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Pharmacological Research

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MicroRNA profiles in serum samples from patients with stable cirrhosis andmiRNA-21 as a predictor of transplant-free survival

Alex Evangelista do Amarala, Michele Patrícia Rodea, Julia Cisilottoa, Telma Erotides da Silvab,Josiane Fischerb, Camila Matiollob, Elayne Cristina de Morais Ratekeb,Janaína Luz Narciso-Schiavonb, Leonardo Lucca Schiavonb,⁎, Tânia Beatriz Creczynski-Pasaa,⁎

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

A R T I C L E I N F O

Keywords:Circulating microRNALiver cirrhosisLiver diseasesmicroRNAPrognosisSurvival time

A B S T R A C T

MicroRNAs (miRNAs) have remarkable potential as diagnostic and prognostic markers because of their roles indisease pathogenesis. miRNAs can be released into the bloodstream, where they are sufficiently stable to bedetected noninvasively. Here, we prospectively evaluated serum levels of miR-21, miR-34a, miR-122, miR-181b,and miR-885-5p in patients with stable cirrhosis. Total RNA was extracted from the sera of patients with cir-rhosis and healthy individuals, and the expression levels of the target miRNAs were analyzed by reverse tran-scription-quantitative polymerase chain reaction. Serum miRNAs levels were correlated with liver functionparameters, etiology, and complications of cirrhosis. Circulating miR-34a, miR-122, and miR-885-5p levels werehigher in patients with cirrhosis than in healthy individuals. These miRNAs were positively correlated withalanine aminotransferase and aspartate aminotransferase levels, and the relative expression levels were higher inhepatitis C virus-infected patients and lower in patients with Child-Pugh C cirrhosis. miR-122 and miR-885-5plevels were also positively correlated with γ-glutamyl transpeptidase concentrations. miR-21 was associated withtransplant-free survival in univariate Cox regression analysis and remained independently associated with sur-vival after adjustment for age, Child-Pugh classification, Model for End-stage Liver Disease score, and history ofprevious decompensation in multivariate Cox regression analysis. These data suggested that miR-34a, miR-122,and miR-885-5p levels may be more related to the inflammatory process and ongoing hepatocyte damage inpatients with cirrhosis. Moreover, miR-21 levels were independently associated with shorter transplant-freesurvival and may be used as a prognostic tool in outpatients with stable cirrhosis.

1. Introduction

Liver cirrhosis is a major global health problem and is mainly causedby alcohol consumption and hepatitis B and C virus (HBV and HCV) in-fections. Mortality related to liver cirrhosis is responsible for a growingand considerable disease burden, with more than one million deaths at-tributed to the disease in 2010 [1]. Individuals with cirrhosis are vul-nerable to several complications, including variceal bleeding, ascites,spontaneous bacterial peritonitis, hepatic encephalopathy, hepatocellularcarcinoma (HCC), hepatorenal syndrome, hepatopulmonary syndrome,

and portal vein thrombosis [2]. However, determining the prognosis ofpatients with liver cirrhosis is challenging because numerous factors, in-cluding the etiology of cirrhosis, intensity of liver dysfunction, presence ofcomplications, and comorbidities, influence disease progression [3]. Thetransition from compensated disease to decompensated cirrhosis occurs ata rate of 5–7% per year and is associated with high mortality and poorquality of life [3,4].

One way to reduce the economic and social impacts of cirrhosis is toimprove our tools for outcome prediction. An ideal prognostic tool shouldbe reproducible, objective, not influenced by other conditions such as

https://doi.org/10.1016/j.phrs.2018.06.019Received 27 March 2018; Received in revised form 14 June 2018; Accepted 19 June 2018

⁎ Corresponding authors.E-mail addresses: alex.amaral@ufsc.br (A.E.d. Amaral), mip.rode@gmail.com (M.P. Rode), juliacisilotto@hotmail.com (J. Cisilotto), telma.gastro@gmail.com (T.E.d. Silva),

josifischer@yahoo.com.br (J. Fischer), cmatiollo@gmail.com (C. Matiollo), elaynecdemorais@gmail.com (E.C.d. Morais Rateke),janaina.narciso@uol.com.br, leo-jf@uol.com.br (L.L. Schiavon), tania.pasa@ufsc.br (T.B. Creczynski-Pasa).

Abbreviations: 95% CI, 95% confidence interval; ALT, alanine aminotransferase; ALP, alkaline phosphatase; AST, aspartate aminotransferase; CHB, chronic hepatitis B; Cq, quantifi-cation cycle; CRP, C-reactive protein; GGT, γ-glutamyl transpeptidase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HR, hazard ratio; INR, internationalnormalized ratio; MELD, model for end-stage liver disease score; miRNA, microRNA; r, Spearman’s correlation coefficient; RT-qPCR, reverse transcription-quantitative polymerase chainreaction; SD, standard deviation; TGF-β, transforming growth factor-beta

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comorbidities and medications, and reliably predict death early in thecourse of the disease [5]. In recent years, microRNAs (miRNAs) have at-tracted attention not only as potential therapeutic targets, but also as bio-markers in several diseases, including chronic liver diseases [6]. miRNAs aresmall noncoding RNAs approximately 22 nucleotides in length that areinvolved in post-transcriptional gene regulation through interaction withcomplementary regions of mRNA [7]. miRNAs can be detected in severaltypes of biological fluids, including serum samples, in a stable and re-producible way among individuals of the same species [8,9]. DifferentmiRNAs have been found in serum samples from patients with liver dis-eases. Among them, miR-21 [10], miR-34a [11], miR-122 [11–13], miR-181b [14], and miR-885-5p [15] have shown promising results in earlierstudies and may be effective prognostic tools in liver cirrhosis.

miR-21 was described as related to liver [16], pulmonary [17], andcardiac fibrosis [18], besides being found to be upregulated in several tu-mors, including HCC [19,20], in patients with HCV [10], and those withautoimmune hepatitis [21]. miR-34a was described as a mediator of p53function and thus may be involved in the development of some cancers[22]. In this sense, miR-34a was found to be both downregulated in humancancers [22] and increased in HCC [23]. miR-122 regulates metabolicpathways in the liver, such as cholesterol biosynthesis [24], and also reg-ulates HCV replication [25,26]. miR-122 was found to be downregulated inpatients with advanced stages of HCC [27,28], and upregulated in patientswith hepatitis C [11,12]. miR-181b was described to be related to liverfibrosis in patients with liver cirrhosis [14,29] in response to transforminggrowth factor-beta 1 (TGF-β1) signaling [14]. miR-885-5p was found to beupregulated in patients with liver cirrhosis and HCC [15]. In addition, miR-885-5p was described as a tumor suppressor both in neuroblastoma(through activation of the p53 pathway) [30] and in HCC (through de-creased activity of the Wnt/β-catenin pathway) [31]. Given the involve-ment of these miRNAs in other related pathologies, in this study, we aimedto investigate the prognostic value of miR-21, miR-34a, miR-122, miR-181b, and miR-885-5p in serum samples from patients with stable cirrhosis.

2. Methods

2.1. Patients

This was a prospective cohort study that included consecutive adultsubjects (≥ 18 years of age) attending the outpatient clinic at theUniversity Hospital of the Federal University of Santa Catarina, Brazil.The diagnosis of cirrhosis was established histologically (when avail-able) or by the combination of clinical, imaging, and laboratory find-ings in patients with evidence of portal hypertension. Cirrhosis wasconsidered stable in outpatients without any sign of acute deteriorationin their condition [32,33]. Patients with HCC, interferon-based therapywithin the last 30 days, or absence of serum under suitable conditionsfor analysis (hemolyzed, icteric or lipemic samples) were excluded fromthe study. Twenty-eight sex- and age-matched healthy individualsevaluated during routine laboratory tests served as a control group.

Informed consent was obtained from each participant (i.e., patientsand healthy volunteers), and the study protocol conformed to theethical guidelines of the 1975 Helsinki Declaration and was approvedby our institutional review board.

The entire cohort was evaluated at the outpatient clinic of theGastroenterology Division from June 2012 to February 2014. The devel-opment of complications, mortality, or liver transplantation was assessed byperiodic phone calls and during outpatient visits. The final re-evaluationwas performed between June and October 2015. The following clinicalvariables were collected: age, sex, race, alcohol abuse, etiology of cirrhosis,history of previous decompensation and hospitalization, diagnosis of eso-phageal varices, presence of ascites, and encephalopathy. All patients un-derwent laboratory evaluation on the same day of clinical evaluation, andthe following tests were performed for this study: aspartate amino-transferase (AST), alanine aminotransferase (ALT), gamma-glutamyl trans-peptidase (GGT), alkaline phosphatase (ALP), albumin, total bilirubin,

international normalized ratio (INR), creatinine, sodium, platelet count, andC-reactive protein (CRP). Current significant alcohol intake was defined asan average overall consumption of 21 or more drinks per week for men and14 or more drinks per week for women during the 4 weeks before enrol-ment (one standard drink is equal to 12 g absolute alcohol) [34]. The samecriterion was used to define previous alcohol abuse, considering the habi-tual alcohol intake pattern before the last 4 weeks. Child-Pugh classification[35] and Model for End-stage Liver Disease (MELD) scores [36] were usedto assess the severity of hepatic disease.

2.2. Blood sampling and clinical chemistry

Peripheral blood (4mL) was collected in serum clot activator tubesat the day of enrollment in the study. The tubes were centrifuged at3000×g for 10min at room temperature (∼25 °C) in a 5702 centrifuge(Eppendorf, Hamburg, Germany). Serum samples were then aliquotedand stored at −80 °C until RNA isolation. Prior to use, serum sampleswere centrifuged at 16,000×g at 4 °C for 10min to remove cell debris[37], and 500 μL of the supernatant was used for RNA isolation. Mar-kers of liver injury and liver function parameters were measured at theClinical Laboratory of the University Hospital/UFSC.

Table 1Characteristics of the included patients.

Variables Patients (n= 186)

Age (years), mean ± SD (median) 54.4 ± 12.4 (54.0)Sex, n (%)Female 59 (31.7)Male 127 (68.3)

Significant current alcoholisma n (%) 12 (6.5)Etiology of cirrhosis,c n (%)Alcohol 101 (54.3)Autoimmune hepatitis 7 (3.8)Hepatitis B 10 (5.4)Hepatitis C 60 (32.3)Other ethiologiesb 16 (8.6)

Diabetes mellitus 52 (28.0)Prior decompensation, n (%)Ascites 90 (48.4)Hepatic encephalopathy 38 (20.4)Gastroesophageal variceal hemorrhage 101 (54.3)

Esophageal varices 148 (79.6)Child-Pugh’s classification, n (%)A 117 (62.9)B 58 (31.2)C 11 (5.9)

MELD score, mean ± SD (median) 10.1 ± 2.5 (9.7)Laboratory parameters, mean ± SD (median)Albumin (g/dL) 3.4 ± 0.5 (3.4)ALT (IU/L) 71.1 ± 52.3 (52.0)ALP (IU/L) 138.8 ± 67.2 (123.0)AST (IU/L) 64.4 ± 45.7 (47.5)Creatinine (mg/dL) 0.9 ± 0.2 (0.9)CRP (mg/L) 6.2 ± 7.4 (3.5)GGT (IU/L) 191.1 ± 306.4 (110.5)Platelets/mm3 103,596.8 ± 67,255.8 (81,000.0)INR 1.2 ± 0.1 (1.2)Sodium (mEq/L) 137.4 ± 2.7 (138.0)Total bilirubin (mg/dL) 1.3 ± 0.9 (1.0)

Abbreviations: ALT, alanine aminotransferase; ALP, alkaline phosphatase; AST,aspartate aminotransferase; CRP, C-reactive protein; GGT, γ-glutamyl trans-peptidase; INR, international normalized ratio; MELD, Model for End-stageLiver Disease; SD, standard deviation.

a Significant current alcoholism: women>168 g/week, men>252 g/week.b Other etiologies: primary biliary cholangitis (n= 7, 3.8%), primary scler-

osing cholangitis (n= 1, 0.5%), hereditary hemochromatosis (n= 2, 1.1%),and nonalcoholic steatohepatitis (n= 6, 3.2%).

c More than one etiologic factor could be present in specific cases.

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2.3. RNA isolation and reverse transcription-quantitative polymerase chainreaction (RT-qPCR)

Total RNA was obtained using amirVana PARIS RNA kit, according tothe manufacturer's instructions (Ambion, Thermo Fisher Scientific,Waltham, MA, USA). At the time of RNA isolation, spike-in 25 fmol cel-

miR-238-3p was added to each serum sample after denaturation forquality control of RNA isolation and RT-qPCR. Total RNA quantificationand quality were assessed by absorbance in a NanoVue Plus spectro-photometer (GE Healthcare Life Sciences, Little Chalfont, UK). A TaqManMicroRNA Reverse Transcription kit (Applied Biosystems [ABI], FosterCity, CA, USA) was used to obtain cDNA from total RNA as specified by

Fig. 1. Comparison of the relative expression levels of the studied miRNAs in patients and controls. The number of patients in each group is indicated in the figure. Inthe boxplot, horizontal lines indicate the median per group, minimum and maximum values. The upper and lower borders of the boxes indicate the 75th and 25thpercentiles, respectively. Statistical differences between groups were calculated using Mann-Whitney U tests. The P values indicate whether there were significantdifferences relative to the healthy controls and this is indicated by asterisks.

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Fig. 2. Correlations between serum miRNA levels and alanine aminotransferase (ALT), aspartate aminotransferase (AST), and γ-glutamyl transpeptidase (GGT) levelsas markers of liver injury in all patients with cirrhosis. Points represent the relative expression levels of miR-21 (A, E, I), miR-34a (B, F, J), miR-122 (C, G, K), andmiR-885-5p (D, H, L) as a function of ALT, AST and GGT. Correlations between the numerical variables were evaluated by using Spearman’s correlation coefficient(r). P values indicate whether there was a significant correlation, and is indicated by asterisks.

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the manufacturer. cel-miR-238-3p, miR-16-5p, miR-21-5p, miR-34a-5p,miR-122-5p, miR-181b-5p, or miR-885-5p levels were assessed byTaqMan Custom Plating (ABI) on a StepOnePlus Real-Time PCR System(ABI). Quantification cycle (Cq), which corresponds to the cycle at whichthe fluorescent signal from amplification exceeds the background fluor-escence in qPCR, was used to calculate the relative expression. The re-sults of qPCR were given as relative expression calculated by2ΔCq(miR−16−5p − miR−target)

[cirrhotic individual] / 2ΔCq(miR−16−5p −

miR−target)[healthy group] [38], yielding the expression level of the target

(miR-21-5p, miR-34a-5p, miR-122-5p, miR-181b-5p, or miR-885-5p) inthe serum of patients with cirrhosis compared with that in healthy in-dividuals.

2.4. Statistical analysis

Data were analyzed using IBM SPSS Statistics version 22 software(IBM, Chicago, IL, USA). Graphs were constructed using GraphPadPrism version 5 software (GraphPad Software, La Jolla, CA, USA). Thenormality of the variable distribution was determined by one-sampleKolmogorov-Smirnov tests. The correlations between numerical vari-ables were evaluated using Spearman’s correlation coefficient.Continuous variables were compared using Mann-Whitney U tests. Acomposite clinical endpoint was defined, composed of liver transplan-tation or liver-related death (transplant-free survival). In cases in whichno endpoint was reached, individuals were censored at the date of studydata collection. Univariate and multivariate Cox regression analyseswere used to investigate the association between the variables andtransplant-free survival. Kaplan-Meier curves were used to illustratetransplant-free survival according to two strata, which were defined bythe cutoff of selected miRNAs. P values were considered significantwhen lower than 0.05. All figures and the graphical abstract were de-signed according to the "Guidelines for preparing color figures for ev-eryone including the colorblind" described by Roskoski [39].

3. Results

3.1. Characteristics of the included patients and miRNAs in the study groups

Between June 2012 and February 2014, 194 patients were eval-uated, and eight were excluded because of inadequate quality of storedsamples. Table 1 shows the main characteristics of the 186 patientsincluded in the analysis. The most common etiological factor was al-cohol abuse (54.3%), followed by chronic HCV infection (32.3%) and

chronic hepatitis B (5.4%). At the time of initial evaluation, 62.9% wereclassified as Child-Pugh A, 31.2% were classified as Child-Pugh B, and5.9% were classified as Child-Pugh C. The mean MELD score was10.1 ± 2.5 (median 9.7).

Levels of miR-21, miR-34a, miR-122, miR-181b, and miR-885-5pwere determined from the sera of 186, 128, 176, 165, and 182 patients,respectively. Serum levels of these miRNAs are reported as relativeexpression, which corresponds to the number of times the expressionlevel was altered compared with that in healthy individuals. Comparedwith healthy controls, there were no significant differences in the ex-pression levels of miR-21 (P= 0.095; Fig. 1A) and miR-181b (P=0.235; Fig. 1D) in patients with cirrhosis. However, patients with cir-rhosis exhibited higher miR-34a (P= 0.001; Fig. 1B), miR-122(P<0.001; Fig. 1C), and miR-885-5p (P<0.001; Fig. 1E) expressionlevels than healthy individuals.

3.2. Serum miRNAs levels according to baseline characteristics of theincluded patients

Spearman’s correlation coefficient (r) was used to identify correla-tions between numerical variables of interest. ALT levels were posi-tively correlated with the expression levels of miR-34a (r= 0.313;P<0.001), miR-122 (r= 0.441; P<0.001), and miR-885-5p(r= 0.480; P<0.001; Fig. 2A–D). Similarly, AST levels were positivelycorrelated with the expression levels of miR-34a (r= 0.384;P<0.001), miR-122 (r= 0.429; P<0.001), miR-885-5p (r= 0.494;P<0.001), and miR-21 (r= 0.157; P= 0.032; Fig. 2E–H). GGT levelswere positively correlated with the expression levels of miR-122(r= 0.188; P= 0.013), miR-885-5p (r = 0.161; P= 0.030; Fig. 2I–L).A negative correlation between miR-885-5p and INR was observed (r =-0.166; P= 0.025). Table 2 shows the correlation analysis between thestudied miRNAs and other baseline numerical variables.

When evaluating only patients with hepatitis C-related cirrhosis,ALT levels were positively correlated with miR-21 (r= 0.337; P =0.009), miR-34a (r= 0.428; P = 0.006), miR-122 (r= 0.594;P<0.001), and miR-885-5p (r= 0.605; P<0.001; Fig. 3A–D). Simi-larly, AST levels were positively correlated with miR-21 (r= 0.262; P= 0.043), miR-34a (r= 0.380; P =0.016), miR-122 (r= 0.585;P<0.001), and miR-885-5p (r= 0.545; P<0.001; Fig. 3E–H). GGTlevels were positively correlated with miR-122 (r= 0.359; P= 0.005)and miR-885-5p (r = 0.305; P= 0.018; Fig. 3I–L).

Spearman’s correlation coefficient (r) was also used to identifycorrelations between the miRNAs studied and ALT, AST, and GGT levels

Table 2Spearman’s correlation coefficient between miRNAs and baseline numerical variables (correlation analysis between miRNAs and other liver enzymes are shown inFigs. 2–4).

Variables miR-21 miR-34a miR-122 miR-181b miR-885-5p

r P r P r P r P r P

Age −0.008 0.911 −0.017 0.847 −0.072 0.344 0.023 0.765 0.014 0.856Albumin −0.094 0.203 −0.157 0.077 0.031 0.686 −0.077 0.325 −0.004 0.953ALP 0.002 0.982 0.026 0.769 −0.004 0.959 0.050 0.527 < 0.001 0.997Creatinine −0.028 0.703 −0.080 0.369 −0.085 0.265 0.103 0.187 0.003 0.965CRP −0.096 0.197 0.013 0.881 −0.145 0.056 −0.136 0.083 −0.096 0.198INR 0.017 0.822 −0.058 0.517 −0.107 0.156 0.007 0.932 -0.166 0.025*MELD score −0.006 0.935 −0.086 0.336 −0.101 0.183 0.026 0.739 −0.092 0.218Platelet count 0.064 0.385 −0.152 0.086 −0.120 0.112 −0.026 0.742 -0.030 0.691Sodium 0.045 0.548 −0.025 0.780 −0.041 0.592 −0.043 0.583 −0.075 0.317Total bilirubin −0.057 0.442 0.051 0.566 −0.010 0.891 −0.029 0.709 -0.058 0.436miR-34a 0.434 < 0.001***miR-122 0.490 < 0.001*** 0.578 < 0.001***miR-181b 0.523 < 0.001*** 0.251 0.005** 0.199 0.011*miR-885-5p 0.557 < 0.001*** 0.576 < 0.001*** 0.791 < 0.001*** 0.308 < 0.001***

Abbreviations: ALP, alkaline phosphatase; CRP, C-reactive protein; INR, international normalized ratio; MELD, Model for End-stage Liver Disease. The correlationsbetween numerical variables were evaluated using Spearman’s correlation coefficient (r). P values indicate whether there was a significant correlation, and this isindicated by asterisks.

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Fig. 3. Correlations between serum miRNA levels and alanine aminotransferase (ALT), aspartate aminotransferase (AST), and γ-glutamyl transpeptidase (GGT) levelsas markers of liver injury in patients with hepatitis C-related cirrhosis. Points represent the relative expression levels of miR-21 (A, E, I), miR-34a (B, F, J), miR-122(C, G, K), and miR-885-5p (D, H, L) as a function of ALT, AST and GGT. Correlations between the numerical variables were evaluated by using Spearman’s correlationcoefficient (r). P values indicate whether there was a significant correlation, and is indicated by asterisks.

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Fig. 4. Correlations between serum miRNA levels and alanine aminotransferase (ALT), aspartate aminotransferase (AST), and γ-glutamyl transpeptidase (GGT) levelsas markers of liver injury in patients classified as Child-Pugh A/B. Points represent the relative expression levels of miR-21 (A, E, I), miR-34a (B, F, J), miR-122 (C, G,K), and miR-885-5p (D, H, L) as a function of ALT, AST and GGT. Correlations between the numerical variables were evaluated by using Spearman’s correlationcoefficient (r). P values indicate whether there was a significant correlation, and is indicated by asterisks.

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among patients with liver cirrhosis classified as Child-Pugh A/B. ALTlevels were positively correlated with the expression levels of miR-34a(r= 0.325; P<0.001), miR-122 (r= 0.450; P<0.001), and miR-885-5p (r= 0.495; P<0.001; Fig. 4A–D). Similarly, AST levels were posi-tively correlated with the expression levels of miR-34a (r= 0.420;P<0.001), miR-122 (r= 0.467; P<0.001), miR-885-5p (r= 0.534;P<0.001), and miR-21 (r= 0.157; P= 0.038; Fig. 4E–H). GGT levelswere positively correlated with the expression levels of miR-122(r= 0.193; P= 0.013), and miR-885-5p (r = 0.171; P= 0.026;Fig. 4I–L).

No differences were observed for miR-34a (P= 0.196), miR-122(P= 0.612), miR-181b (P= 0.497), and miR-885-5p (P= 0.184)expression levels, based on sex. There was a trend towards higher miR-21 expression in men (P= 0.066). Similarly, there was a trend towardshigher miR-34a among patients with current significant alcohol con-sumption (P= 0.054), whereas no differences were observed for miR-21 (P = 0.518), miR-122 (P= 0.685), miR-181b (P= 0.702), andmiR-885-5p (P= 0.202) expression levels. Regarding the etiology ofcirrhosis, miR-34a (P= 0.006; Fig. 5B), miR-122 (P= 0.011; Fig. 5C),and miR-885-5p (P<0.001; Fig. 5D) showed significantly higher re-lative expression in HCV-related cirrhosis than in the other etiologies,but no differences were observed for miR-21 (P= 0.139; Fig. 5A) andmiR-181b (median: 1.6 vs. 1.5, respectively; P= 0.700) expressionlevels. However, patients with HCV-related cirrhosis showed higherrelative expression levels of miR-21 (P = 0.020; Fig. 5A), miR-34a(P < 0.001; Fig. 5B), miR-122 (P < 0.001; Fig. 5C), and miR-885-5p

(P < 0.001; Fig. 5D) than healthy individuals, but no difference wasobserved for miR-181b (median: 1.6 vs. 1.3, respectively; P= 0.197).In addition, only miR-34a (P= 0.005; Fig. 5B), miR-122 (P < 0.001;Fig. 5C), and miR-885-5p (P = 0.001; Fig. 5D) levels were found to bedifferent between healthy individuals and patients with non HCV-re-lated cirrhosis; no difference was observed for miR-181b (median: 1.3vs. 1.5, respectively; P= 0.317). There was a trend towards lower miR-181b expression among patients with HBV infection (P = 0.057),whereas no differences were observed for miR-21 (P= 0.564), miR-34a (P= 0.625), miR-122 (P= 0.972), and miR-885-5p (P= 0.226)expression levels.

Patients with previous ascites exhibited lower miR-122 relativeexpression than those without history of ascites (P= 0.022). Therewere no significant differences in the relative expression levels of thestudied miRNAs according to the presence of any complications ofcirrhosis and history of previous decompensation [ascites: miR-21 (P=0.826), miR-34a (P= 0.734), miR-181b (P= 0.091), miR-885-5p(P= 0.130); hepatic encephalopathy: miR-21 (P= 0.930), miR-34a(P= 0.097), miR-122 (P= 0.562), miR-181b (P= 0.987), miR-885-5p (P= 0.365); gastroesophageal variceal hemorrhage: miR-21 (P=0.236), miR-34a (P= 0.220), miR-122 (P= 0.364), miR-181b (P=0.104), miR-885-5p (P= 0.500); presence of esophageal varices: miR-21 (P= 0.475), miR-34a (P= 0.553), miR-122 (P= 0.582), miR-181b (P= 0.734), miR-885-5p (P= 0.970)]. When the Child-Pughclassification was dichotomized as A/B and C, patients classified asChild-Pugh C showed lower relative expression levels of miR-34a (P=

Fig. 5. Serum miR-21, miR-34a, miR-122, and miR-885-5p levels in patients with cirrhosis with or without hepatitis C. The number of patients in each group isindicated in the figure. In the boxplot, horizontal lines indicate the median per group, minimum and maximum values. The upper and lower borders of the boxesindicate the 75th and 25th percentiles, respectively. Statistical differences between groups were calculated using Mann-Whitney U tests. P values indicate whetherthere were significant differences relative to the healthy controls or between patients with non-HCV cirrhosis (HCV−) and patients with HCV-cirrhosis (HCV+), andthis is indicated by asterisks.

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0.010; Fig. 6B), miR-122 (P= 0.011; Fig. 6C), and miR-885-5p (P=0.035; Fig. 6D) as compared to patients classified as Child-Pugh A/B,but no differences were observed for miR-21 (P= 0.380; Fig. 6A) andmiR-181b (median: 2.4 vs. 1.5, respectively; P= 0.852). In addition,miR-21 (P = 0.914; Fig. 6A), miR-34a (P= 0.835; Fig. 6B), miR-122(P= 0.247; Fig. 6C), miR-181b (median: 2.4 vs. 1.3, respectively; P=0.450), and miR-885-5p (P= 0.949; Fig. 6D) levels were not differentbetween patients with cirrhosis classified as Child-Pugh C and healthyindividuals. However, patients classified as Child-Pugh A/B showedhigher relative expression levels of miR-34a (P < 0.001; Fig. 6B), miR-122 (P < 0.001; Fig. 6C), and miR-885-5p (P < 0.001; Fig. 6D) thanhealthy individuals, but no differences were observed for miR-21 (P=0.080; Fig. 6A) and miR-181b (median: 1.5 vs. 1.3, respectively; P=0.246).

When considering only patients with HCV-related cirrhosis, thoseclassified as Child-Pugh A/B showed higher relative expression levels ofmiR-21 (P= 0.018; Fig. 7A), miR-34a (P<0.001; Fig. 7B), miR-122(P<0.001; Fig. 7C), and miR-885-5p (P<0.001; Fig. 7D) than healthyindividuals, but no difference was observed for miR-181b (median: 1.5vs. 1.3, respectively; P= 0.291). However, miR-21 (P = 0.837;Fig. 7A), miR-34a (P= 0.173; Fig. 7B), miR-122 (P= 0.279; Fig. 7C),and miR-885-5p (P= 0.966; Fig. 7D) levels were not different betweenpatients with HCV-related cirrhosis classified as Child-Pugh C andhealthy individuals, whereas a difference was observed for miR-181b(median: 26.8 vs. 1.3, respectively; P= 0.009). In addition, patientsclassified as Child-Pugh A/B showed higher relative expression levels of

miR-34a (P= 0.003; Fig. 7B) and lower relative expression of miR-181b (median: 1.5 vs. 26.8, respectively; P= 0.036) than patientsclassified as Child-Pugh C, but no difference was observed for miR-21(P= 0.498; Fig. 7A), miR-122 (P= 0.182; Fig. 7C), and miR-885-5p(P= 0.452; Fig. 7D).

3.3. Circulating miRNAs as predictors of transplant-free survival inoutpatients with liver cirrhosis

Univariate Cox regression analysis was performed for each miRNAto investigate the relationship with transplant-free survival. In thisanalysis, miR-21 was associated with significantly shorter survival(hazard ratio [HR]= 1.069, 95% confidence interval [CI]:1.013–1.127, P = 0.014). No associations with survival were found forother miRNAs: miR-34a (HR=1.003, 95% CI: 0.999–1.006, P=0.115), miR-122 (HR = 0.999, 95% CI: 0.997–1.002, P= 0.497), miR-181b (HR = 1.015, 95% CI: 0.990–1.040, P= 0.237), and miR-885-5p(HR = 1.001, 95% CI: 0.991–1.012, P= 0.821). Subsequently, weconducted a multiple Cox regression analysis including miR-21, age,and other variables classically associated with reduced survival (Child-Pugh classification, MELD, and history of previous decompensation).Higher circulating miR-21 (HR = 1.065, 95% CI: 1.010–1.122; P=0.019) and MELD score (HR = 1.330, 95% CI: 1.157–1.528; P <0.001) were independently associated with reduced event-free survival(Table 3). Additional multiple Cox regression analyses were conductedseparating patients according to the Child-Pugh classification (only

Fig. 6. Comparison of serum miR-21, miR-34a, miR-122, and miR-885-5p levels between control group and patients with cirrhosis categorized according to Child-Pugh classification. The number of patients in each group is indicated in the figure. In the boxplot, horizontal lines indicate the median per group, minimum andmaximum values. The upper and lower borders of the boxes indicate the 75th and 25th percentiles, respectively. Statistical differences between groups werecalculated using Mann-Whitney U tests. P values indicate whether there were significant differences relative to the healthy controls or between Child-Pugh A/B andChild-Pugh C patients, and this is indicated by asterisks.

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Child-Pugh A or B) and etiology (HCV vs. non-HCV) (Table 3). Survivalcurves for miR-21 classified according to the best cutoff were plottedusing the Kaplan-Meier method. Transplant-free survival was sig-nificantly lower in patients with miR-21 relative expression of greaterthan or equal to 8 (22.2 months; 95% CI: 14.1–30.3) compared withthose with relative expression less than 8 (32.3 months; 95% CI:30.6–34.0; P= 0.029; Fig. 8A). Other cutoff values for miR-21, such as1.75 (P= 0.739), 3.5 (P= 0.916), 5 (P= 0.551), and 6 were in-vestigated in the entire cohort. However, transplant-free survival wasfound significantly lower only when miR-21 relative expression was≥6(24.4 months; 95% CI: 17.3–31.6; n = 18) than in those with relativeexpression<6 (32.2 months; 95% CI: 30.5–34.0; n = 168; P= 0.044).Nevertheless, the cutoff of 8 performed better than other values toidentify patients with worse prognosis and therefore was chosen forfurther evaluation.

Similar results were obtained when only patients classified as Child-Pugh A/B were considered, with significantly lower survival observedin patients with miR-21 expression ≥8 (23.3 months; 95% CI:14.9–31.7) than in those with expression<8 (33.3 months; 95% CI:31.7–34.9; P= 0.038; Fig. 8B). Given the observed impact of HCVinfection on circulating miR-21 relative expression, survival analysiswas performed comparing HCV-related cirrhosis and non-HCV cir-rhosis. Among those with HCV infection, no difference was observed inthe survival in patients with miR-21 expression ≥8 (31.8 months; 95%CI: 22.7–40.9) and<8 (32.5 months; 95% CI: 29.9–35.1; P= 0.616;Fig. 8D). On the other hand, in patients with non HCV-related cirrhosis,

transplant-free survival was significantly lower in those with miR-21relative expression ≥8 (17.0 months; 95% CI: 7.3–26.7) than in thosewith relative expression< 8 (32.2 months; 95% CI: 30.0–34.5; P=0.001; Fig. 8C).

4. Discussion

Although the natural history of cirrhosis may be affected by severalfactors, the need for prognostic markers and scoring systems is crucialto manage individuals facing different therapeutic options. Even thoughthe Child-Pugh classification and MELD scores are very useful in clinicalpractice, they are subject to various limitations. The Child-Pugh clas-sification includes subjective variables, such as hepatic encephalopathyand ascites, increasing the risk of misclassification [40]. In addition,variables and cutoffs included in the model were selected arbitrarily[40]. As MELD scores include only laboratory parameters, limitationsinvolving subjective evaluation are avoided. However, the variablesincluded in the model were also empirically selected and importantcomplications of cirrhosis were not included [41]. In addition, eachcomponent of a MELD score is subject to limitations, such as increasesin indirect bilirubin levels not related to liver disease, effect of sex oncreatinine levels, and inter-laboratory variability in INR reading [41].Circulating miRNAs are emerging as promising biomarkers in severalmedical conditions, including liver diseases. In this study, we aimed toinvestigate the prognostic value of miR-21, miR-34a, miR-122, miR-181b, and miR-885-5p in serum samples from patients with stable

Fig. 7. Comparison of serum miR-21, miR-34a, miR-122, and miR-885-5p levels between control group and patients with HCV-related cirrhosis categorized ac-cording to the Child-Pugh classification. The number of patients in each group is indicated in the figure. In the boxplot, horizontal lines indicate the median pergroup, minimum and maximum values. The upper and lower borders of the boxes indicate the 75th and 25th percentiles, respectively. Statistical differences betweengroups were calculated using Mann-Whitney U tests. P values indicate whether there were significant differences relative to the healthy controls or between patientswith HCV-related cirrhosis classified as Child-Pugh A/B and Child-Pugh C, and this is indicated by asterisks.

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cirrhosis. When considering the entire cohort, no significant differenceswere found in the expression levels of serum miR-181b in patients withliver cirrhosis compared with those in healthy individuals. In addition,miR-181b was not related to liver function parameters. Patients withHCV-related cirrhosis classified as Child-Pugh C exhibited higher miR-181b expression than healthy controls and those classified as Child-Pugh A/B. However, these results should be evaluated with caution asonly two patients were classified as Child-Pugh C among HCV-infectedindividuals. Wang et al. found that serum miR-181b levels were upre-gulated in liver cirrhosis and suggested that TGF-β1 could induce theexpression of miR-181b which, in turn, could inhibit the cell cycleregulator p27, thereby promoting the growth of hepatic stellate cells[14], important mediators of liver fibrosis [42]. In an independentvalidation cohort, Chen et al. reported that plasma miR-181b was up-regulated in patients with chronic hepatitis B (CHB) cirrhosis comparedwith that in healthy individuals [29]. The discrepancies between ourfindings and the Chinese cohorts may be explained by the distinctetiologic factors for liver cirrhosis across studies because only a fewpatients included in our study had HBV infection. Regarding our find-ings among HCV-infected patients with Child-Pugh C cirrhosis, morestudies including a larger number of patients are needed to confirmthese results and to address serum miR-181b as a prognostic marker insubjects with more advanced liver disease.

In this study, serum miR-885-5p levels were upregulated in patientswith cirrhosis compared to healthy individuals. Positive correlationswere found between miR-885-5p and ALT, AST, and GGT, whereas anegative correlation was observed with INR. In addition, patientsclassified as Child-Pugh C showed lower relative expression levels ofmiR-885-5p as compared Child-Pugh A/B subjects. In an independentvalidation cohort, Gui et al. found increased expression of serum miR-885-5p in patients with liver cirrhosis, CHB, or HCC compared tohealthy individuals; furthermore, ATP binding cassette subfamily Amember 1, an important regulator of cholesterol and phospholipidmetabolism, was found to be a potential target of miR-885-5p, sug-gesting that this miRNA could have a metabolic role in liver cellularfunction [15]. In this sense, Raitoharju et al. found that miR-885-5plevels were inversely associated with extra-large high-density lipopro-tein particle lipids, cholesterol levels, and oxysterol-binding protein-

related protein 2, a protein that affects cholesterol metabolism [43].These data suggest that miR-885-5p may be related to liver damage andnecro-inflammation and plays a role in liver fat accumulation throughinterference with the expression of proteins related to the transport oflipids in hepatocytes. In addition, our finding of lower miR-885-5prelative expression among Child-Pugh C cirrhosis indicate that thismiRNA may be useful prognostic marker, but this still must be eval-uated in further studies including a larger number of patients withdecompensated cirrhosis.

Our results showed that serum miR-34a levels were increased inpatients with HCV-induced cirrhosis compared with those in patientswith non-HCV cirrhosis and healthy individuals. In addition, patientsclassified as Child-Pugh C showed lower relative expression levels ofmiR-34a than those classified as Child-Pugh A/B. In an independentvalidation cohort, Cermelli et al. found increased expression of serummiR-34a in patients with HCV and nonalcoholic fatty liver diseasecompared to healthy individuals; moreover, this miRNA was un-detectable in serum from healthy subjects [11], which may indicate aneed for a pre-amplification step since serum miR-34a was also notdetectable in eight out of 28 healthy individuals and in 58 out of 186patients with liver cirrhosis in our current study. Notably, miR-34a canbe induced following activation of p53, a tumor-suppressor protein[22,44,45], and has been shown to be upregulated in HCC [23]. Ad-ditionally, Navarro and Lieberman reported that miR-34a, in turn,could enhance p53 function by inhibiting negative regulators of p53and increasing p53 protein stability or inhibit p53 function by directlyinhibiting TP53, the gene encoding the p53 protein, and many p53-activated genes; thus, the effects of miR-34a on p53 will depend on thebalance of these pathways enabling a fine adjustment of the p53 re-sponse to genotoxic stress [46]. These data suggested that the liverinflammatory process related to HCV infection may stimulate miR-34aexpression via p53 in order to reduce inflammation and prevent pro-gression of tumors.

In this study, serum miR-122 was upregulated in patients with cir-rhosis compared with that in healthy individuals, and higher expressionwas also observed in patients with HCV-induced cirrhosis compared tocirrhosis induced by other etiologies. In addition, positive correlationswere observed among serum miR-122 and ALT, AST, or GGT. miR-122relative expression was significantly lower among patients with ascitesand those classified as Child-Pugh C cirrhosis. Several studies havedemonstrated increased expression of miR-122 in patients with hepa-titis C and positive correlations with aminotransferases [12,47–49].Moreover, in an independent validation cohort, Waidmann et al. foundthat serum miR-122 levels were downregulated in patients with de-compensated liver cirrhosis compared to patients with compensateddisease. Additionally, miR-122 was associated with survival [13]. Fur-thermore, because liver damage may be a major source of hepatocyte-derived miRNAs [50,51], lower miR-122 levels in patients with ad-vanced-stage disease may be a marker of liver functional capacity re-lated to hepatocyte loss or could be caused by a higher volume dis-tribution in patients with ascites, making serum miR-122 a potentialmarker of liver function or prognosis for patients with liver cirrhosis[13].

In this study, patients with HCV-related cirrhosis showed higher relativeexpression levels of miR-21 than healthy individuals. This difference wasnot observed when HCV-negative subjects were compared to controls.Previous studies reported that miR-21 was upregulated in patients withHCV infection [10] and in other hepatic diseases associated with significantliver inflammation, such as hepatitis B-related acute-on-chronic liver failure[52] and autoimmune hepatitis [21]. Here, serum miR-21 levels were po-sitively correlated with ALT and AST in patients with HCV-related cirrhosis.Similarly, Bihrer et al. [10] and Migita et al. [21] found that miR-21 waspositively correlated with aminotransferases. In the present study, trans-plant-free survival was significantly lower in patients with higher miR-21levels. Higher miR-21 levels were significantly associated with lower sur-vival, even after adjustment for age, Child-Pugh classification, MELD, and

Table 3Multiple Cox regression analysis of parameters associated with overall survival.

Variables Multivariate analysis

HR 95% CI P value

Entire cohort of patients with cirrhosis (n= 186)miR-21 1.065 1.010–1.122 0.019Age 1.002 0.979–1.025 0.895Prior decompensation 1.512 0.666–3.435 0.323Child-Pugh score (A/B) 1.309 0.451–3.797 0.620MELD score 1.330 1.157–1.528 < 0.001

Patients classified as Child-Pugh A/B (n=175)miR-21 1.069 1.015–1.126 0.011Age 0.983 0.956–1.012 0.248Prior decompensation 1.921 0.744–4.959 0.177MELD score 1.400 1.216–1.612 < 0.001

Patients with non HCV-related cirrhosis (n= 126)miR-21 1.114 1.038–1.196 0.003Age 0.993 0.966–1.021 0.620Prior decompensation 0.980 0.386–2.489 0.967Child-Pugh score (A/B) 1.219 0.330–4.493 0.766MELD score 1.316 1.106–1.566 0.002

Patients with HCV-related cirrhosis (n= 60)miR-21 1.009 0.899–1.132 0.884Age 1.019 0.962–1.080 0.521Prior decompensation 5.802 0.724–46.467 0.098Child-Pugh score (A/B) 1.764 0.173–18.022 0.632MELD score 1.386 1.092–1.759 0.007

Abbreviations: CI, confidence interval; HR, hazard ratio; MELD, Model for End-stage Liver Disease score.

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history of previous decompensation. Marquez et al. suggested that miR-21could stimulate TGF-β signaling to increase fibrogenesis by targetingSMAD7, a negative regulator of TGF-β signaling [16]. Moreover, Ng et al.reported that after hepatectomy, miR-21 promotes liver regeneration bystimulating cyclin D1 translation via direct inhibition of Ras homolog genefamily member B expression [53]. Thus, we hypothesized that in advancedstages of liver cirrhosis, higher miR-21 levels could occur in order to induceliver tissue regeneration. In this way, serum miR-21 could be a relevantbiomarker of liver cirrhosis to complement Child-Pugh score and MELDscore in clinical practice. When evaluated according to etiology of cirrhosis,miR-21 was related to shorter survival only in patients without HCV-relatedcirrhosis. One possible explanation for this fact is a more intense necroin-flammatory activity in patients with hepatitis C, which may have affectedmiR-21 expression and hampered its prognostic ability. Novel studies ex-ploring miR-21 as a prognostic marker in larger cohorts of patients withoutHCV infection as well as in HCV patients treated with direct antiviral agents(DAAs) would be of interest. In fact, our data were collected before theavailability of DAAs in Brazil. Although this is a strength of the study, asDAAs are now known to influence the natural history of HCV-related cir-rhosis and could be a confounder, the improvement in liver inflammationafter HCV clearance might positively impact miR-21 as a prognostic marker[54].

5. Conclusions

In conclusion, in patients with cirrhosis, miR-34a, miR-122, andmiR-885-5p levels were positively correlated with aminotransferasesand were higher in HCV-infected patients and lower in patients withChild-Pugh C. These findings indicated that these three miRNAs wererelated to the inflammatory process and ongoing hepatocyte damage. Incontrast, miR-21 levels were associated with shorter transplant-freesurvival independent of Child-Pugh classification and MELD scores.This prognostic ability seems to be restricted to patients without HCV-related cirrhosis, possibly because of the influence of liver inflammationon miR-21 expression in HCV-infected subjects.

Source of funding

This work was supported by grants from Conselho Nacional deDesenvolvimento Científico e Tecnológico (CNPq, grant no. 457373/2013-0), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior(CAPES), and Fundação de Amparo à Pesquisa e Inovação do Estado deSanta Catarina (FAPESC). This study is a part of the PhD thesis of AlexE. Amaral.

Fig. 8. Transplant-free survival in patients with cirrhosis according to serum miR-21 levels in the entire cohort (A), in patients with cirrhosis classified as Child-PughA/B (B), in patients with non HCV-related cirrhosis (C), and in patients with HCV-related cirrhosis (D). The number of patients in each group is indicated in the figure.The entire cohort was evaluated from June 2012 to February 2014 and the final re-evaluation was performed between June and October 2015 (median follow-up of32 months). Duration of transplant-free survival was significantly lower in patients with miR-21 relative expression ≥8 than in those with relative expression< 8.This difference was observed for the entire cohort, patients with cirrhosis classified as Child-Pugh A/B, and in those with non HCV-related cirrhosis, but not in HCV-infected patients. Survival curves were calculated using the Kaplan-Meier method, and survival differences between groups were compared using log-rank tests andthis is indicated by asterisks.

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Conflict of interest

None.

Acknowledgements

We would like to thank to Albrecht Piiper (Department of MedicineI, Goethe University, Frankfurt/Main, Germany) for technical assis-tance.

References

[1] A.A. Mokdad, A.D. Lopez, S. Shahraz, R. Lozano, A.H. Mokdad, J. Stanaway,C.J. Murray, M. Naghavi, Liver cirrhosis mortality in 187 countries between 1980and 2010: a systematic analysis, BMC Med. 12 (2014) 145, http://dx.doi.org/10.1186/s12916-014-0145-y.

[2] E.A. Tsochatzis, J. Bosch, A.K. Burroughs, Liver cirrhosis, Lancet 383 (9930) (2014)1749–1761, http://dx.doi.org/10.1016/S0140-6736(14)60121-5.

[3] G. D’Amico, A. Morabito, M. D’Amico, L. Pasta, G. Malizia, P. Rebora,M.G. Valsecchi, New concepts on the clinical course and stratification of compen-sated and decompensated cirrhosis, Hepatol. Int. 12 (1) (2017) 34–43, http://dx.doi.org/10.1007/s12072-017-9808-z.

[4] G. D’Amico, G. Garcia-Tsao, L. Pagliaro, Natural history and prognostic indicatorsof survival in cirrhosis: a systematic review of 118 studies, J. Hepatol. 44 (1) (2006)217–231, http://dx.doi.org/10.1016/j.jhep.2005.10.013.

[5] F. Durand, D. Valla, Assessment of the prognosis of cirrhosis: Child-Pugh versusMELD, J. Hepatol. 42 (Suppl. (1)) (2005) S100–S107, http://dx.doi.org/10.1016/j.jhep.2004.11.015.

[6] A.E. do Amaral, J. Cisilotto, T.B. Creczynski-Pasa, L. de Lucca Schiavon, CirculatingmiRNAs in nontumoral liver diseases, Pharmacol. Res. 128 (2017) 274–287, http://dx.doi.org/10.1016/j.phrs.2017.10.002.

[7] V. Ambros, The functions of animal microRNAs, Nature 431 (7006) (2004)350–355, http://dx.doi.org/10.1038/nature02871.

[8] S.S. Chim, T.K. Shing, E.C. Hung, T.Y. Leung, T.K. Lau, R.W. Chiu, Y.M. Lo,Detection and characterization of placental microRNAs in maternal plasma, Clin.Chem. 54 (3) (2008) 482–490, http://dx.doi.org/10.1373/clinchem.2007.097972.

[9] E. van Rooij, The art of microRNA research, Circ. Res. 108 (2) (2011) 219–234,http://dx.doi.org/10.1161/CIRCRESAHA.110.227496.

[10] V. Bihrer, O. Waidmann, M. Friedrich-Rust, N. Forestier, S. Susser, J. Haupenthal,M. Welker, Y. Shi, J. Peveling-Oberhag, A. Polta, M. von Wagner, H.H. Radeke,C. Sarrazin, J. Trojan, S. Zeuzem, B. Kronenberger, A. Piiper, Serum microRNA-21as marker for necroinflammation in hepatitis C patients with and without hepato-cellular carcinoma, PLoS One 6 (10) (2011) e26971, http://dx.doi.org/10.1371/journal.pone.0026971.

[11] S. Cermelli, A. Ruggieri, J.A. Marrero, G.N. Ioannou, L. Beretta, CirculatingmicroRNAs in patients with chronic hepatitis C and non-alcoholic fatty liver dis-ease, PLoS One 6 (8) (2011) e23937, http://dx.doi.org/10.1371/journal.pone.0023937.

[12] V. Bihrer, M. Friedrich-Rust, B. Kronenberger, N. Forestier, J. Haupenthal, Y. Shi,J. Peveling-Oberhag, H.H. Radeke, C. Sarrazin, E. Herrmann, S. Zeuzem,O. Waidmann, A. Piiper, Serum miR-122 as a biomarker of necroinflammation inpatients with chronic hepatitis C virus infection, Am. J. Gastroenterol. 106 (9)(2011) 1663–1669, http://dx.doi.org/10.1038/ajg.2011.161.

[13] O. Waidmann, V. Koberle, F. Brunner, S. Zeuzem, A. Piiper, B. Kronenberger, SerummicroRNA-122 predicts survival in patients with liver cirrhosis, PLoS One 7 (9)(2012) e45652, http://dx.doi.org/10.1371/journal.pone.0045652.

[14] B. Wang, W. Li, K. Guo, Y. Xiao, Y. Wang, J. Fan, miR-181b promotes hepaticstellate cells proliferation by targeting p27 and is elevated in the serum of cirrhosispatients, Biochem. Biophys. Res. Commun. 421 (1) (2012) 4–8, http://dx.doi.org/10.1016/j.bbrc.2012.03.025.

[15] J. Gui, Y. Tian, X. Wen, W. Zhang, P. Zhang, J. Gao, W. Run, L. Tian, X. Jia, Y. Gao,Serum microRNA characterization identifies miR-885-5p as a potential marker fordetecting liver pathologies, Clin. Sci. (Lond.) 120 (5) (2011) 183–193, http://dx.doi.org/10.1042/CS20100297.

[16] R.T. Marquez, S. Bandyopadhyay, E.B. Wendlandt, K. Keck, B.A. Hoffer, M.S. Icardi,R.N. Christensen, W.N. Schmidt, A.P. McCaffrey, Correlation between microRNAexpression levels and clinical parameters associated with chronic hepatitis C viralinfection in humans, Lab. Invest. 90 (12) (2010) 1727–1736, http://dx.doi.org/10.1038/labinvest.2010.126.

[17] G. Liu, A. Friggeri, Y. Yang, J. Milosevic, Q. Ding, V.J. Thannickal, N. Kaminski,E. Abraham, miR-21 mediates fibrogenic activation of pulmonary fibroblasts andlung fibrosis, J. Exp. Med. 207 (8) (2010) 1589–1597, http://dx.doi.org/10.1084/jem.20100035.

[18] T. Thum, C. Gross, J. Fiedler, T. Fischer, S. Kissler, M. Bussen, P. Galuppo, S. Just,W. Rottbauer, S. Frantz, M. Castoldi, J. Soutschek, V. Koteliansky, A. Rosenwald,M.A. Basson, J.D. Licht, J.T. Pena, S.H. Rouhanifard, M.U. Muckenthaler, T. Tuschl,G.R. Martin, J. Bauersachs, S. Engelhardt, MicroRNA-21 contributes to myocardialdisease by stimulating MAP kinase signalling in fibroblasts, Nature 456 (7224)(2008) 980–984, http://dx.doi.org/10.1038/nature07511.

[19] F. Meng, R. Henson, H. Wehbe-Janek, K. Ghoshal, S.T. Jacob, T. Patel, MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocel-lular cancer, Gastroenterology 133 (2) (2007) 647–658, http://dx.doi.org/10.

1053/j.gastro.2007.05.022.[20] H. Varnholt, The role of microRNAs in primary liver cancer, Ann. Hepatol. 7 (2)

(2008) 104–113.[21] K. Migita, A. Komori, H. Kozuru, Y. Jiuchi, M. Nakamura, M. Yasunami,

H. Furukawa, S. Abiru, K. Yamasaki, S. Nagaoka, S. Hashimoto, S. Bekki,H. Kamitsukasa, Y. Nakamura, H. Ohta, M. Shimada, H. Takahashi, E. Mita,T. Hijioka, H. Yamashita, H. Kouno, M. Nakamuta, K. Ario, T. Muro, H. Sakai,K. Sugi, H. Nishimura, K. Yoshizawa, T. Sato, A. Naganuma, T. Komatsu, Y. Oohara,F. Makita, M. Tomizawa, H. Yatsuhashi, Circulating microRNA profiles in patientswith type-1 autoimmune hepatitis, PLoS One 10 (11) (2015) e0136908, http://dx.doi.org/10.1371/journal.pone.0136908.

[22] H. Hermeking, p53 enters the microRNA world, Cancer Cell 12 (5) (2007) 414–418,http://dx.doi.org/10.1016/j.ccr.2007.10.028.

[23] Y. Mizuguchi, T. Mishima, S. Yokomuro, Y. Arima, Y. Kawahigashi, K. Shigehara,T. Kanda, H. Yoshida, E. Uchida, T. Tajiri, T. Takizawa, Sequencing and bioinfor-matics-based analyses of the microRNA transcriptome in hepatitis B-related hepa-tocellular carcinoma, PLoS One 6 (1) (2011) e15304, http://dx.doi.org/10.1371/journal.pone.0015304.

[24] Q. Wu, J.V. Li, F. Seyfried, C.W. le Roux, H. Ashrafian, T. Athanasiou, W. Fenske,A. Darzi, J.K. Nicholson, E. Holmes, N.J. Gooderham, Metabolic phenotype-microRNA data fusion analysis of the systemic consequences of Roux-en-Y gastricbypass surgery, Int. J. Obes. (Lond.) 39 (7) (2015) 1126–1134, http://dx.doi.org/10.1038/ijo.2015.33.

[25] J.I. Henke, D. Goergen, J. Zheng, Y. Song, C.G. Schuttler, C. Fehr, C. Junemann,M. Niepmann, microRNA-122 stimulates translation of hepatitis C virus RNA,EMBO J. 27 (24) (2008) 3300–3310, http://dx.doi.org/10.1038/emboj.2008.244.

[26] G. Randall, M. Panis, J.D. Cooper, T.L. Tellinghuisen, K.E. Sukhodolets, S. Pfeffer,M. Landthaler, P. Landgraf, S. Kan, B.D. Lindenbach, M. Chien, D.B. Weir,J.J. Russo, J. Ju, M.J. Brownstein, R. Sheridan, C. Sander, M. Zavolan, T. Tuschl,C.M. Rice, Cellular cofactors affecting hepatitis C virus infection and replication,Proc. Natl. Acad. Sci. U. S. A. 104 (31) (2007) 12884–12889, http://dx.doi.org/10.1073/pnas.0704894104.

[27] C. Coulouarn, V.M. Factor, J.B. Andersen, M.E. Durkin, S.S. Thorgeirsson, Loss ofmiR-122 expression in liver cancer correlates with suppression of the hepaticphenotype and gain of metastatic properties, Oncogene 28 (40) (2009) 3526–3536,http://dx.doi.org/10.1038/onc.2009.211.

[28] C. Zeng, R. Wang, D. Li, X.J. Lin, Q.K. Wei, Y. Yuan, Q. Wang, W. Chen,S.M. Zhuang, A novel GSK-3 beta-C/EBP alpha-miR-122-insulin-like growth factor 1receptor regulatory circuitry in human hepatocellular carcinoma, Hepatology 52(5) (2010) 1702–1712, http://dx.doi.org/10.1002/hep.23875.

[29] Y.J. Chen, J.M. Zhu, H. Wu, J. Fan, J. Zhou, J. Hu, Q. Yu, T.T. Liu, L. Yang, C.L. Wu,X.L. Guo, X.W. Huang, X.Z. Shen, Circulating microRNAs as a fingerprint for livercirrhosis, PLoS One 8 (6) (2013) e66577, http://dx.doi.org/10.1371/journal.pone.0066577.

[30] E.A. Afanasyeva, P. Mestdagh, C. Kumps, J. Vandesompele, V. Ehemann,J. Theissen, M. Fischer, M. Zapatka, B. Brors, L. Savelyeva, V. Sagulenko,F. Speleman, M. Schwab, F. Westermann, MicroRNA miR-885-5p targets CDK2 andMCM5, activates p53 and inhibits proliferation and survival, Cell Death Differ. 18(6) (2011) 974–984, http://dx.doi.org/10.1038/cdd.2010.164.

[31] Z. Zhang, J. Yin, J. Yang, W. Shen, C. Zhang, W. Mou, J. Luo, H. Yan, P. Sun, Y. Luo,Y. Tian, R. Xiang, miR-885-5p suppresses hepatocellular carcinoma metastasis andinhibits Wnt/beta-catenin signaling pathway, Oncotarget 7 (46) (2016)75038–75051, http://dx.doi.org/10.18632/oncotarget.12602.

[32] G. Mehta, R.P. Mookerjee, V. Sharma, R. Jalan, Systemic inflammation is associatedwith increased intrahepatic resistance and mortality in alcohol-related acute-on-chronic liver failure, Liver Int. 35 (3) (2015) 724–734, http://dx.doi.org/10.1111/liv.12559.

[33] N.J. Taylor, G.K. Manakkat Vijay, R.D. Abeles, G. Auzinger, W. Bernal, Y. Ma,J.A. Wendon, D.L. Shawcross, The severity of circulating neutrophil dysfunction inpatients with cirrhosis is associated with 90-day and 1-year mortality, Aliment.Pharm. Ther. 40 (6) (2014) 705–715, http://dx.doi.org/10.1111/apt.12886.

[34] A.J. Sanyal, E.M. Brunt, D.E. Kleiner, K.V. Kowdley, N. Chalasani, J.E. Lavine,V. Ratziu, A. McCullough, Endpoints and clinical trial design for nonalcoholicsteatohepatitis, Hepatology 54 (1) (2011) 344–353, http://dx.doi.org/10.1002/hep.24376.

[35] R.N. Pugh, I.M. Murray-Lyon, J.L. Dawson, M.C. Pietroni, R. Williams, Transectionof the oesophagus for bleeding oesophageal varices, Br. J. Surg. 60 (8) (1973)646–649, http://dx.doi.org/10.1002/bjs.1800600817.

[36] P.S. Kamath, R.H. Wiesner, M. Malinchoc, W. Kremers, T.M. Therneau,C.L. Kosberg, G. D’Amico, E.R. Dickson, W.R. Kim, A model to predict survival inpatients with end-stage liver disease, Hepatology 33 (2) (2001) 464–470, http://dx.doi.org/10.1053/jhep.2001.22172.

[37] J. Zhou, L. Yu, X. Gao, J. Hu, J. Wang, Z. Dai, J.F. Wang, Z. Zhang, S. Lu, X. Huang,Z. Wang, S. Qiu, X. Wang, G. Yang, H. Sun, Z. Tang, Y. Wu, H. Zhu, J. Fan, PlasmamicroRNA panel to diagnose hepatitis B virus-related hepatocellular carcinoma, J.Clin. Oncol. 29 (36) (2011) 4781–4788, http://dx.doi.org/10.1200/JCO.2011.38.2697.

[38] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method, Methods 25 (4) (2001)402–408, http://dx.doi.org/10.1006/meth.2001.1262.

[39] R. Roskoski Jr., Guidelines for preparing color figures for everyone including thecolorblind, Pharmacol. Res. 119 (2017) 240–241, http://dx.doi.org/10.1016/j.phrs.2017.02.005.

[40] H.J. Kim, H.W. Lee, Important predictor of mortality in patients with end-stage liverdisease, Clin. Mol. Hepatol. 19 (2) (2013) 105–115, http://dx.doi.org/10.3350/cmh.2013.19.2.105.

A.E.d. Amaral et al. Pharmacological Research 134 (2018) 179–192

191

[41] A.K. Singal, P.S. Kamath, Model for end-stage liver disease, J. Clin. Exp. Hepatol. 3(1) (2013) 50–60, http://dx.doi.org/10.1016/j.jceh.2012.11.002.

[42] Y. Popov, D. Schuppan, Targeting liver fibrosis: strategies for development andvalidation of antifibrotic therapies, Hepatology 50 (4) (2009) 1294–1306, http://dx.doi.org/10.1002/hep.23123.

[43] E. Raitoharju, I. Seppala, L.P. Lyytikainen, J. Viikari, M. Ala-Korpela, P. Soininen,A.J. Kangas, M. Waldenberger, N. Klopp, T. Illig, J. Leiviska, B.M. Loo, N. Oksala,M. Kahonen, N. Hutri-Kahonen, R. Laaksonen, O. Raitakari, T. Lehtimaki, Bloodhsa-miR-122-5p and hsa-miR-885-5p levels associate with fatty liver and relatedlipoprotein metabolism-the young Finns study, Sci. Rep. 6 (2016) 38262, http://dx.doi.org/10.1038/srep38262.

[44] T.C. Chang, E.A. Wentzel, O.A. Kent, K. Ramachandran, M. Mullendore, K.H. Lee,G. Feldmann, M. Yamakuchi, M. Ferlito, C.J. Lowenstein, D.E. Arking, M.A. Beer,A. Maitra, J.T. Mendell, Transactivation of miR-34a by p53 broadly influences geneexpression and promotes apoptosis, Mol. Cell 26 (5) (2007) 745–752, http://dx.doi.org/10.1016/j.molcel.2007.05.010.

[45] N. Raver-Shapira, E. Marciano, E. Meiri, Y. Spector, N. Rosenfeld, N. Moskovits,Z. Bentwich, M. Oren, Transcriptional activation of miR-34a contributes to p53-mediated apoptosis, Mol. Cell 26 (5) (2007) 731–743, http://dx.doi.org/10.1016/j.molcel.2007.05.017.

[46] F. Navarro, J. Lieberman, miR-34 and p53: new insights into a complex functionalrelationship, PLoS One 10 (7) (2015) e0132767, http://dx.doi.org/10.1371/journal.pone.0132767.

[47] S. Kumar, Y.K. Chawla, S. Ghosh, A. Chakraborti, Severity of hepatitis C virus(genotype-3) infection positively correlates with circulating microRNA-122 in pa-tients sera, Dis. Mark. 2014 (2014) 435476, http://dx.doi.org/10.1155/2014/435476.

[48] T.H. Su, C.H. Liu, C.J. Liu, C.L. Chen, T.T. Ting, T.C. Tseng, P.J. Chen, J.H. Kao,D.S. Chen, Serum microRNA-122 level correlates with virologic responses to pe-gylated interferon therapy in chronic hepatitis C, Proc. Natl. Acad. Sci. U. S. A. 110(19) (2013) 7844–7849, http://dx.doi.org/10.1073/pnas.1306138110.

[49] J.H. Wang, D. Jiang, H.Y. Rao, J.M. Zhao, Y. Wang, L. Wei, Absolute quantificationof serum microRNA-122 and its correlation with liver inflammation grade andserum alanine aminotransferase in chronic hepatitis C patients, Int. J. Infect. Dis. 30(2015) 52–56, http://dx.doi.org/10.1016/j.ijid.2014.09.020.

[50] M. Lagos-Quintana, R. Rauhut, A. Yalcin, J. Meyer, W. Lendeckel, T. Tuschl,Identification of tissue-specific microRNAs from mouse, Curr. Biol. 12 (9) (2002)735–739, http://dx.doi.org/10.1016/S0960-9822(02)00809-6.

[51] K. Wang, S. Zhang, J. Weber, D. Baxter, D.J. Galas, Export of microRNAs andmicroRNA-protective protein by mammalian cells, Nucleic Acids Res. 38 (20)(2010) 7248–7259, http://dx.doi.org/10.1093/nar/gkq601.

[52] Q.F. Zheng, J.Y. Zhang, J.S. Wu, Y. Zhang, M. Liu, L. Bai, J. Zhao, Y. Chen,Z.P. Duan, S.J. Zheng, Upregulation of miRNA-130a represents good prognosis inpatients with HBV-related acute-on-chronic liver failure: a prospective study,Medical (Baltim.) 95 (6) (2016) e2639, http://dx.doi.org/10.1097/MD.0000000000002639.

[53] R. Ng, G. Song, G.R. Roll, N.M. Frandsen, H. Willenbring, A microRNA-21 surgefacilitates rapid cyclin D1 translation and cell cycle progression in mouse liver re-generation, J. Clin. Invest. 122 (3) (2012) 1097–1108, http://dx.doi.org/10.1172/JCI46039.

[54] I. Grgurevic, T. Bozin, A. Madir, Hepatitis C is now curable, but what happens withcirrhosis and portal hypertension afterwards? Clin. Exp. Hepatol. 3 (4) (2017)181–186, http://dx.doi.org/10.5114/ceh.2017.71491.

A.E.d. Amaral et al. Pharmacological Research 134 (2018) 179–192

192