Hepatology 2014;59(6):2188–2195 Wieckowska A, McCullough AJ, Feldstein AE. Noninvasive diagnosis and monitoring of nonalcoholic steatohepatitis: present and future. Hepatology 2007;46(2):582–589 Eguchi A, Povero D, Alkhouri N, Feldstein AE. Novel therapeutic targets for nonalcoholic fatty liver disease. Expert Opin Ther Targets 2013;17(7):773–779 Clarke JD, Sharapova T, Lake AD, Blomme E, Maher J, Cherrington NJ. Circulating microRNA 122 in the methionine and cholinedeficient mouse model of non-alcoholic steatohepatitis. J Appl Toxicol 2014;34(6):726–732 Yamada H, Suzuki K, Ichino N, et al. Associations between circulating microRNAs (miR-21, miR-34a, miR-122 and miR451) and non-alcoholic fatty liver. Clin Chim Acta 2013; 424:99–103 Longchamps RJ, Abey SK, Martino AC, Henderson WA. Letter: gender-associated cell-free microRNA profiles in non-alcoholic fatty liver disease. Aliment Pharmacol Ther 2014;39(9):997–998 Vincent R, Sanyal A. Recent advances in understanding of NASH: MicroRNAs as both biochemical markers and players. Curr Pathobiol Rep 2014;2:109–116 Shrivastava S, Mukherjee A, Ray RB. Hepatitis C virus infection, microRNA and liver disease progression. World J Hepatol 2013; 5(9):479–486 Singaravelu R, Russell RS, Tyrrell DL, Pezacki JP. Hepatitis C virus and microRNAs: miRed in a host of possibilities. Curr Opin Virol 2014;7:1–10 Randall G, Panis M, Cooper JD, et al. Cellular cofactors affecting hepatitis C virus infection and replication. Proc Natl Acad Sci U S A 2007;104(31):12884–12889 Shwetha S, Gouthamchandra K, Chandra M, Ravishankar B, Khaja MN, Das S. Circulating miRNA profile in HCV infected serum: novel insight into pathogenesis. Sci Rep 2013;3:1555 Bihrer V, Friedrich-Rust M, Kronenberger B, et al. Serum miR-122 as a biomarker of necroinflammation in patients with chronic Seminars in Liver Disease

Vol. 35

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This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

19 György B, Szabó TG, Pásztói M, et al. Membrane vesicles, current

Arrese et al.

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62

63

64

65 66

67 68

69

70

71

72

73

74 75

76 77

78

79

80

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hepatitis C virus infection. Am J Gastroenterol 2011;106(9): 1663–1669 van der Meer AJ, Farid WR, Sonneveld MJ, et al. Sensitive detection of hepatocellular injury in chronic hepatitis C patients with circulating hepatocyte-derived microRNA-122. J Viral Hepat 2013;20(3):158–166 Shrivastava S, Petrone J, Steele R, Lauer GM, Di Bisceglie AM, Ray RB. Up-regulation of circulating miR-20a is correlated with hepatitis C virus-mediated liver disease progression. Hepatology 2013;58(3):863–871 Abdalla MA, Haj-Ahmad Y. Promising candidate urinary MicroRNA biomarkers for the early detection of hepatocellular carcinoma among high-risk hepatitis C virus Egyptian patients. J Cancer 2012;3:19–31 Thirion M, Ochiya T. Roles of microRNAs in the hepatitis B virus infection and related diseases. Viruses 2013;5(11):2690–2703 Wei YF, Cui GY, Ye P, Chen JN, Diao HY. MicroRNAs may solve the mystery of chronic hepatitis B virus infection. World J Gastroenterol 2013;19(30):4867–4876 Zhang X, Hou J, Lu M. Regulation of hepatitis B virus replication by epigenetic mechanisms and microRNAs. Front Genet 2013;4:202 Novellino L, Rossi RL, Bonino F, et al. Circulating hepatitis B surface antigen particles carry hepatocellular microRNAs. PLoS ONE 2012;7(3):e31952 Ji F, Yang B, Peng X, Ding H, You H, Tien P. Circulating microRNAs in hepatitis B virus-infected patients. J Viral Hepat 2011;18(7): e242–e251 Zhang H, Li QY, Guo ZZ, et al. Serum levels of microRNAs can specifically predict liver injury of chronic hepatitis B. World J Gastroenterol 2012;18(37):5188–5196 Arataki K, Hayes CN, Akamatsu S, et al. Circulating microRNA-22 correlates with microRNA-122 and represents viral replication and liver injury in patients with chronic hepatitis B. J Med Virol 2013;85(5):789–798 Waidmann O, Bihrer V, Pleli T, et al. Serum microRNA-122 levels in different groups of patients with chronic hepatitis B virus infection. J Viral Hepat 2012;19(2):e58–e65 Winther TN, Heiberg IL, Bang-Berthelsen CH, Pociot F, Hogh B. Hepatitis B surface antigen quantity positively correlates with plasma levels of microRNAs differentially expressed in immunological phases of chronic hepatitis B in children. PLoS ONE 2013; 8(11):e80384 Forner A, Llovet JM, Bruix J. Hepatocellular carcinoma. Lancet 2012;379(9822):1245–1255 Singal AG, Marrero JA, Yopp A. Screening process failures for hepatocellular carcinoma. J Natl Compr Canc Netw 2014;12(3): 375–382 Forner A, Bruix J. Biomarkers for early diagnosis of hepatocellular carcinoma. Lancet Oncol 2012;13(8):750–751 Li X, Yang W, Lou L, Chen Y, Wu S, Ding G. microRNA: a promising diagnostic biomarker and therapeutic target for hepatocellular carcinoma. Dig Dis Sci 2014;59(6):1099–1107 Otsuka M, Kishikawa T, Yoshikawa T, et al. The role of microRNAs in hepatocarcinogenesis: current knowledge and future prospects. J Gastroenterol 2014;49(2):173–184 Braconi C, Henry JC, Kogure T, Schmittgen T, Patel T. The role of microRNAs in human liver cancers. Semin Oncol 2011;38(6): 752–763 Qi P, Cheng SQ, Wang H, Li N, Chen YF, Gao CF. Serum microRNAs as biomarkers for hepatocellular carcinoma in Chinese patients with chronic hepatitis B virus infection. PLoS ONE 2011;6(12): e28486 Qu KZ, Zhang K, Li H, Afdhal NH, Albitar M. Circulating microRNAs as biomarkers for hepatocellular carcinoma. J Clin Gastroenterol 2011;45(4):355–360

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85

86 87

88

89 90 91

92

93

94

95

96

97

98

99

100

101 102

microRNA 215 as potential biomarkers for the diagnosis of chronic hepatitis and hepatocellular carcinoma. Diagn Pathol 2014;9:135 Liu AM, Yao TJ, Wang W, et al. Circulating miR-15b and miR-130b in serum as potential markers for detecting hepatocellular carcinoma: a retrospective cohort study. BMJ Open 2012;2(2): e000825 Li LM, Hu ZB, Zhou ZX, et al. Serum microRNA profiles serve as novel biomarkers for HBV infection and diagnosis of HBV-positive hepatocarcinoma. Cancer Res 2010;70(23):9798–9807 Zhou J, Yu L, Gao X, et al. Plasma microRNA panel to diagnose hepatitis B virus-related hepatocellular carcinoma. J Clin Oncol 2011;29(36):4781–4788 Yang Y, Zhu R. Diagnostic value of circulating microRNAs for hepatocellular carcinoma. Mol Biol Rep 2014;41(10):6919–6929 Wang H, Hou L, Li A, Duan Y, Gao H, Song X. Expression of serum exosomal microRNA-21 in human hepatocellular carcinoma. Biomed Res Int 2014;2014:864894 Köberle V, Kronenberger B, Pleli T, et al. Serum microRNA-1 and microRNA-122 are prognostic markers in patients with hepatocellular carcinoma. Eur J Cancer 2013;49(16):3442–3449 Marin JJ, Bujanda L, Banales JM. MicroRNAs and cholestatic liver diseases. Curr Opin Gastroenterol 2014;30(3):303–309 Wang XW, Heegaard NH, Orum H. MicroRNAs in liver disease. Gastroenterology 2012;142(7):1431–1443 Shen WJ, Dong R, Chen G, Zheng S. microRNA-222 modulates liver fibrosis in a murine model of biliary atresia. Biochem Biophys Res Commun 2014;446(1):155–159 Yamaura Y, Nakajima M, Takagi S, Fukami T, Tsuneyama K, Yokoi T. Plasma microRNA profiles in rat models of hepatocellular injury, cholestasis, and steatosis. PLoS ONE 2012;7(2):e30250 Woolbright BL, Antoine DJ, Jenkins RE, Bajt ML, Park BK, Jaeschke H. Plasma biomarkers of liver injury and inflammation demonstrate a lack of apoptosis during obstructive cholestasis in mice. Toxicol Appl Pharmacol 2013;273(3):524–531 Ninomiya M, Kondo Y, Funayama R, et al. Distinct microRNAs expression profile in primary biliary cirrhosis and evaluation of miR 505-3p and miR197-3p as novel biomarkers. PLoS ONE 2013; 8(6):e66086 Qin B, Huang F, Liang Y, Yang Z, Zhong R. Analysis of altered microRNA expression profiles in peripheral blood mononuclear cells from patients with primary biliary cirrhosis. J Gastroenterol Hepatol 2013;28(3):543–550 Katsushima F, Takahashi A, Sakamoto N, Kanno Y, Abe K, Ohira H. Expression of micro-RNAs in peripheral blood mononuclear cells from primary biliary cirrhosis patients. Hepatol Res 2014;44(10): E189–E197 Zahm AM, Hand NJ, Boateng LA, Friedman JR. Circulating microRNA is a biomarker of biliary atresia. J Pediatr Gastroenterol Nutr 2012;55(4):366–369 O’Hara SP, Gradilone SA, Masyuk TV, Tabibian JH, LaRusso NF. MicroRNAs in cholangiopathies. Curr Pathobiol Rep 2014;2(3): 133–142 Elfimova N, Schlattjan M, Sowa JP, Dienes HP, Canbay A, Odenthal M. Circulating microRNAs: promising candidates serving as novel biomarkers of acute hepatitis. Front Phys 2012;3:476 Waidmann O, Köberle V, Brunner F, Zeuzem S, Piiper A, Kronenberger B. Serum microRNA-122 predicts survival in patients with liver cirrhosis. PLoS ONE 2012;7(9):e45652 Chen YJ, Zhu JM, Wu H, et al. Circulating microRNAs as a fingerprint for liver cirrhosis. PLoS ONE 2013;8(6):e66577 Gehrau RC, Mas VR, Maluf DG. Hepatic disease biomarkers and liver transplantation: what is the potential utility of microRNAs? Expert Rev Gastroenterol Hepatol 2013;7(2):157–170

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