ReviewCirculating miRNAs in nontumoral liver diseases
Graphical abstract
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.
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