Hepatology Snapshot

Circulating microRNAs as markers of liver inflammation, fibrosis and cancer Christoph Roderburg1, Tom Luedde1,* Department of Medicine III, University Hospital RWTH Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany *E-mail address: [email protected] *Corresponding author. Address: Department of Medicine III, University Hospital RWTH Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany Tel.: +49-241-80-80329. E-mail address: [email protected] (T. Luedde). 1

Key facts Since their discovery in 1993, miRNAs have emerged as a new class of small RNAs that control intracellular gene expression networks at a post-transcriptional level. In the liver, miRNAs play fundamental roles in the regulation of physiological and pathological processes.

Immune cells

Exportin 5

Hepatocytes

Endothelial cells

Stellate cells

Drosha

Pre-miRNA

Chronic hepatic diseases Dicer Pre-miRNA Pre-miR

Pri-miRNA RISC DNA A

Release

mature m ature miRNA

Nucleus Nu Nuc uccleu le s

Cytoplasm

Passive release (cell death)

Microvesicles

Active release (e.g. secretion)

?

Nucleus

RNA-binding proteins Exosomes

Nucleus

Apoptotic bodies

High density lipoprotein particles

Conclusion Despite their obvious potential as biomarkers, there are striking inter-study variances of miRNAregulation patterns in patients with hepatic diseases. As key prerequisites for a future clinical use- wellstandardized protocols have to be implemented for the analysis of miRNAs from patients’ serum.

miRNAs

Keywords: miRNA, microRNA, Biomarker, Liver, Fibrosis, Hepatocellular carcinoma, Exosomal miRNA, Inflamation. Received 18 March 2014; received in revised form 21 May 2014; accepted 02 July 2014.

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Table 1. Selected studies on miRNA-serum alterations in human liver diseases.

miRNA biomarker

Etiology

Normalization

Message

[Ref.]

Up: miR-25, -92a, let7f, miR-375

HBV (n ≈ 150)

None

miR-375 is HBV specific and predicts HCC

Li et al., Cancer Res. 2010 Dec 1;70(23):9798-807

Up: miR-122

HBV (n = 83)

U6 RNA

Increase of miR-122 in HBV

Zhang et al., Clin Chem. 2010 Dec;56(12):1830-8

Up: miR-122

HCV (n = 68)

None

miR-122 reflects liver damage

Bihrer et al., Am J Gastroenterol. 2011 Sep;106(9):1663-9

Up: miR-122, -34

NAFLD; HCV (n ≈ 35)

Spike-in RNA

miR-122 reflects liver damage and fibrosis

Cermelli et al., PLoS One. 2011;6(8): e23937

Up: miR-885-5p

HBV, HCC (n ≈ 100)

U6 RNA

Increase in HCC

Gui et al., Clin Sci (Lond). 2011 Mar;120(5):183-93

Up: miR-122, -222, -223 Down: miR-21

CHB, HCC (n = 118)

miR-16

Deregulation in HCC

Qi et al., PLoS One. 2011;6(12):e28486

Down: miR-29a Up: miR-133a

HCV, EtOH (n = 69)

Spike-in RNA

miR-29a reflects progression

Roderburg et al., Hepatology. 2011 Jan;53(1):20918., J Hepatol. 2013 Apr;58(4):736-42

Up: miR-122, -192

Acetaminophen (n = 53)

U6 RNA

Increase reflects liver damage

Starkey Lewis et al., Hepatology. 2011 Nov;54(5):1767-76

Up: miR-21, -122, -223

HBV, HCC (n ≈ 150)

miR-181

miRNAs are increased increased in HBV, HCC

Xu et al., Mol Carcinog. 2011 Feb;50(2):136-42

Up: miR-21 -192, -801 Down: miR-26a, -27a, -122, -223

HBV, HCC (n ≈ 800)

miR-1228

Differentiation between healthy, HBV and HCC

Zhou et al., J Clin Oncol. 2011 Dec 20;29(36):4781-8

Up: miR-122, -148a, -192

Liver transplant (n =107)

Spike-in RNA

miRNAs reflects transplantate rejection

Farid et al., Liver Transpl. 2012 Mar;18(3):290-7

Up: miR-571 Down: miR-652

HCV, EtOH (n = 68)

Spike-in RNA

miR-29a reflects progression

Roderburg et al., PLoS One. 2012;7(3):e32999

Up: miR-21

HBV, HCV, HCC (n = 166)

miR-16

miR-21 indicates HCC

Tomimaru et al., J Hepatol. 2012 Jan;56(1):167-75

Up: miR-106b Down: miR181b

HBV (n = 62)

miR-16

miRNAs reflects HBV infection

Chen et al., PLoS One. 2013 Jun 21;8(6):e66577

Down: miR-197-3p, -505-3p

HBV/HCV, PBC (n = 20)

None

Differentiation healthy, HBV/ HCV and PBC

Ninomiya et al., PLoS One. 2013 Jun 12;8(6):e66086

Up: miR-20a, -92a

HCV (n = 58)

Spike-in RNA

miRNAs for early detection of HCV

Shrivastava et al., Hepatology. 2013 Sep;58(3):86371

Up: miR-122

HCV (n = 164)

Spike-in RNA

miR-122 reflects inflamm. and damage

Trebicka et al., J Hepatol. 2013 Feb;58(2):234-9

Up/down: 16 miRNA panel

HBV in children (n = 60)

None

Panel of miRNAs reflects HBV in children

Winther et al., PLoS One. 2013 Nov 11;8(11):e80384

Up: miR-125-5p Down: miR-223-3p

HBV, HCC (n = 66)

Spike-in RNA

miRNAs reflect HCC and CLD-

Giray et al., Mol Biol Rep. 2014 Mar 5

Up: miR-122

HCV (n = 25)

U6 RNA

miR-122 reflects HCV infection

Kumar et al., Dis Markers. 2014;2014:435476

Background Since their discovery in 1993, miRNAs have emerged as a new class of small RNAs that control intracellular gene expression networks at a posttranscriptional level, thereby regulating biological processes like inflammation, fibrogenesis or carcinogenesis. miRNAs can also be detected and quantified in the blood, where they are found in nucleated and non-nucleated blood cells as well as in plasma and serum [1–4]. Circulating miRNAs are extraordinarily stable towards conditions that usually would degrade most RNAs. Recent studies have reported that plasma miRNAs exist in at least two different “protected” conditions: they can either be associated with RNA-binding proteins and lipoprotein complexes or they can be packaged into microparticles (exosomes, microvesicles and apoptotic bodies, reviewed in [4,5]). The exact distribution and function of miRNAs in these respective compartments has been controversially debated: a recent study suggested that the majority of miRNAs in serum or plasma are concentrated in exosomes [6], while, in contrast, other groups provided evidence that up to 90% of circulating miRNAs are associated with proteins [7]. By performing mass spectrometry, Wang et al. identified a panel of twelve binding partners for miRNAs, such as nucleophosmin

1 and nucleolin, which have both been implicated in the nuclear export from ribosomes [8]. In contrast, the group of Arroyo provided evidence that most circulating miRNAs are associated with only one protein, namely Ago2, which is involved in miRNA mediated RNA silencing (reviewed in [4,5]). Different miRNAs were suggested to be enriched in specific extracellular compartments. However, these seem to vary between specific pathological conditions: In alcoholic liver disease and inflammation, increases in circulating miR-122 and miR-155 were restricted to the exosome-rich fraction, while in contrast, these miRNAs were predominantly increased in the protein-rich fractions during drug induced liver injury, suggesting that the shift of miRNAs from the proteinbound to the exosomal compartment (and vice versa) might represent a cellular adaptation mechanism towards different disease conditions [9]. In line with this hypothesis, it was shown that exosomal miRNA profiles and concentrations do not necessarily reflect miRNA expression profiles found in donor cells, highlighting that miRNAs might actively be retained in cells or released by exosomes. Some studies even provided evidence that these exosomes can donate their miRNAs to recipient cells and thereby influence their gene expression [5,10], which would represent a novel mechanism of intercellular communication.

Fig. 1. Cellular release of miRNAs during chronic hepatic diseases. After transcription from DNA and cleavage by the RNase III enzyme Drosha, so called pre-miRNAs are transported into the cytoplasm where they can be further cleaved into 19- to 23-nucleotide mature miRNA duplexes and loaded into the RNA-induced silencing complex (RISC) (to prevent translation of the mRNA into proteins). In the cytoplasm, pre-miRNAs as well as mature miRNAs may be incorporated into microparticles such as exosomes, apoptotic bodies or microvesicles and can be released from cells. Besides being included into vesicular structures, miRNAs are also present in a microparticle free compartment. These miRNAs are associated with high density lipoproteins or RNA-binding proteins (indicated as orange sphere, for details see text). Presently, it is only poorly understood how these miRNA-protein complexes are released from cells. In the course of hepatic diseases, miRNAs may be released passively (e.g., cell death), or actively, through secretion in vesicular structures or associated to proteins through interaction with specific membrane channels or proteins.

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Hepatology Snapshot Considering these novel findings, miRNAs in body fluids have been investigated in a wide variety of patient samples and animal models and found considerable attention as biomarkers for disease conditions such as inflammation, fibrosis and cancer. Specifically in the field of hepatology, easily accessible serum based miRNAs might provide a novel tool in diagnosis and monitoring of liver diseases and might represent an alternative to invasive diagnostic procedures. Functions of miRNAs in liver diseases In the liver, miRNAs play fundamental functional roles in the regulation of physiological and pathological processes [11]. As such, miR-122 is the most abundant miRNAs in the liver, accounting for up to 72% of all hepatic miRNAs [12]. On a functional level, it was demonstrated that miR-122 is essential for liver homeostasis and its loss promotes steatosis, inflammation, fibrosis and liver cancer by regulating hepatic networks of genes involved in cell cycle regulation, lipid metabolism, inflammation and oncogenesis [12–14]. In humans, miR-122 expression was associated with hepatocarcinogenesis and miR-122 loss was associated with a poor prognosis of patients with hepatocellular carcinoma (HCC), while miR-122 overexpression sensitized HCC cells to chemotherapy and was associated with longer survival times [12]. Moreover, the stability of hepatitis C virus (HCV) is dependent on a functional interaction between the HCV genome and miR-122. By blocking this interaction with a specific antisense oligonucleotide, it was possible to inhibit viral replication in HCV patients [15], thus demonstrating the potential of miRNAs as therapeutic targets in liver disease. Besides miR-122, members of the miR-29 and miR-199 families have also been shown to be involved in several regulatory steps along the cascade leading from inflammation to fibrosis and HCC [16,17]. Alterations of circulating miRNAs in liver injury, inflammation and fibrosis Based on their aberrant expression patterns in liver disease, levels of circulating microRNAs were analysed in the context of liver inflammation, fibrosis and cancer (Summarized in Table 1). Pilot studies in mice showed that miR-122 serum levels were elevated upon acetaminophen induced acute liver injury and were earlier markers of hepatocellular destruction than e.g., serum transaminases in this setting. In patients with chronic liver disease and liver fibrosis, array-based studies identified a whole panel of miRNAs, including miR-29a, miR-197-3p, miR-505-3p, miR-652, miR-20a, miR-21, miR-34a, miR-122, miR-133a, miR-223, miR-571, let7 and others that were deregulated in the serum compared to healthy controls, several of which also correlated with the degree of liver fibrosis. Of note, in most instances the functional link between the regulation of miRNA serum-levels and their roles in the specific cell compartments involved in fibrogenesis have remained unclear. In line, lower serum levels of miR-29 correlated with a downregulation in human hepatic stellate cells in liver cirrhosis, while alterations in serum levels of miR-652 reflected a concordant downregulation of this miRNA in circulating monocytes of cirrhosis patients. Moreover, elevated miR-122 serum levels correlate with hepatic cell death and necroinflammatory activity in patients with HCV infection but not with fibrosis stage or liver function. Finally, several reports suggested that alterations in serum levels of specific circulating miRNAs might allow a differential diagnosis between the different aetiologies of chronic liver disease [18]. Alterations of circulating miRNAs in hepatocellular carcinoma Alterations in miRNA serum levels have been linked with various cancers, including HCC. Several miRNAs were suggested to be potential diagnostic and prognostic markers. However, most studies were limited by a small sample size, heterogeneous patients’ collectives or a small number of miRNAs explored. Moreover, the value of the miRNA signatures was often not prospectively evaluated and no external validation of the diagnostic accuracy of the deregulated miRNAs was conducted. Zhou and co-workers identified a panel of deregulated miRNAs with diagnostic potential featuring miR-21, miR122, miR-192, miR-223, miR-26a, miR-27a, and miR-801 in the serum 1436

of a large and well-defined cohort of HCC patients (Table 1). While the diagnostic accuracy of single markers was insufficient, the whole panel had a much better diagnostic performance to differentiate patients with HCC from control groups (AUROC 0.88). Another study in a large cohort of patients with HBV- or HCV-associated HCCs revealed that the presence of HCC was associated with simultaneous elevation of miR-25, let-7f, and miR-375 in the serum and that miR-375 elevations are specifically linked with HBV-related hepatocarcinogenesis. These and other miRNAs might therefore be useful to monitor the cascade from liver injury to HCC. Discussion and outlook Despite their obvious potential as biomarkers, there are striking interstudy variances of miRNA-regulation patterns in patients with hepatic diseases. Although miRNAs are very stable molecules in serum or plasma, it is likely that a lack in standardization of sample collection, data normalization and analysis might have biased many studies. Thus, for better comparability and reproducibility- as key prerequisites for a future clinical use- well-standardized protocols have to be implemented for the analysis of miRNAs from patients’ serum. If these current limitations can be overcome, the whole new cosmos of more than 1600 miRNAs might offer highly attractive options to establish new biomarkers in clinical hepatology. © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Financial support Work in the lab of T.L. and C.R. was supported by a Starting Grant from the European Research Council within the FP 7 (ERC 2007-Stg/208237-Luedde-Med3-Aachen), the German Research Foundation (SFB TRR57/P06), the German Cancer Aid (Deutsche Krebshilfe Grant 110043), the EMBO Young Investigator Program, the Ernst Jung Foundation Hamburg and a grant from the Medical Faculty Aachen. Competing interest The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Acknowledgements The authors would like to express their gratitude to Michaela Roderburg-Goor, Dr. Jane Beger-Luedde, Dr. Jeremie Gautheron, and Dr. Mihael Vucur for helpful discussions.

References [1] Mitchell PS, Parkin RK, Kroh EM, et al. Circulating microRNAs as stable bloodbased markers for cancer detection. Proc Natl Acad Sci U S A 2008;105:10513–8. [2] Lawrie CH, Gal S, Dunlop HM, et al. Detection of elevated levels of tumourassociated microRNAs in serum of patients with diffuse large B-cell lymphoma. Br J Haematol 2008;141:672–5. [3] Chen X, Ba Y, Ma L, et al. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res 2008;18:997– 1006. [4] Creemers EE, Tijsen AJ, Pinto YM. Circulati ng microRNAs: novel biomarkers and extracellular communicators in cardiovascular disease? Circ Res;110:483–95. [5] Cortez MA, Bueso-Ramos C, Ferdin J, et al. MicroRNAs in body fluids--the mix of hormones and biomarkers. Nat Rev Clin Oncol;8:467–77. [6] Gallo A, Tandon M, Alevizos I, et al. The majority of microRNAs detectable in serum and saliva is concentrated in exosomes. PLoS One;7:e30679. [7] Arroyo JD, Chevillet JR, Kroh EM, et al. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci U S A;108:5003–8. [8] Wang K, Zhang S, Weber J, et al. Export of microRNAs and microRNA-protective protein by mammalian cells. Nucleic Acids Res;38:7248–59.

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[9] Bala S, Petrasek J, Mundkur S, et al. Circulating microRNAs in exosomes indicate hepatocyte injury and inflammation in alcoholic, drug-induced, and inflammatory liver diseases. Hepatology;56:1946–57. [10] Valadi H, Ekstrom K, Bossios A, et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 2007;9:654–9. [11] Szabo G, Bala S. MicroRNAs in liver disease. Nat Rev Gastroenterol Hepatol;10:542–52. [12] Hu J, Xu Y, Hao J, et al. MiR-122 in hepatic function and liver diseases. Protein Cell;3:364–71. [13] Tsai WC, Hsu SD, Hsu CS, et al. MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis. J Clin Invest;122:2884–97. [14] Hsu SH, Wang B, Kutay H, et al. Hepatic loss of miR-122 predisposes mice to hepatobiliary cyst and hepatocellular carcinoma upon diethylnitrosamine exposure. Am J Pathol;183:1719–30.

[15] Janssen HL, Reesink HW, Lawitz EJ, et al. Treatment of HCV infection by targeting microRNA. N Engl J Med;368:1685–94. [16] Hou J, Lin L, Zhou W, et al. Identification of miRNomes in human liver and hepatocellular carcinoma reveals miR-199a/b-3p as therapeutic target for hepatocellular carcinoma. Cancer Cell;19:232–43. [17] Xiong Y, Fang JH, Yun JP, et al. Effects of microRNA-29 on apoptosis, tumorigenicity, and prognosis of hepatocellular carcinoma. Hepatology;51:836–45. [18] Li LM, Hu ZB, Zhou ZX, et al. Serum microRNA profiles serve as novel biomarkers for HBV infection and diagnosis of HBV-positive hepatocarcinoma. Cancer Res;70:9798–807.

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Circulating microRNAs as markers of liver inflammation, fibrosis and cancer.

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