Elsevier

Pharmacological Research

Volume 128, February 2018, Pages 274-287
Pharmacological Research

Review
Circulating miRNAs in nontumoral liver diseases

https://doi.org/10.1016/j.phrs.2017.10.002Get rights and content

Abstract

In recent years, there has been increasing interest in finding new biomarkers for diagnosis and prognostication of liver diseases. MicroRNAs (miRNAs) are small noncoding RNA molecules involved in the regulation of gene expression and have been studied in relation to several conditions, including liver disease. Mature miRNAs can reach the bloodstream by passive release or by incorporation into lipoprotein complexes or microvesicles, and have stable and reproducible concentrations among individuals. In this review, we summarize studies involving circulating miRNAs sourced from the serum or plasma of patients with nontumoral liver diseases in attempt to bring insights in the use of miRNAs as biomarkers for diagnosis, as well as for prognosis of such diseases. In addition, we present pre-analytical aspects involving miRNA analysis and strategies for normalization of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) data related to the studies evaluated.

Introduction

The main causes of liver disease are alcohol abuse, viral hepatitis, nonalcoholic fatty liver disease (NAFLD), hemochromatosis, autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), and primary sclerosing cholangitis. Alcohol and the hepatitis B and C viruses (HBV and HCV) are the most important causes of liver cirrhosis and hepatocellular carcinoma (HCC) [1], [2], [3], [4]. Mortality related to liver disease is growing, with more than one million deaths from liver cirrhosis worldwide in 2010 [5]. When deaths from liver cirrhosis are combined with deaths from liver cancer and acute hepatitis, the annual number of fatalities due to liver disease can exceed two million [6]. In Europe, liver cirrhosis is responsible for around 170,000 deaths per year, and more than 5500 liver transplants are performed each year [1]. Chronically infected HBV and HCV patients are at high risk of developing cirrhosis (∼20% of patients) [7], [8], [9] and HCC (∼25% and 7% for HBV and HCV, respectively) [8], [10]. HBV and HCV infections are responsible for a mortality rate of approximately 3 and 11 deaths, respectively, per 100,000 inhabitants per year in Spain [11]. Although alcohol control policies, vaccination, and prevention strategies have contributed to decreased incidence of chronic liver diseases, these diseases remain public health problems because of their associated complications. In addition, there is a high cost related to antiviral therapy for HBV and HCV chronic infection [12], [13].

The health, social, and economic impacts associated with liver diseases could be reduced by improving diagnosis and prognostication. New biomarkers have been investigated for this purpose, including circulating microRNAs (miRNAs). MicroRNAs are small RNAs about 22 nucleotides (nt) long that regulate gene expression by binding to complementary regions of messenger RNA (mRNA) [14]. Circulating miRNAs can be detected in human serum and plasma, and have stable and reproducible concentrations among individuals [15], [16]. The purpose of this review is to highlight recent advances in the study of circulating miRNAs as markers of nontumoral liver damage, and to provide an overview of the biological properties of these nucleic acids and analytical challenges associated with their quantitation.

MicroRNAs are endogenous, noncoding RNAs about 22 nt long that regulate gene expression post-transcriptionally by binding to complementary regions of mRNA, resulting in repression of translation or mRNA degradation [14]. Fig. 1 gives a brief overview of miRNA biogenesis. Initially, (1) DNA is transcribed by RNA polymerase II (Pol II), yielding a transcript called primary RNA (pri-miRNA) [17], [18], which usually has a stem-loop structure and a length over 1 kb. After that, (2) the RNase III Drosha cleaves the hairpin ∼11 base pairs (bp) from the basal junction and ∼22 bp from the apical junction connected to the terminal loop [19], [20], forming a hairpin RNA ∼65 nt long, termed pre-miRNA [21]. Then, (3) pre-miRNA forms a transport complex with the protein exportin-5 and the Ran-guanosine-5′-triphosphate complex (Ran•GTP), allowing export into the cytoplasm. Once in the cytoplasm, (4) pre-miRNA is cleaved by the RNase III Dicer near the terminal loop, releasing a small RNA duplex [22], [23], [24], [25], [26], which is loaded into an Argonaute (Ago) protein, forming the pre-RNA-induced silencing complex (pre-RISC) [27], [28], [29]. Next, the duplex is unwound, which usually causes degradation of the passenger strand (miRNA*) and the formation of mature miRNA [30].

Once integrated into the RISC, mature miRNA is functional and able to regulate gene expression. This regulation may occur by: (I) inhibition of translation, the most common mechanism in humans, which is dependent on the Ago proteins Ago1, Ago3, or Ago4, and involves imperfect base pairing between the miRNA and the 3′-untranslated region (UTR) of mRNA [31]; (II) cleavage of mRNA, an Ago2-dependent mechanism, which involves perfect base pairing between the miRNA and the 3′-UTR of mRNA; and (III) translation activation, which involves interaction of miRNA with the 5ʹ-UTR of mRNA [32], [33], [34], [35], [36], [37]. The mature miRNA can reach the bloodstream by (A) active release (by incorporation into lipoprotein complexes, microvesicles, or exosomes) or (B) passive release (caused by cell death; by incorporation into apoptotic bodies) [38], [39], [40]. In associated form, circulating miRNAs are resistant to nuclease activity, making them a promising source of diagnostic or prognostic biomarkers [41].

Although miRNAs were discovered in 1993 by Lee et al. [43] while studying the development of the free-living nematode Caenorhabditis elegans, the term “miRNA” started to be used in mid-2001 [28], [44], [45], [46]. Since the early 2000s, when a correlation between miRNA levels and human disease was demonstrated [47], [48], [49], the number of studies on the applications of miRNAs as biomarkers and therapeutics has progressively increased (Fig. 2). In 2015, there were 11,216 new citations related to miRNA in PubMed (Fig. 2A), of which 978 (9%) related to both miRNA and the liver (Fig. 2B). In 2016, although the number of new citations related to miRNA was lower than in 2015 (9022 citations), the proportion of these 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 annotated with the MeSH term “liver.”

MicroRNAs have been implicated in the regulation of many important biological processes, such as cell growth and differentiation, development, apoptosis, and modulation of the host response to viral infection [50]. For example, miR-122 plays an important role in liver physiology, since it is involved in acquisition and maintenance of the hepato-specific phenotype, and participates in the regulation of cholesterol and fatty acids in hepatic metabolism [51], [52]. Moreover, the propagation of HCV RNA occurs after binding of miR-122 to the 5ʹ-noncoding region of the HCV genome [51], [53], [54]. The following section describes other circulating miRNAs, beyond miR-122, that have been described in patients with liver disease.

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

Table 1 summarizes the content covered in the subsequent sections. In the present review, impact factor (IF) was not used as a criterion for the selection of research articles because it does not necessarily reflect the quality of studies [57]. In addition, only 9 studies (listed in Table 1) developed independent validation cohort: liver cirrhosis [58], [59], CHB cirrhosis [60], HBV-HCC [61], HCV [62], [63], [64], NAFLD [65], and PBC [66]. For this reason, we sought to include, in this review, studies involving miRNA expression in serum or plasma samples from humans with non-tumoral liver diseases, even though independent validation cohort studies were not available in majority of the cases. Although the focus of the review is circulating miRNAs (serum or plasma) in nontumoral liver diseases, we also address some studies involving HCC, which is associated with several chronic liver diseases.

Section snippets

Liver cirrhosis

MicroRNAs are fundamental in the pathogenesis of various diseases, including cancer and other chronic liver diseases. In addition, miRNAs detected in patients’ serum may serve as biomarkers and represent a novel approach for diagnostic blood screening [92]. The expression of various miRNAs is altered in the serum of patients with liver disease; for example, miR-146a, miR-215, miR-224, miR-574-3p, and miR-885-5p were shown to be upregulated in the serum of patients with HCC and liver cirrhosis.

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 healthy subjects (n = 10) within 72 h of storage. The authors demonstrated that 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. In addition, hemolysis (up to 600 mg/dL and 1200 mg/dL hemoglobin) did not alter miR-24 and miR-122, respectively, but 25 mg/dL hemoglobin caused a significant increase in miR-15b and

miRNAs as biomarkers: methodological considerations for clinical use

In addition to other considerations, clinical use of miRNAs as biomarkers should account for differences in sample types and methods for normalization of RT-qPCR data. While some research has shown that there is a strong correlation between serum and plasma miRNA profiles [81], [106], other studies have reported that some miRNAs may occur at different levels in these sample types [105], [113]. Therefore, caution should be exercised when comparing results from different studies. Another

Conclusions

Liver diseases are common and remain an important public health problem, with increasing mortality and healthcare costs. New biomarkers to improve the diagnosis and prognostication of liver diseases could lead to a better management of these challenging conditions, reducing health, social, and economic impacts associated with them. Over the last decade, the amount of published data on circulating miRNAs as biomarkers for liver disease has increased substantially. Some miRNAs, such as miR-122,

Conflict of interest

None.

Acknowledgements

The authors wish to thanks Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant n°457373/2013-0) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support. This work makes part of the thesis of Alex E. Amaral.

References (116)

  • S. Bandiera et al.

    miR-122—a key factor and therapeutic target in liver disease

    J. Hepatol.

    (2015)
  • B. Wang et al.

    miR-181b promotes hepatic stellate cells proliferation by targeting p27 and is elevated in the serum of cirrhosis patients

    Biochem. Biophys. Res. Commun.

    (2012)
  • J.H. Wang et al.

    Absolute quantification of serum microRNA-122 and its correlation with liver inflammation grade and serum alanine aminotransferase in chronic hepatitis C patients

    Int. J. Infect. Dis.

    (2015)
  • X. Yang et al.

    Potential of extracellular microRNAs as biomarkers of acetaminophen toxicity in children

    Toxicol. Appl. Pharmacol.

    (2015)
  • O. Waidmann et al.

    Pretreatment serum microRNA-122 is not predictive for treatment response in chronic hepatitis C virus infection

    Dig. Liver Dis.

    (2012)
  • G. Sygitowicz et al.

    Circulating microribonucleic acids miR-1, miR-21 and miR-208a in patients with symptomatic heart failure: preliminary results

    Arch. Cardiovasc. Dis.

    (2015)
  • R.E. Mann et al.

    The epidemiology of alcoholic liver disease

    Alcohol Res. Health

    (2003)
  • J. Rehm et al.

    Alcohol as a risk factor for liver cirrhosis: a systematic review and meta-analysis

    Drug Alcohol Rev.

    (2010)
  • W.A. Zatonski et al.

    Liver cirrhosis mortality in Europe, with special attention to Central and Eastern Europe

    Eur. Addict. Res.

    (2010)
  • A.A. Mokdad et al.

    Liver cirrhosis mortality in 187 countriesbetween 1980 and 2010: a systematic analysis

    BMC Med.

    (2014)
  • P. Byass

    The global burden of liver disease: a challenge for methods and for public health

    BMC Med.

    (2014)
  • M.J. Alter

    Epidemiology of hepatitis C

    Hepatology

    (1997)
  • J.F. Cadranel et al.

    Epidemiology of chronic hepatitis B infection in France: risk factors for significant fibrosis–results of a nationwide survey

    Aliment. Pharmacol. Ther.

    (2007)
  • A. Mota et al.

    Chronic liver disease and cirrhosis among patients with hepatitis B virus infection in northern Portugal with reference to the viral genotypes

    J. Med. Virol.

    (2011)
  • A. Hatzakis et al.

    The state of hepatitis B and C in Europe: report from the hepatitis B and C summit conference*

    J. Viral Hepat.

    (2011)
  • A. Garcia-Fulgueiras et al.

    Hepatitis C and hepatitis B-related mortality in Spain

    Eur. J. Gastroenterol. Hepatol.

    (2009)
  • S. Liu et al.

    New protease inhibitors for the treatment of chronic hepatitis C: a cost-effectiveness analysis

    Ann. Intern. Med.

    (2012)
  • V. Ambros

    The functions of animal microRNAs

    Nature

    (2004)
  • S.S. Chim et al.

    Detection and characterization of placental microRNAs in maternal plasma

    Clin. Chem.

    (2008)
  • E. van Rooij

    The art of microRNA research

    Circ. Res.

    (2011)
  • X. Cai et al.

    Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs

    RNA

    (2004)
  • Y. Lee et al.

    MicroRNA genes are transcribed by RNA polymerase II

    EMBO J.

    (2004)
  • Y. Zeng et al.

    Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha

    EMBO J.

    (2005)
  • Y. Lee et al.

    The nuclear RNase III Drosha initiates microRNA processing

    Nature

    (2003)
  • E. Bernstein et al.

    Role for a bidentate ribonuclease in the initiation step of RNA interference

    Nature

    (2001)
  • G. Hutvagner et al.

    A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA

    Science

    (2001)
  • R.F. Ketting et al.

    Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans

    Genes Dev.

    (2001)
  • S.W. Knight et al.

    A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans

    Science

    (2001)
  • S.M. Hammond et al.

    Argonaute2, a link between genetic and biochemical analyses of RNAi

    Science

    (2001)
  • Z. Mourelatos et al.

    miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs

    Genes Dev.

    (2002)
  • M. Ha et al.

    Regulation of microRNA biogenesis

    Nat. Rev. Mol. Cell Biol.

    (2014)
  • H. Su et al.

    Essential and overlapping functions for mammalian Argonautes in microRNA silencing

    Genes Dev.

    (2009)
  • M.V. Iorio et al.

    MicroRNA dysregulation in cancer: diagnostics, monitoring and therapeutics. A comprehensive review

    EMBO Mol. Med

    (2012)
  • J.R. Lytle et al.

    Target mRNAs are repressed as efficiently by microRNA-binding sites in the 5' UTR as in the 3' UTR

    Proc. Natl. Acad. Sci. U. S. A.

    (2007)
  • F. Moretti et al.

    Mechanism of translational regulation by miR-2 from sites in the 5' untranslated region or the open reading frame

    RNA

    (2010)
  • W. Qin et al.

    miR-24 regulates apoptosis by targeting the open reading frame (ORF) region of FAF1 in cancer cells

    PLoS One

    (2010)
  • S. Vasudevan et al.

    Switching from repression to activation: microRNAs can up-regulate translation

    Science

    (2007)
  • M.A. Cortez et al.

    MicroRNAs in body fluids–the mix of hormones and biomarkers

    Nat. Rev. Clin. Oncol.

    (2011)
  • E.E. Creemers et al.

    Circulating microRNAs: novel biomarkers and extracellular communicators in cardiovascular disease?

    Circ. Res.

    (2012)
  • L.S. Enache et al.

    Circulating RNA molecules as biomarkers in liver disease

    Int. J. Mol. Sci.

    (2014)
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