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Contents lists available at ScienceDirect

Pharmacological Research

journa l homepage: www.e lsev ier .com/ locate /yphrs

eview

irculating miRNAs in nontumoral liver diseases

lex Evangelista do Amaral a, Júlia Cisilotto a, Tânia Beatriz Creczynski-Pasa a,∗,eonardo de Lucca Schiavon b,∗

Department of Pharmaceutical Sciences, Federal University of Santa Catarina, Delfino Conti Street, 88040-370 Florianopolis, SC, BrazilDepartment of Internal Medicine, Division of Gastroenterology, Federal University of Santa Catarina, Maria Flora Pausewang Street, 88036-800lorianopolis, SC, Brazil

r t i c l e i n f o

rticle history:eceived 11 July 2017eceived in revised form 11 October 2017ccepted 12 October 2017vailable online xxx

a b s t r a c t

In recent years, there has been increasing interest in finding new biomarkers for diagnosis and prog-nostication of liver diseases. MicroRNAs (miRNAs) are small noncoding RNA molecules involved in theregulation of gene expression and have been studied in relation to several conditions, including liver dis-ease. Mature miRNAs can reach the bloodstream by passive release or by incorporation into lipoproteincomplexes or microvesicles, and have stable and reproducible concentrations among individuals. In this

eywords:irculating miRNAiver cirrhosisiver diseases

review, we summarize studies involving circulating miRNAs sourced from the serum or plasma of patientswith nontumoral liver diseases in attempt to bring insights in the use of miRNAs as biomarkers for diag-nosis, as well as for prognosis of such diseases. In addition, we present pre-analytical aspects involvingmiRNA analysis and strategies for normalization of reverse transcription-quantitative polymerase chain

icroRNAiRNA

reaction (RT-qPCR) data related to the studies evaluated.© 2017 Elsevier Ltd. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 001.1. Overview of miRNA biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

2. Circulating miRNA profile in patients with liver disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.1. Liver cirrhosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.2. Hepatitis B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.3. Hepatitis C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.4. Hepatotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.5. Nonalcoholic fatty liver disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.6. Other liver diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.7. MicroRNA across distinct disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

3. Pre-analytical aspects involving miRNA analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004. miRNAs as biomarkers: methodological considerations for clinical use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .005. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Please cite this article in press as: A.E. do Amaral, et al., Circulatinhttps://doi.org/10.1016/j.phrs.2017.10.002

Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: Ago, Argonaute; AFP, �-fetoprotein; AH, alcoholic hepatitis; AIH, autominotransferase; AMA, antimitochondrial antibody; AP, alkaline phosphatase; APAP,

UROC, area under the receiver operating characteristic (curve); CHB, chronic hepatitindex; HBV, hepatitis B virus; HBV-ACLF, hepatitis B-related acute-on-chronic liver failuCV, hepatitis C virus; HSC, hepatic stellate cells; INR, international normalized ratio;

icrovesicle; NAFLD, nonalcoholic fatty liver disease; NAS, NAFLD activity score; NASH,

icroRNA; RISC, RNA-induced silencing complex; RT-qPCR, reverse transcription-quantesponse; TGF-�, transforming growth factor-beta; UTR, untranslated region.∗ Corresponding authors.

E-mail addresses: alex.amaral@ufsc.br (A.E. do Amaral), juliacisilotto@hotmail.com (J.L. de Lucca Schiavon).

ttps://doi.org/10.1016/j.phrs.2017.10.002043-6618/© 2017 Elsevier Ltd. All rights reserved.

g miRNAs in nontumoral liver diseases, Pharmacol Res (2017),

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .00

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

immune hepatitis; ALD, alcoholic liver disease; ALI, acute liver injury; ALT, alanineacetaminophen; ASH, alcoholic steatohepatitis; AST, aspartate aminotransferase;s B; GC, gastric cancer; GGT, �-glutamyl transpeptidase; HAI, histologic activityre; HBV-ASC, chronic asymptomatic HBV carriers; HCC, hepatocellular carcinoma;MELD, Model for End-Stage Liver Disease; MeSH, Medical Subject Headings; MV,nonalcoholic steatohepatitis; PBC, primary biliary cholangitis; pri-miRNA, primaryitative polymerase chain reaction; SS, simple steatosis; SVR, sustained virological

Cisilotto), tania.pasa@ufsc.br (T.B. Creczynski-Pasa), leo-jf@uol.com.br

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. Introduction

The main causes of liver disease are alcohol abuse, viral hep-titis, nonalcoholic fatty liver disease (NAFLD), hemochromatosis,utoimmune hepatitis (AIH), primary biliary cholangitis (PBC), andrimary sclerosing cholangitis. Alcohol and the hepatitis B and Ciruses (HBV and HCV) are the most important causes of liver cir-hosis and hepatocellular carcinoma (HCC) [1–4]. Mortality relatedo liver disease is growing, with more than one million deathsrom liver cirrhosis worldwide in 2010 [5]. When deaths from liverirrhosis are combined with deaths from liver cancer and acute hep-titis, the annual number of fatalities due to liver disease can exceedwo million [6]. In Europe, liver cirrhosis is responsible for around70,000 deaths per year, and more than 5500 liver transplants areerformed each year [1]. Chronically infected HBV and HCV patientsre at high risk of developing cirrhosis (∼20% of patients) [7–9]nd HCC (∼25% and 7% for HBV and HCV, respectively) [8,10]. HBVnd HCV infections are responsible for a mortality rate of approx-mately 3 and 11 deaths, respectively, per 100,000 inhabitants perear in Spain [11]. Although alcohol control policies, vaccination,nd prevention strategies have contributed to decreased incidencef chronic liver diseases, these diseases remain public health prob-

ems because of their associated complications. In addition, theres a high cost related to antiviral therapy for HBV and HCV chronicnfection [12,13].

Please cite this article in press as: A.E. do Amaral, et al., Circulatinhttps://doi.org/10.1016/j.phrs.2017.10.002

The health, social, and economic impacts associated with liveriseases could be reduced by improving diagnosis and prognosti-ation. New biomarkers have been investigated for this purpose,

ig. 1. Schematic representation of miRNA biogenesis (see text for details). This figurereparing color figures for everyone including the colorblind” described by Roskoski [42]

PRESSl Research xxx (2017) xxx–xxx

including circulating microRNAs (miRNAs). MicroRNAs are smallRNAs about 22 nucleotides (nt) long that regulate gene expressionby binding to complementary regions of messenger RNA (mRNA)[14]. Circulating miRNAs can be detected in human serum andplasma, and have stable and reproducible concentrations amongindividuals [15,16]. The purpose of this review is to highlight recentadvances in the study of circulating miRNAs as markers of nontu-moral liver damage, and to provide an overview of the biologicalproperties of these nucleic acids and analytical challenges associ-ated with their quantitation.

1.1. Overview of miRNA biogenesis

MicroRNAs are endogenous, noncoding RNAs about 22 nt longthat regulate gene expression post-transcriptionally by binding tocomplementary regions of mRNA, resulting in repression of trans-lation or mRNA degradation [14]. Fig. 1 gives a brief overviewof miRNA biogenesis. Initially, (1) DNA is transcribed by RNApolymerase II (Pol II), yielding a transcript called primary RNA (pri-miRNA) [17,18], which usually has a stem-loop structure and alength over 1 kb. After that, (2) the RNase III Drosha cleaves the hair-pin ∼11 base pairs (bp) from the basal junction and ∼22 bp fromthe apical junction connected to the terminal loop [19,20], forminga hairpin RNA ∼65 nt long, termed pre-miRNA [21]. Then, (3) pre-

g miRNAs in nontumoral liver diseases, Pharmacol Res (2017),

miRNA forms a transport complex with the protein exportin-5 andthe Ran-guanosine-5′-triphosphate complex (Ran•GTP), allowingexport into the cytoplasm. Once in the cytoplasm, (4) pre-miRNAis cleaved by the RNase III Dicer near the terminal loop, releasing

as well as the graphical abstract were designed according to the “Guidelines for.

ARTICLE ING ModelYPHRS-3702; No. of Pages 14

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a((cm

abhAaobttbt([no

wC2mnawoIw

Fig. 2. Number of publications related to miRNA (see text for details).

small RNA duplex [22–26], which is loaded into an ArgonauteAgo) protein, forming the pre-RNA-induced silencing complexpre-RISC) [27–29]. Next, the duplex is unwound, which usuallyauses degradation of the passenger strand (miRNA*) and the for-ation of mature miRNA [30].

Once integrated into the RISC, mature miRNA is functionalnd able to regulate gene expression. This regulation may occury: (I) inhibition of translation, the most common mechanism inumans, which is dependent on the Ago proteins Ago1, Ago3, orgo4, and involves imperfect base pairing between the miRNAnd the 3′-untranslated region (UTR) of mRNA [31]; (II) cleavagef mRNA, an Ago2-dependent mechanism, which involves perfectase pairing between the miRNA and the 3′-UTR of mRNA; and (III)ranslation activation, which involves interaction of miRNA withhe 5′-UTR of mRNA [32–37]. The mature miRNA can reach theloodstream by (A) active release (by incorporation into lipopro-ein complexes, microvesicles, or exosomes) or (B) passive releasecaused by cell death; by incorporation into apoptotic bodies)38–40]. In associated form, circulating miRNAs are resistant touclease activity, making them a promising source of diagnosticr prognostic biomarkers [41].

Although miRNAs were discovered in 1993 by Lee et al. [43]hile studying the development of the free-living nematode

aenorhabditis elegans, the term “miRNA” started to be used in mid-001 [28,44–46]. Since the early 2000s, when a correlation betweeniRNA levels and human disease was demonstrated [47–49], the

umber of studies on the applications of miRNAs as biomarkersnd therapeutics has progressively increased (Fig. 2). In 2015, there

Please cite this article in press as: A.E. do Amaral, et al., Circulatinhttps://doi.org/10.1016/j.phrs.2017.10.002

ere 11,216 new citations related to miRNA in PubMed (Fig. 2A),f which 978 (9%) related to both miRNA and the liver (Fig. 2B).

n 2016, although the number of new citations related to miRNAas lower than in 2015 (9022 citations), the proportion of these

PRESSl Research xxx (2017) xxx–xxx 3

citations that also related to the liver was similar (685/9022, 8%).Considering all 53,503 citations related to miRNA up to 2016, 3883(7%) related to both miRNA and the liver, that is, approximately one-fourteen of citations identified using the Medical Subject Headings(MeSH) search terms “microRNA” and “miRNA” were also anno-tated with the MeSH term “liver.”

MicroRNAs have been implicated in the regulation of manyimportant biological processes, such as cell growth and differentia-tion, development, apoptosis, and modulation of the host responseto viral infection [50]. For example, miR-122 plays an importantrole in liver physiology, since it is involved in acquisition and main-tenance of the hepato-specific phenotype, and participates in theregulation of cholesterol and fatty acids in hepatic metabolism[51,52]. Moreover, the propagation of HCV RNA occurs after bind-ing of miR-122 to the 5′-noncoding region of the HCV genome[51,53,54]. The following section describes other circulating miR-NAs, beyond miR-122, that have been described in patients withliver disease.

The names of miRNAs are composed of a three-letter prefix thatidentifies the source organism, the prefix “miR,” which denotesmature miRNA, and a unique identification number, which is simplysequential. The 5′ and 3′ arms are denoted by the suffixes 5p and 3p,respectively. For example, “hsa-miR-122-5p” corresponds to the5′ arm of mature miR-122 in Homo sapiens (hsa) [55,56] (www.mirbase.org). Since this work focuses on human miRNA, the prefixspecifying the organism will be omitted.

Table 1 summarizes the content covered in the subsequent sec-tions. In the present review, impact factor (IF) was not used asa criterion for the selection of research articles because it doesnot necessarily reflect the quality of studies [57]. In addition, only9 studies (listed in Table 1) developed independent validationcohort: liver cirrhosis [58,59], CHB cirrhosis [60], HBV-HCC [61],HCV [62–64], NAFLD [65], and PBC [66]. For this reason, we soughtto include, in this review, studies involving miRNA expression inserum or plasma samples from humans with non-tumoral liver dis-eases, even though independent validation cohort studies were notavailable in majority of the cases. Although the focus of the review iscirculating miRNAs (serum or plasma) in nontumoral liver diseases,we also address some studies involving HCC, which is associatedwith several chronic liver diseases.

2. Circulating miRNA profile in patients with liver disease

2.1. Liver cirrhosis

MicroRNAs are fundamental in the pathogenesis of various dis-eases, including cancer and other chronic liver diseases. In addition,miRNAs detected in patients’ serum may serve as biomarkers andrepresent a novel approach for diagnostic blood screening [92]. Theexpression of various miRNAs is altered in the serum of patientswith liver disease; for example, miR-146a, miR-215, miR-224, miR-574-3p, and miR-885-5p were shown to be upregulated in theserum of patients with HCC and liver cirrhosis. In the same study,the expression of these miRNAs was also evaluated in the serumof patients with chronic hepatitis B (CHB) or gastric cancer (GC);among these miRNAs, in the independent validation cohort, miR-885-5p presented higher expression in patients with HCC (n = 46),liver cirrhosis (n = 26), and CHB (n = 23), compared with GC patients(n = 17) and healthy controls (n = 24). In addition, the area underthe receiver operating characteristic (AUROC) curve for miR-885-5p was 0.904, with sensitivity and specificity of 90.5% and 79.2%,

g miRNAs in nontumoral liver diseases, Pharmacol Res (2017),

respectively, in distinguishing patients with liver disease fromthe control group. However, miR-885-5p did not correlate withthe hepatic function markers �-fetoprotein (AFP), alanine amino-transferase (ALT), aspartate aminotransferase (AST), or �-glutamyl

Please cite this article in press as: A.E. do Amaral, et al., Circulating miRNAs in nontumoral liver diseases, Pharmacol Res (2017),https://doi.org/10.1016/j.phrs.2017.10.002

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4 A.E. do Amaral et al. / Pharmacological Research xxx (2017) xxx–xxx

Table 1Circulating miRNAs in nontumoral liver diseases (serum or plasma).

miRNA Expression Reference group Disease Variables correlated Ref.

miR-885-5p up Healthy controls LC, CHB, HCC NC AFP, ALT, AST, GGT [58]miR-122 down Compensated LC Decompensated LC − Creatinine, INR, MELD [59]

+ AFP, ALT, AST, GGTNC Albumin, bilirubin, total protein

miR-181b up Healthy controls LC NA [67]miR-571 up Healthy controls LC − Albumin, PCHE [68]

+ Child-PughNC Creatinine

miR-106b down Healthy controls CHB cirrhosis NC Bilirubin, Albumine,ALT, INR, PT

[60]miR-181b up16 miRNAs1 up Healthy children CHB + HBV DNA [69]miR-29 down Healthy controls LC + HBV DNA [70]miR-29 up CHB NC ALTmiR-122 up CHB, HCCmiR-15b up Healthy controls HBV-HCC NA [61]miR-130b upmiR-15b down Healthy controls CHB NA [71]miR-15b up HBV-HCCmiR-125b up CHBmiR-125b down HBV-HCCmiR-130b down CHBmiR-130b up HBV-HCClet-7b up Healthy controls HBV-ASC, CHB,

HBV-ACLF– Age (miR-122, miR-194) [72]

miR-16 up NC ALT, HBV DNA (miR-122, miR-194)miR-20a upmiR-92a upmiR-106a upmiR-194 upmiR-122 up HBV-ASC, CHBmiR-19b up CHB, HBV-ACLFmiR-223 upmiR-21 up Healthy controls HBV-ACLF − INR, MELD, PT (miR-130a) [73]

+ PTA (miR-130a)miR-122 upmiR-130a upmiR-143 upmiR-148a upmiR-192 upmiR-200a upmiR-486-5p upmiR-210 up Healthy controls CHB-M, CHB-S,

HBV-ACLF− PTA [74]+ ALT, AST, bilirubin

miR-122 up Healthy controls HCV + ALT, AST, HAI [75]NC Albumin, bilirubin, HCV RNA, INR,

liver fibrosismiR-21 up Healthy controls HCV, HCV-HCC + ALT, AST, Bilirubin, GGT, HAI, INR [62]

NC Albumin, HCV RNAmiR-122 up Healthy controls HCV + ALT, AST, HCV RNA [76]miR-122 up Non-SVR patients HCV + ALT, HAI [64]

NC HCV RNA, liver fibrosis (Metavir)miR-122 up Healthy controls HCV NC ALT, HCV RNA, Ishak score [77]miR-155 up Healthy controls HCV, HCV-HCC + IP-10 [78]miR-143 up Healthy controls HCV, HCC NA [79]miR-215 upmiR-122 up Healthy controls APAP overdose + ALT (miR-122) [80]miR-192 up NC ALT (miR-192); bilirubin,

creatinine, PT (miR-122)miR-122 up Healthy controls APAP overdose + ALT [81]miR-122

up Healthy children APAP overdose + APAP protein adducts [82]NC ALT

miR-122 up Baseline Cholestyraminetreatment

NC ALT [83]

miR-122 up Healthy controls Paraquat poisoning + ALT [84]miR-16 up Healthy controls NAFLD, HCV + inflammation activity, fibrosis

stage (miR-34a, −122);[63]

miR-34a up ALT, AST, LDL, total cholesterol(miR-122)

miR-122 up NC HCV RNAmiR-122 up Mild steatosis patients NAFLD − fibrosis stage [85]

+ liver miR-122 levelNC ALP, ALT, AST, total cholesterol,

GGT, NAS, TGmiR-122 up Healthy controls NAFLD + ALT, AST, GGT

(miR-122)[65]

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Table 1 (Continued)

miRNA Expression Reference group Disease Variables correlated Ref.

miR-192 upmiR-375 upmiR-21 up Healthy controls AIH − fibrosis stage [86]miR-122 up + ALT, AST;

necroinflammation(miR-21)

NC type IV collagen;necroinflammation(miR-122)

miR-197-3p down Healthy controls PBC NA [87]miR-505-3p downmiR-26b-5p down Healthy controls PBC NA [66]miR-122 upmiR-141-3p upmiR-135b up Healthy controls ASH NA [88]miR-490 upmiR-761 upmiR-203 downmiR-214 downmiR-182 up Healthy controls AH NC liver miR-182 level,

disease severity,mortality

[89]

miR-223 up Healthy controls Alcoholics NA [90]sma-miR-277 up Egg-negative

individualsSchistosomiasis(Schistosoma mansoni)

NA [91]sma-miR-3479-3p upsma-bantam up

AH, alcoholic hepatitis; AIH, autoimmune hepatitis; APAP, acetaminophen (N-acetyl-p-aminophenol); ASH, alcoholic steatohepatitis; CHB, chronic hepatitis B; CHB-M, mildchronic hepatitis B; CHB-S, severe chronic hepatitis B; HBV, hepatitis B virus; HBV-ACLF, hepatitis B virus-related acute-on-chronic liver failure; HBV-ASC, hepatitis B virus-chronic asymptomatic carriers; HBV-HCC, hepatitis B virus-related hepatocellular carcinoma; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HCV-HCC, hepatitis Cvirus-related hepatocellular carcinoma; LC, liver cirrhosis; NAFLD, nonalcoholic fatty liver disease; PBC, primary biliary cholangitis; sma, Schistosoma mansoni; SVR, sustainedvirological response. −, negative correlation; +, positive correlation; NA, not applicable; NC, no significant correlation; AFP, �-fetoprotein; ALT, alanine aminotransferase;AST, aspartate aminotransferase; GGT, �-glutamyl transpeptidase; HAI, histologic activity index; INR, international normalized ratio; IP-10, interferon-gamma-induciblep DiseasP R-99am 247.

td

t(lpcpwiws(

mc1glto1r1apsamim

rotein of 10 kDa; LDL, low-density lipoprotein; MELD, Model for End-Stage Liver

TA, prothrombin time activity; TG, triglyceride. 1, 16 miRNAs were upregulated: miiR-215, miR-365, miR-455-5p, miR-455-3p, miR-483-3p, miR-885-5p, and miR-1

ranspeptidase (GGT). Despite this, miR-885-5p exhibited betteriagnostic performance than ALT (AUROC of 0.742) [58].

On the other hand, other authors have found a positive correla-ion between miR-122 and ALT, AST, GGT, and alkaline phosphataseALP) in patients with liver cirrhosis. In this study, serum miR-122evels were evaluated in patients with compensated and decom-ensated liver cirrhosis, which included 107 patients in a testohort and 143 patients in a validation cohort. Patients with decom-ensated liver cirrhosis presented lower miR-122 levels than thoseith compensated liver cirrhosis both in the cohort test and in the

ndependent validation cohort. Furthermore, lower miR-122 levelsere associated with shorter survival in the Cox regression analy-

is, even after adjusting for the Model for End-Stage Liver DiseaseMELD) score [59].

In addition to the miRNAs described above, the expression ofiR-181a and miR-181b in serum from 22 patients with liver

irrhosis has been evaluated by Wang et al. [67]. While miR-81a showed no significant difference compared with the controlroup (n = 17), miR-181b was significantly higher in patients withiver cirrhosis. In addition, the authors demonstrated, in vitro, thatransforming growth factor-beta 1 (TGF-�1) induced expressionf miR-181b in hepatic stellate cells-T6 (HSC-T6). In turn, miR-81b promoted HSC-T6 proliferation by targeting the cell cycleegulator p27. Thus, TGF-�1 can induce the expression of miR-81b and promote the proliferation of hepatic stellate cells, whichre important mediators of liver fibrosis. Therefore, miR-181b is aotential diagnostic biomarker for liver cirrhosis [67]. In anothertudy, Roderburg et al. demonstrated that isolated liver cells (hep-tocytes and hepatic stellate cells) increased the expression of

Please cite this article in press as: A.E. do Amaral, et al., Circulatinhttps://doi.org/10.1016/j.phrs.2017.10.002

iR-571 in response to treatment with TGF-� [68]. These authorsdentified changes in three miRNAs (miR-513-3p, miR-571, and

iR-652) in 67 patients with liver cirrhosis induced by alcohol or

e; NAS, NAFLD activity score; PCHE, pseudocholinesterase; PT, prothrombin time;, miR-100, miR-122, miR-122*, miR-125b, miR-192, miR-192*, miR-193b, miR-194,

hepatitis C, compared with the control group (n = 17). These threemiRNAs showed altered serum levels, miR-513-3p and miR-571being upregulated and miR-652 downregulated. The AUROC formiR-571 was 0.91, but when miR-571 was combined with the othertwo miRNAs (miR-513-3p and miR-652) the AUROC was approxi-mately 0.97, showing even greater power to discriminate patientswith chronic liver disease and liver cirrhosis from healthy controls.However, among these three miRNAs, only miR-571 was associatedwith disease stage, which was classified according to the Child-Pugh classification; miR-571 was significantly elevated in patientswith Child C cirrhosis compared with those with Child A cirrhosis[68].

2.2. Hepatitis B

In a three-phase study (exploration, training, and validation),Chen et al. demonstrated that plasma miR-106b and miR-181bwere downregulated and upregulated, respectively, in CHB-relatedcirrhosis (training phase, n = 90), compared with healthy controls(n = 60) [60]. The authors used miR-106b and miR-181b to con-struct a miRNA panel with an AUROC of 0.882 (sensitivity: 0.86;specificity; 0.75) that could discriminate CHB cirrhosis patientsfrom healthy individuals better than miR-106b (AUROC: 0.715) ormiR-181b (AUROC: 0.833) alone. In addition, they demonstratedthat there was no significant difference in miR-106b and miR-181b levels when comparing patients with CHB-related cirrhosisand non-CHB-related cirrhosis (such as alcoholic, schistosomiasis,autoimmune, and biliary), suggesting that variations in the plasma

g miRNAs in nontumoral liver diseases, Pharmacol Res (2017),

levels of these miRNAs are independent of etiology [60].Additionally, when juvenile CHB patients were compared with

healthy children (n = 60), 16 plasma miRNAs were found to beupregulated: miR-99a, miR-100, miR-122, miR-122*, miR-125b,

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iR-192, miR-192*, miR-193b, miR-194, miR-215, miR-365, miR-55-5p, miR-455-3p, miR-483-3p, miR-885-5p, and miR-1247 [69].ll 16 miRNAs presented higher expression in Hepatitis B Antigen

HBeAg)-positive children (n = 34) than in HBeAg-negative childrenn = 26). In addition, the authors found a strong positive corre-ation between all miRNAs and viral load (HBV DNA) suggestinghat circulating miRNAs might play a role in the establishmentnd maintenance of CHB in children [69]. Jinato et al. evaluatediRNA expression profile in liver biopsies collected from patientsith hepatitis B virus genotype C (HBeAg positive) both before and

fter treatment with pegylated-interferon alpha-2a for 48 weeksn = 10). After the treatment, patients were sorted as respondersith HBsAg clearance (n = 3), responders without HBsAg clearance

n = 3), and non-responders (n = 4). Out of these three groups, onlyatients classified as “responders with HBsAg clearance” presentedownregulated miR-185-5p and miR-186-5p levels suggesting thathese miRNAs might indicate the response to treatment [93].

Xing et al. recently evaluated serum miR-29 and miR-122 levelsn HCC (n = 20), liver cirrhosis (n = 20), and CHB patients (n = 29), andBV inactive carriers (n = 20) [70]. The authors demonstrated that

erum miR-122 levels were upregulated in HCC and CHB patientsompared with healthy controls (n = 20). On the other hand, miR-9 was downregulated in liver cirrhosis patients and upregulated

n CHB patients compared with healthy controls. In addition, aositive correlation was found between serum miR-29 and miR-22 levels and viral load (HBV DNA) in CHB patients. However,he increased miR-122 expression was not associated with ALT inHB patients. The authors suggest that downregulation of miR-29xpression may be related to hepatic fibrosis [70].

In patients with HBV-related HCC (HBV-HCC), Liu et al. foundhat serum miR-15b and miR-130b levels were upregulated [61].his study was divided into three phases: exploration, selection,nd validation. The validation phase included 30 healthy individu-ls, 29 CHB carriers, and 57 HCC patients. In this phase, the authorssed miR-15b and miR-130b to construct a miRNA panel to distin-uish between HCC patients and non-cancerous controls (healthyndividuals and CHB carriers), which had an AUROC of 0.981 (sen-itivity: 98.3%; specificity: 91.5%). In addition, they demonstratedhat this miRNA panel was useful for diagnosing HCC in subjectsith low serum AFP level (lower than 20 ng/mL) and that it could

etect cases of HCC in the early stages of development [61].Interestingly, Li et al. studied plasma microvesicles (MV) miR-

As in patients with CHB (n = 354) and HBV-HCC (n = 265) [71]. Theuthors found 219 miRNAs expressed only in CHB MVs, 189 miR-As expressed only in HCC MVs, and 53 miRNAs coexpressed in

hese two groups. The following miRNAs that target genes related tohe inflammatory response were expressed in both groups: miR-1,

iR-17, miR-20a, miR-23a, miR-30b, miR-128, miR-188-5p, miR-94, miR-377, miR-410, miR-432, miR-494, miR-936, and miR-939.

n addition, miR-15b and miR-130b were downregulated in CHBVs, but upregulated in HCC MVs [71], which is in accordance with

he result of Liu et al. described previously [61]. miR-125b, whichcts as a tumor suppressor, was upregulated in CHB MVs and down-egulated in HCC MVs. Thus, these results indicate that miRNAs mayerve as biomarkers for HCC screening and risk stratification in CHB71].

Ji et al. compared the expression of serum-circulating miRNAs inhronic asymptomatic HBV carriers (HBV-ASC) (n = 9), CHB (n = 21),BV-related acute-on-chronic liver failure (HBV-ACLF) (n = 21), andealthy controls (12) [72]. Using an array of 377 miRNAs, a total of7 miRNAs were detected in the control samples, while 77, 101,nd 135 miRNAs were detected in the HBV-ASC, CHB, and HBV-

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CLF samples, respectively. Interestingly, the amount of miRNAetected increased according to disease severity. Moreover, expres-ion of miR-122, -194, -223, -16, -92a, -19b, -20a, -106a, let-7bas confirmed by reverse transcription-quantitative polymerase

PRESSl Research xxx (2017) xxx–xxx

chain reaction (RT-qPCR). These miRNAs were upregulated in mostof the HBV patients, compared with healthy controls. Expressionlevels of miR-223 and miR-19b were similar between the HBV-ASCand control groups but increased significantly in the CHB and HBV-ACLF groups. Furthermore, expression of miR-122 was significantlyhigher in the HBV-ASC and CHB groups, but not the HBV-ACLFgroup, compared with the control group [72]. Conversely, Zhenget al. found that miR-122 expression was significantly higher inpatients with HBV-ACLF (n = 39) than in healthy controls (n = 20),but did not observe a significant difference in miR-194 expres-sion [73]. Increased levels of miR-21, -486-5p, -130a, -192, -148a,-143, and -200a were also observed in HBV-ACLF patients (P < 0.05).Moreover, expression of these miRNAs was evaluated in patientswho had recovered (n = 20) and not recovered (n = 19) from HBV-ACLF; low levels of miR-130a and miR-486-5p were observed inthe serum of the patients who had not recovered from the disease.Thus, miR-130a and miR-486-5p were suggested to be related toprognosis [73]. To investigate markers of disease progression, Songet al. evaluated miR-210 expression in the serum of CHB subjects[74]; this miRNA was chosen because its expression had alreadybeen described in inflammation and hypoxia. In this study, 30mild CHB patients, 30 severe CHB patients, 30 HBV-ACLF patients,and 30 healthy controls were included. Serum miR-210 expressionwas shown to increase significantly with the severity of HBV-related chronic liver disease. Serum miR-210 levels in the threegroups were higher than in the control group (P < 0.01). Severe CHBand HBV-ACLF serum samples showed higher expression of miR-210 than mild CHB serum samples (P < 0.01), and miR-210 levelswere higher in the HBV-ACLF group than in the severe CHB group(P < 0.05). Serum miR-210 was found to be positively correlatedwith ALT, AST, and total bilirubin, and negatively correlated withprothrombin activity. The authors suggested that increased serummiR-210 expression may be related to the severity of hepatitis,whereas increased liver miR-210 may be a response of hepatic cellsto hypoxia during liver inflammation [74].

In summary, serum miRNA expression profiles in HBV patientsvaried across studies. Expression of some miRNAs varied dependingon the severity of the disease and the presence of HCC, suggestingthat these miRNAs might have potential as biomarkers for diseaseprogression and cancer screening. Further studies are necessary toestablish a panel of specific miRNAs for each disease stage, allowingclinical use of miRNAs with high specificity and sensitivity.

2.3. Hepatitis C

miR-122 has also been studied in patients with chronic hep-atitis C virus (HCV) infection. Bihrer et al. demonstrated that thesepatients (n = 68) had higher serum miR-122 levels than healthy sub-jects (n = 19) [75]. Although no correlation was observed betweenmiR-122 and international normalized ratio (INR), liver fibrosis,or viral load (serum HCV RNA), miR-122 was correlated with ALT,AST, histologic activity index (HAI), and necroinflammatory activ-ity. In addition, the authors demonstrated that miR-122 levels didnot differ between healthy subjects and patients with normal ALT.Therefore, increased concentrations of circulating miR-122 mayreflect inflammatory activity in patients with chronic HCV infec-tion [75]. In another study, Bihrer et al. found that serum miR-21was upregulated in chronic HCV (n = 62) and in HCV-associated HCC(n = 29) patients compared with healthy controls (n = 19) [62]. How-ever, miR-21 was not able to differentiate between HCV patientswith and without HCC, because its levels were similar in bothgroups. On the other hand, miR-21 correlated with HAI, ALT, AST,

g miRNAs in nontumoral liver diseases, Pharmacol Res (2017),

bilirubin, GGT, and INR. In order to evaluate the correlation betweenmiR-21 and necroinflammatory activity, a validation study wasperformed using a validation cohort of patients with chronic HCV(n = 47), and it was demonstrated that miR-21 was able to differ-

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ntiate between minimal and mild to severe necroinflammationAUROC: 0.76; sensitivity: 53.3%; specificity: 95.2%) [62].

Recently, Kumar et al. evaluated miR-122 expression in serumrom 25 patients with HCV genotype 3 and viral load greater than0,000 IU/mL [76]. These patients exhibited increased miR-122xpression compared with the control group (n = 25). In addition,iR-122 correlated positively with ALT, AST, and viral load. For

his group of patients, the AUROC was 0.929 with sensitivity andpecificity of 92.0% and 84.0%, respectively, for distinguishing HCVatients from healthy controls [76]. In another study, Su et al. eval-ated the potential of miR-122 in predicting virological response toegylated interferon and ribavirin, retrospectively in 126 patientsnd prospectively in another 51 patients (this group correspondedo an independent validation cohort) with chronic HCV infec-ion by quantifying circulating miR-122 both before and duringreatment [64]. miR-122 was positively correlated with hepaticecroinflammation. Patients who achieved sustained virologicalesponse (SVR), especially in genotype 2 infection, had higher miR-22 levels in the pre-treatment phase than those with non-SVR.

n addition, a strong correlation was found between serum andepatic miR-122 expression [64]. Murakami el al. demonstrated

n the liver biopsies of patients with HCV genotype 1b (n = 99) thathe expression level of 9 miRNAs (miR-18a, miR-27b, miR-34b*,

iR-122, miR-143, miR-145, miR-378, miR-422b*, miR-652) wasignificantly different in the SVR (n = 46) and non-responder (NR)n = 25) groups [94]. Waidmann et al. evaluated miR-122 expressionn the serum of patients with chronic hepatitis C virus genotype

infection treated with a combination of pegylated interferon-�nd ribavirin (n = 52). There was no significant difference in miR-22 expression in the serum from patients who showed sustainedirological response (n = 28) in comparison to that from the non-esponse group (n = 24), suggesting that this miRNA may not be auitable marker for response prediction of the treatment [95]. How-ver, Butt et al. evaluated the expression of liver and serum miR-122n patients with CHC genotype 3 (n = 123). The authors found liver

iR-122 downregulated and serum miR-122 upregulated in thoseatients in relation to healthy individuals (n = 60), demonstrating aignificant inverse correlation. In addition, the expression of serumiR-122 before the pre-treatment with pegylated-interferon plus

ibavirin was upregulated in patients with SVR (n = 70) comparedo that in the non-responders or relapsers (n = 53), and it remainedndependently associated with treatment responses as obtainedrom multivariate analysis, hence suggesting that serum miR-122as the potential for predicting response to treatment [96].

Waring et al. evaluated the expression of miR-122 in serumamples from patients with HCV (n = 86, 45 being genotype 1; 20,enotype 2; and 21, genotype 3) before (baseline), during, and 8eeks after the end of treatment with paritaprevir plus ritonavir

ombined with dasabuvir or ombitasvir, which can be associated orot with ribavirin. In patients with genotype 1 who achieved SVRn = 33) miR-122 expression decreased over the treatment periodnd remained below the baseline level 8 weeks after the end of thereatment. On the other hand, in non-responders (n = 7) the expres-ion of miR-122 initially decreased but returned to the baselinefter the second week of the treatment. Regarding relapsers (n = 5),lthough they had decreased miR-122 expression throughout thereatment, the level returned to the baseline 8 weeks after the endf the treatment. The miR-122 expression profile was similar forll patients with genotype 2 [SVR (n = 14), relapsers (n = 2), non-esponders (n = 2), and lost to follow-up (n = 2)]. In all the patientsith genotype 3, miR-122 expression decreased with treatment

nd did not return to the baseline even 8 weeks after the end of the

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reatment [SVR (n = 6), relapsers (n = 3), non-responders (n = 11),nd lost to follow-up (n = 1)]. In addition, the authors demonstratedhat miR-122 expression in patients with SVR 8 weeks after the

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treatment completion was greater in relation to healthy subjects(n = 30) [97].

Wang et al. evaluated serum miR-122 levels in 105 patientswith chronic HCV infection and 11 patients with acute hepatitis(six with hepatitis E, four with hepatitis B, and one with hepatitisA) [77]. They found that miR-122 levels were significantly higher inchronic HCV and acute hepatitis patients than in healthy individ-uals (n = 33). A strong correlation was found between ALT activityand hepatic inflammation, which was graded using the Ishak scor-ing system, but there was no correlation between miR-122 andALT serum levels or liver inflammatory activity in chronic HCVpatients [77]. Additionally, Zhang et al. reported that HCV-infectedpatients (HCV, n = 34; HCV-HCC, n = 10) had higher miR-155 expres-sion in hepatic tissue than healthy controls (n = 7) and nonalcoholicsteatohepatitis (NASH) patients (n = 12) [78]. They also demon-strated that, before interferon plus ribavirin therapy, circulatingmiR-155 levels were positively correlated with the levels of plasmainterferon-gamma-inducible protein of size 10 kDa (IP-10), whichis a chemokine with increased expression in inflammatory diseases.Patients who achieved SVR had lower expression of circulatingmiR-155, indicating that this miRNA has potential as a responsepredictor [78]. In a different study, Zhang et al. compared expres-sion of serum miR-143 and miR-215 in 118 chronic HCV and 95 HCCpatients with expression in healthy controls (n = 127) [79]. ThesemiRNAs showed increased expression in the case of both chronicHCV and HCC. The AUROC was 0.617 (sensitivity: 78.0%; speci-ficity: 64.0%) for miR-143 and 0.802 (sensitivity: 78.0%; specificity:89.0%) for miR-215 in distinguishing HCV patients from healthycontrols. For distinguishing between the HCC group and the controlgroup, the AUROC was 0.795 (sensitivity: 73.0%; specificity: 83.0%)for miR-143 and 0.816 (sensitivity: 80.0%; specificity: 91.0%) formiR-215 [79].

2.4. Hepatotoxicity

The expression of miRNAs in the serum and plasma ofacetaminophen (APAP)-intoxicated patients was evaluated byStarkey Lewis et al. [80]. These authors found that miR-122 andmiR-192 levels were upregulated in APAP-induced acute liverinjury (APAP-ALI) patients (n = 53) compared with healthy individ-uals (n = 25) and chronic kidney disease patients (n = 22). Althoughno significant correlation was found between miR-122 and pro-thrombin time, total serum bilirubin, or serum creatinine levels,serum miR-122 levels were correlated with ALT in APAP-ALIpatients [80]. In another study, Ward et al. evaluated miRNA expres-sion in both plasma and serum of 49 patients with APAP overdoseand seven patients with ischemic hepatitis [81]. In this work, morethan 100 miRNAs were found to be upregulated at least eight-fold inAPAP compared with a control group that did not use APAP in theprevious week (n = 12). Among these miRNAs, seven (miR-21-5p,miR-27b-3p, miR-122-5p, miR-125b-5p, miR-193a-5p, miR-194-5p, and miR-1290) were found to be able to differentiate betweenischemic hepatitis and APAP-induced liver injury. In this study, theAUROC for miR-122 was 0.897 for distinguishing between APAP-induced and ischemic liver injury. In addition to these miRNAs,miR-23b-5p, miR-28-3p, miR-1247-5p, and miR-4524a-3p wereshown to compose a miRNA recovery profile capable of differen-tiating patients with APAP overdose from those with ischemic liverinjury during N-acetylcysteine treatment, since these miRNAs nor-malized after successful N-acetylcysteine treatment [81].

Likewise, Yang et al. studied miRNA expression in serum andurine samples of children hospitalized for APAP overdose [82]. The

g miRNAs in nontumoral liver diseases, Pharmacol Res (2017),

authors found 147 serum miRNAs that were upregulated in eightchildren hospitalized for APAP overdose, compared with 10 healthychildren (who had not taken APAP for 14 days) and 10 hospital-ized children receiving therapeutic doses of APAP. Among these

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iRNAs, miR-122 stood out as being strongly correlated with theeak serum level of APAP-protein adducts, which is an indicatorf APAP oxidative metabolism. However, no significant correlationas observed between serum miR-122 and ALT. Despite this, it

s interesting to note that these miRNAs were able to differenti-te between children with APAP overdose and patients receivingherapeutic doses [82].

Recently, Singhal et al. attempted to determine whether newiomarkers that have been proposed for hepatotoxicity, includ-

ng miR-122 as well as sorbitol dehydrogenase, cytokeratin 18,igh-mobility group box-1, and glutamate dehydrogenase, would

ncrease during ALT elevation related to cholestyramine treatment,hich is a cholesterol-lowering drug not associated with clini-

ally significant liver damage. In 11 patients out of a total of 67,n ALT elevation greater than three times the upper normal limitas observed and accompanied by an increase in serum miR-122

reater than 20-fold, suggesting a considerable degree of specificiver injury [83].

Ding et al., studying a different kind of poisoning, assessed thexpression of miR-122 in the serum of 36 patients with acute hep-totoxicity caused by paraquat (N,N′-dimethyl-4,4′-bipyridiniumichloride), a widely used herbicide [84]. The authors demon-trated that serum miR-122 levels were significantly elevated inhis group of patients compared with the control group (n = 19), and

positive correlation was found between miR-122 and ALT greaterhan 600 U/I. In addition, daily monitoring of miR-122 expressionhroughout treatment showed that there was a reduction in both

iR-122 and ALT with treatment evolution [84].Although further studies are required to validate the clinical

pplication of miRNAs, in general, miR-122 has stood out amongll miRNAs related to hepatotoxicity as a potential marker of acuteiver injury. Future studies exploring miR-122 and other miRNAs inon-APAP liver toxicity are needed to discover new diagnostic andrognostic tools in this often-neglected area.

.5. Nonalcoholic fatty liver disease

Cermelli et al. evaluated the expression of miR-16, miR-21, miR-4a, and miR-122 in chronic HCV infection and nonalcoholic fatty

iver disease (NAFLD) [63]. They demonstrated that both in theohort test and in the independent validation cohort the expressionf miR-16, miR-34a, and miR-122 was higher in both HCV (n = 53)nd NAFLD (n = 34) patients than in the control group (n = 19), andhat miR-21 expression was not altered in either group. In addition,

iR-34a and miR-122 correlated positively with ALT, AST, fibro-is stage, and inflammatory activity. In NAFLD patients, miR-122lso correlated with serum lipids (total cholesterol level and low-ensity lipoprotein). miR-34a and miR-122 could also be used toiscriminate between NAFLD patients with NAFLD-simple steatosisNAFLD-SS) and nonalcoholic steatohepatitis (NASH), which wereistinguished using the NAFLD activity score (NAS); for distinguish-

ng between these groups, miR-34a had an AUROC of 0.75 andiR-122 had an AUROC of 0.70. Thus, miR-34a and miR-122 might

epresent useful diagnostic biomarkers in NAFLD [63].Likewise, Miyaaki et al. evaluated miR-122 expression in both

iver biopsy (n = 67) and serum samples (52 from 67 samples) fromAFLD patients, and found a significant correlation between serumiR-122 and liver miR-122 [85]. In addition, patients with mild

teatosis (less than 33%) had lower serum miR-122 expression thanatients with severe steatosis (greater than 33%). There was also

ower expression of serum miR-122 in patients with severe fibrosisgreater than F1) than in patients with mild fibrosis (F0 or F1). The

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uthors constructed an ROC curve for serum miR-122 as a predictorf liver fibrosis and found an AUROC of 0.820, which was greaterhan the AUROCs for hyaluronic acid and type IV collagen, both

arkers associated with liver fibrosis [85].

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Recently, Pirola et al. demonstrated, through an independentvalidation cohort, that serum miR-122, miR-192, and miR-375 lev-els were upregulated in patients with NAFLD (both SS and NASH)(n = 65) [65]. These miRNAs correlated with the severity of diseaseand had greater expression in NASH patients than in SS patients.Despite this, these miRNAs were not related to the severity offatty liver infiltration and lobular inflammation. On the other hand,miR-122 correlated with advanced fibrosis, AST, ALT, and GGT. TheAUROCs for differentiating between the advanced stages of the dis-ease were similar for miR-122, miR-192, and miR-375 (∼0.7). Inaddition, miR-122 has been shown to be able to distinguish a NASscore greater than five to the same extent as hepatic transaminases(AST and ALT) [65].

2.6. Other liver diseases

Serum miR-21 and miR-122 expressions were evaluated byMigita et al. in 46 patients with type 1 autoimmune hepatitis(AIH) [86]. miR-21 and miR-122 were found to be upregulated inAIH patients as compared with chronic HCV patients (n = 40) andhealthy controls (n = 13). Both miR-21 and miR-122 were positivelycorrelated with ALT and AST, but there was no significant cor-relation with type IV collagen. Only miR-21 presented a positivecorrelation with liver necroinflammation grading, although bothmiR-21 and miR-122 presented lower expression in patients withadvanced fibrosis. In addition, it was demonstrated that these miR-NAs are upregulated in untreated AIH patients, but downregulatedfour weeks after corticosteroid therapy (n = 34, with paired serumsamples from the same subjects) [86].

Ninomiya et al. assessed miRNA expression in patients withprimary biliary cholangitis (PBC), an autoimmune disease char-acterized by progressive destruction of bile ducts and positiveantimitochondrial antibody (AMA) [87]. In the validation phaseof the study, the authors found that miR-197-3p and miR-505-3pwere downregulated in PBC patients compared with healthy indi-viduals, CHB patients, and chronic HCV patients (each of the fourgroups consisted of 10 samples) [87]. In addition, Tan et al. found,in the validation phase of their study, that serum miR-122 and miR-141-3p levels were upregulated, and miR-26b-5p downregulated,in PBC patients (n = 82) compared with healthy individuals (n = 60)[66]. The authors constructed a miRNA panel with these three miR-NAs and obtained an AUROC of 0.905 (sensitivity: 80.5%; specificity:88.3%) for distinguishing between PBC patients and healthy indi-viduals. In addition, this miRNA panel presented better diagnosticperformance than ALP and antinuclear antibody, but lower sensi-tivity and specificity than AMA [66].

Alcoholic liver disease (ALD) is one of the major causes ofcirrhosis and can present histologically as steatosis, steatohepati-tis, and cirrhosis. Chen et al. identified a serum miRNA patternthat could be used to distinguish alcoholic steatohepatitis patients(ASH) (n = 3) from healthy subjects (n = 3) [88]. Some of the miR-NAs included in this profile were miR-135b, miR-490, miR-761 (allupregulated), miR-203, and miR-214 (both downregulated) [88].Blaya et al. found that serum miR-182 levels were upregulatedin 28 patients with alcoholic hepatitis (AH) compared with thecontrol group (n = 8), but there was no association with diseaseseverity and mortality. In contrast, miR-182 expression in the livershowed association with disease severity and mortality; thus, theauthors suggested that serum miR-182 levels might not be a suit-able biomarker for AH [89]. Li et al. found that serum miR-223levels were upregulated in alcoholics (n = 300, including 140 with

g miRNAs in nontumoral liver diseases, Pharmacol Res (2017),

recent excessive drinking and 160 without recent excessive drink-ing) compared with healthy individuals (n = 45) [90]. The authorsfurther demonstrated that miR-223 is important for limiting hep-atic neutrophil infiltration that occurs in alcoholic liver disease [90].

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Interestingly, Hoy et al. evaluated the expression of miRNAsn serum from patients infected with Schistosoma mansoni in theiida community of Uganda, an area of high endemicity (n = 20),nd in the Chiredzi community of Zimbabwe, an area of lowndemicity (n = 5) [91]. The control groups were composed of indi-iduals from the same areas whose feces did not present parasiteggs (n = 10 for the Piida community and n = 9 for the Chiredziommunity). The authors demonstrated that infected individualsould not be discriminated from uninfected individuals based onerum host-derived miRNAs, but could be discriminated basedn parasite-derived miRNAs found in patients’ serum. The threearasite-derived miRNAs found in patient serum were sma-miR-77, sma-miR-3479-3p, and sma-bantam. The authors used thesehree miRNAs to construct a miRNA panel that presented an AUROCf 0.845 (sensitivity: 90.0%; specificity: 80.0%) for the Piida commu-ity and an AUROC of 0.933 (sensitivity: 80.0%; specificity: 89.0%)

or the Chiredzi community for distinguishing infected individualsrom the control group [91].

.7. MicroRNA across distinct disorders

Out of the studies presented in Table 1, 19 reportedncreased expression of circulating miR-122 in patients with HBV69,70,72,73], HCV [63,64,75–77], HCC [70], NAFLD [63,65,85],IH [86], PBC [66], LC [59], APAP overdose [80–82], cholestyra-ine treatment [83], or paraquat poisoning [84]. Nine of these

tudies showed a positive correlation between miR-122 and ALT59,63–65,75,76,80,81,84], while six others showed not so sig-ificant correlation [70,72,77,82,83,85]. Adding to the fact thatiR-122 is considered hepatospecific [52], miR-122 could be

otent as a marker for liver injury. In addition to miR-122, otheriRNAs have also been described as organ-specific, such as miR-

08a and miR-208b (heart) [98–100], or miR-9 and miR-124 (brain)101–103] that may be potential diagnostic markers since they areound in serum or plasma samples after a tissue injury [100,101].ther miRNAs, such as miR-192 (liver) [80], miR-1 and miR-499

heart) [80,100], and miR-218 (brain) [80], have been described asrgan-enriched, that is, abundant in certain organs but not spe-ific to them; they can also be released into circulation when thergan is injured. This points at the possibility of using both organ-pecific and organ-enriched miRNAs to improve the sensitivity andpecificity of identification of the specific cause of a liver disease80,100].

In comparison to the other miRNAs presented in Table 1, therere not enough studies yet to assign their expression to a spe-ific disease or a disease stage. However, it is clearly observedhat different circulating miRNAs are altered for the same etiol-gy: miR-21 (upregulated: HBV [73], HCV, HCV-HCC [62], and AIH86]), miR-29a (downregulated: LC [70]; upregulated: CHB [70]),

iR-34a (upregulated: NAFLD and HCV [63]), miR-92a (upregu-ated: HBV [72]), miR-106a (upregulated: HBV [72]), miR-106bdownregulated: CHB cirrhosis [60]), miR-130a (upregulated: HBV73]), miR-130b (downregulated: CHB [71]; upregulated: HBV-CC [61,71]), miR-181b (upregulated: CHB cirrhosis [60], and LC

67]), miR-192 (upregulated: HBV [73], APAP overdose [80], andAFLD [65]), miR-215 (upregulated: CHB [69], HCV, and HCC [79]),iR-223 (upregulated: alcoholics [90], and HBV [72]), miR-885-5p

upregulated: CHB [58,69], LC, and HCC [58]). Table 2 presents someiRNAs, arranged in ascending order of miRNA nomenclature, in

rder to provide insights into the different etiologies in which aiven miRNA might be altered. For example, miR-125b, describeds a tumor suppressor by targeting Bcl-2 and stimulating apoptosis

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71,104], has been found to be upregulated in microvesicles isolatedrom plasma samples of patients with CHB, but downregulatedn HCC, thereby suggesting that a lower expression of miR-125b

ight result in loss of tumor suppression, hence causing HCC

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development [71]. It is important to note that similar independentstudies have reported concordant results. For example, circulat-ing miR-122 has been described as upregulated in patients withHCV [63,64,75–77], NAFLD [63,65,85], or APAP overdose [80–82];the results of these studies have been confirmed by independentcohort validation for HCV [63,64] and NAFLD [63,65]. In this regard,confirmation of these results by independent research groups iscritical for validating the potential of circulating miRNAs in prog-nosis and diagnosis of diseases. Thus, further studies are necessaryto evaluate whether an expression profile in a particular liver dis-ease could be correlated to the initial liver injury, inflammation,establishment, and progression of the disease, eventually hintingat the strategies for cure or even to characterize the etiology.

3. Pre-analytical aspects involving miRNA analysis

McDonald et al. evaluated the expression of miR-15b, miR-16,miR-24, and miR-122 in serum and plasma samples from healthysubjects (n = 10) within 72 h of storage. The authors demonstratedthat these miRNAs were stable for up to 72 h if refrigerated (4 ◦C)or frozen (−20 ◦C), and up to 24 h if kept at room temperature. Inaddition, hemolysis (up to 600 mg/dL and 1200 mg/dL hemoglobin)did not alter miR-24 and miR-122, respectively, but 25 mg/dLhemoglobin caused a significant increase in miR-15b and miR-16concentrations indicating that these miRNAs may be released fromerythrocytes [105]. In another study, the expression of miR-15b,miR-16, and miR-24 in plasma samples (n = 2) were found stablewhen kept at room temperature for up to 24 h or subjected toeight cycles of freeze-thaw. In addition, the authors demonstratedthat unlike naked miRNAs that are susceptible to degradation byRNase activity in plasma, the levels of endogenous miRNAs miR-15b, miR-16, and miR-24 were not significantly altered, indicatingthat circulating miRNAs exist in a form resistant to plasma RNaseactivity [106].

Blondal et al. evaluated the expression of 119 miRNAs mostcommonly found in serum and plasma samples, including let-7b,miR-21, miR-23a, miR-29a, miR-92a, miR-106a, miR-106b, miR-130a, miR-192, miR-215, miR-223, miR-451 among others [107].The serum and plasma samples were grouped as hemolyzed andnon-hemolyzed, each group consisting of 381 samples, resultingin an n > 1500. The authors suggested two methods for hemolysisdetection: absorbance and delta Cq (miR-23a – miR-451). In thefirst method, the hemolyzed samples showed an absorbance peakat a wavelength of 414 nm corresponding to oxyhemoglobin, whilethe non-hemolyzed samples did not. Since as miR-23a expressionwas stable in serum and plasma samples and remained unaffectedby hemolysis, and miR-451 is known to be enriched in erythrocytes,in the second method, the authors found that delta Cq (miR-23a –miR-451) > 5 could be an indicator of possible erythrocyte miRNAcontamination, and the delta Cq (miR-23a – miR-451) ≥ 7 indicateda high risk of hemolysis. The authors carefully draw attention tothe fact that many miRNAs found in serum and plasma samplesremain unaffected by hemolysis and therefore may remain associ-ated as disease biomarkers, but hemolysis may introduce changesin the miRNA expression profile causing systematic error in the dataanalysis [107].

Grasedieck et al. evaluated the stability and amount of miRNA(pg/�L) using Bioanalyzer Small RNA kit (Agilent Technologies,Santa Clara, CA) and identified individual miRNA (miR-24, miR-93, miR-223, and miR-451) by RT-qPCR in serum samples fromhealthy subjects (n = 3) and patients with multiple myeloma (n = 3)

g miRNAs in nontumoral liver diseases, Pharmacol Res (2017),

for short- (10 days), intermediate- (≤20 months), and long-termstorages (≤10 years) at −80 ◦C and −20 ◦C; short-term storage sam-ples of serum from healthy subjects were also evaluated at roomtemperature. The authors demonstrated that there was no differ-

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Table 2Different liver disorders in which a given circulating miRNA might be altered.

miRNA Disease Expression Observation References

miR-15b CHB down Potential marker of HBV-positive HCC [71]HBV-HCC up [61,71]

miR-16 HBV-ASC, CHB, HBV-ACLF up Possibly associated with HBeAg production in patients withCHB and potential marker for early stages of CHC and NAFLD

[72]NAFLD, HCV up [63]

miR-21 AIH up Possibly involved in the mediation of inflammation in AIHand potential prognostic and injury marker of HBV-ACLFand CHC

[86]HBV-ACLF up [73]HCV, HCV-HCC up [62]

miR-122 Decompensated LC down Possible role as a prognostic marker [59]miR-122 AIH up Hepato-specific, potential marker for liver injury [86]

APAP overdose up [80–82]CHB, HCC up [70]HBV-ACLF up [73]HBV-ASC up [72]HCV up [64,75–77]NAFLD up [63,65,85]Paraquat poisoning up [84]PBC up [66]

miR-125b HBV-HCC down Potential marker of HCC [71]miR-125b CHB up Possibly associated with HBeAg production and maintenance of CHB [69,71]miR-192 APAP overdose up Liver-enriched miRNA, potential markers of liver injury,

possibly associated with HBeAg production, andassociation with NAFLD severity

[80]CHB up [69]HBV-ACLF up [73]NAFLD up [65]

miR-885-5p CHB up Possibly associated with HBeAg production andmaintenance of CHB, and potential markers for liver injury

[69]LC, CHB, HCC up [58]

AIH, autoimmune hepatitis; APAP, acetaminophen (N-acetyl-p-aminophenol); CHB, chronic hepatitis B; CHC, chronic hepatitis C; HBeAg, hepatitis B e antigen; HBV, hepatitisB virus; HBV-ACLF, hepatitis B virus-related acute-on-chronic liver failure; HBV-ASC, hepatitis B virus-chronic asymptomatic carriers; HBV-HCC, hepatitis B virus-relatedh HCV-Hn

eass9apmesaymitcBm3fct2taTmms

2s1ma

epatocellular carcinoma; HCC, hepatocellular carcinoma; HCV, hepatitis C virus;

onalcoholic fatty liver disease; PBC, primary biliary cholangitis.

nce between short-term storage at −80 ◦C and −20 ◦C, but storaget room temperature and repeated freeze-thaw cycles did lead to aignificant reduction in miRNA levels compared to the continuoustorage samples at −80 ◦C, although expression of miR-24, miR-3, miR-223, and miR-451 remained detectable by RT-qPCR. Theuthors observed no difference in the miRNA concentration in sam-les stored for intermediate-term at −80 ◦C and −20 ◦C, except foriR-93 that showed a significant decrease, hence suggesting differ-

nces in the stability of the individual serum miRNAs. In long-termtorage conditions, the authors reported a small reduction in themount of miRNA within 4 years and a significant reduction after 6ears; these results were confirmed by RT-qPCR for all of miR-24,iR-93, miR-223, and miR-451. Thus, the authors emphasize the

mportance of consistency of storage conditions for the samples andhe stability of miRNAs for use as biomarkers even under conditionsonsidered unsuitable for messenger RNA (mRNA) [108]. Likewise,alzano et al. demonstrated that the expression of miR-21-5p,iR-125b-5p, miR-126-3p, miR-200b-5p, miR-200c-3p, miR-212-

p, miR-425-5p, and miR-579-3p were stable in plasma samplesrom healthy subjects (n = 5) even 12 months of storage at −80 ◦C,ompared to that of the same samples before freezing. In addition,he expression of these miRNAs were evaluated in another set of4 plasma samples from healthy subjects stored at −80 ◦C for upo 14 years demonstrating that all miRNAs remained detectable,lthough only miR-212-3p expression did not significantly reduce.he authors suggest that the presence of AU nucleotide sequencesay contribute to the reduced miRNA stability. Overall, none of theiRNAs showed any change in stability over a period of 4 years of

torage [109].Köberle et al. evaluated the expression of miR-1, miR-16, miR-

1, miR-122, and miR-142-3p in serum samples from healthyubjects (n ≥ 3) incubated at room temperature for up to 24 h; miR-

Please cite this article in press as: A.E. do Amaral, et al., Circulatinhttps://doi.org/10.1016/j.phrs.2017.10.002

and miR-122 levels decreased significantly after 3 h, whereasiR-16, miR-21, and miR-142-3p levels were reduced only slightly

fter 5 h, suggesting that endogenous miRNAs exhibit different sta-

CC, hepatitis C virus-related hepatocellular carcinoma; LC, liver cirrhosis; NAFLD,

bilities. In addition, they demonstrated that RNase A, B, and Cinhibitor (0.3 U/�L) minimized the loss of miRNAs in serum andwhole blood samples (n = 3). The authors also showed that theexpression of miR-122, which is not expressed in blood cells, inhemolyzed serum samples were more stable compared to thatin non-hemolyzed samples (n = 3) suggesting that hemolysis maycontribute to the stability of circulating miRNAs due to the releaseof RNase inhibitors from blood cells, and that vesicle-associatedmiRNAs exhibited increased resistance to RNase A activity in rela-tion to non-vesicle-associated miRNAs [110].

Although it has been demonstrated that miRNAs have adequatestability to be used as biomarkers, collection, preparation, han-dling, and storage of the samples should be standardized to avoidconfounding factors that might influence the results [111,112].

4. miRNAs as biomarkers: methodological considerationsfor clinical use

In addition to other considerations, clinical use of miRNAs asbiomarkers should account for differences in sample types andmethods for normalization of RT-qPCR data. While some researchhas shown that there is a strong correlation between serum andplasma miRNA profiles [81,106], other studies have reported thatsome miRNAs may occur at different levels in these sample types[105,113]. Therefore, caution should be exercised when comparingresults from different studies. Another important technical consid-eration is the normalization of RT-qPCR data to reduce variation inthe quantification of gene expression among samples [114]. Vari-ous methods have been proposed for the normalization of miRNAexpression data, including the use of a spike-in (an exogenous syn-thetic control not present in humans) [68,74,106], RNU6B (U6)

g miRNAs in nontumoral liver diseases, Pharmacol Res (2017),

[58,67,70,71], miR-16 [59,60,62,75], and a combination of threeor more miRNAs that exhibit stable expression between analyzedsamples [69,115,116]. It may be most appropriate to use a combi-nation of three or more miRNAs, because this method may reduce

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A.E. do Amaral et al. / Pharmaco

ariation between technical replicates and minimize the number ofalse-positive downregulated miRNAs being possible the detectionf the real changes in patient samples profiles in function of theisease evolution. In contrast, the use of spike-in controls shoulde limited to controlling for the efficiency of RNA extraction andeverse transcription, because this method does not account for allypes of experimental variability [116].

. Conclusions

Liver diseases are common and remain an important publicealth problem, with increasing mortality and healthcare costs.ew biomarkers to improve the diagnosis and prognostication of

iver diseases could lead to a better management of these chal-enging conditions, reducing health, social, and economic impactsssociated with them. Over the last decade, the amount of pub-ished data on circulating miRNAs as biomarkers for liver diseaseas increased substantially. Some miRNAs, such as miR-122, haveemonstrated potential as diagnostic and prognostic tools for mul-iple liver diseases. Other miRNAs, such as miR-34a, miR-192, and

iR-885-5p, appear to be more disease-specific, and the potentialf these miRNAs to be used as biomarkers needs further investiga-ion with adequate methodology and sample size. In addition, weighlight the fact that suitable methodological and independenttudies with sufficient sample size, preferably involving indepen-ent validation cohort, are still required to evaluate the usefulnessf miRNAs as biomarkers in clinical practice.

onflict of interest

None.

cknowledgements

The authors wish to thanks Conselho Nacional de Desenvolvi-ento Científico e Tecnológico (CNPq, grant n◦457373/2013-0)

nd Coordenac ão de Aperfeic oamento de Pessoal de Nível SuperiorCAPES) for financial support. This work makes part of the thesis oflex E. Amaral.

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