Importance of MicroRNAs in Hepatitis B and C Diagnostics and Treatment Importance of MicroRNAs in Hepatitis B and C Diagnostics and Treatment Importance of MicroRNAs in Hepatitis B and C Diagnostics and Treatment

MicroRNAs (miRNAs) are small‐sized RNAs with ability to regulate gene expression and have been recently discovered as promising diagnostic and therapeutic biomarkers in the field of clinical medicine and microbiology, specifically in viral diseases. Infections with hepatitis B virus (HBV) or hepatitis C virus (HCV) often lead to chronic infections and development of liver hepatocellular carcinoma (HCC). Challenges in early diag ‐ nosis of HCC and rapid development of novel HCV antivirals call for identification of novel miRNA biomarkers. An extensive selection of single miRNAs and miRNA pan ‐ els has been provided by accumulating studies, discovering miRNA potentials in HBV and HCV diagnostics and treatment. Currently, the diagnostic potential of miRNAs in HBV and HCV has not been established yet. However, a promising HCV treatment drug Miravirsen, a locked nucleic acid, complementary to miRNA‐122, has entered a human clinical trial recently. In this review, we outline the role of miRNAs in HBV and HCV pathogenesis and differences in up‐ and downregulation of miRNAs upon HBV and HCV infection and HCC development.


Introduction
Hepatitis B and C viruses (HBV and HCV) are globally spread hepatotropic pathogens and major etiological factors of liver cirrhosis and hepatocellular carcinoma (HCC), infecting millions of people worldwide. HBV and HCV significantly differ in structure, genomic characteristics, and pathogenesis [1,2].
MicroRNAs (miRNAs) are small noncoding RNAs that control gene expression and participate in complex cellular pathways and pathogenesis of various viral infections and cancers [3][4][5].
Despite the availability of prophylactic HBV vaccine and recently improved HCV therapy strategies, early diagnosis of HBV/HCV-related HCC, viral reactivation, resistance, drug interactions, and viral interferences in HBV/HCV co-infected patients remain major obstacles in currently available HBV and HCV diagnostics and treatments [6,7].
Multiple studies have proposed specific miRNA or miRNA panels to be used as biomarkers for HBV/HCV-related liver disease, staging of liver disease progression, and anti-HBV/HCV therapeutic options.
In this chapter, we briefly outline the HBV and HCV biology and basis of miRNA expression in liver and HCC. Subsequently, we summarize recently described and proposed miRNAs for HBV-and HCV-associated diagnostics, particularly HBV/HCV-related HCC.

Hepatitis B virus
Hepatitis B virus is an enveloped, partially double-stranded DNA virus, classified in the Hepadnaviridae family. HBV genome is approximately 3.2 kb long and contains four RNA transcripts (P, S, C, and X), of which the S transcript encodes the surface antigen HBsAg, the main indicator of HBV infection [1]. Upon infection, cellular polymerase converts HBV DNA into covalently closed circular DNA (cccDNA), which represents a constant template for pregenomic RNA and mRNA transcription. Due to the stable state of HBV cccDNA in hepatocytes, HBV can reactivate after immunosuppression [1,8].
HBV is classified into eight genotypes (designated A through H). Most chronic infections are related to infection with HBV genotypes B and C; however, in Europe, genotype D has been shown to be more often associated with active liver disease [1].
Globally, two billion people are infected with HBV, with majority of HBV infections occurring in South-Eastern Asia and sub-Saharan Africa [9,10]. Approximately, one-fourth of HBV-infected individuals suffer from liver cirrhosis and 70-90% of HCC develop from cirrhotic liver [1,10]. In developing countries, HBV infection accounts for about 60% of the total liver cancer and in developed countries for about 23% [11]. In endemic areas, HBV infections occur mostly by vertical and perinatal transmission and in more than 90% lead to chronic HBV infections. In low prevalence countries, HBV infections occur mostly through a horizontal transfer (sexual transmission), which in more than 90% lead to acute HBV infections with spontaneous viral clearance [10]. Despite improved HBV antiviral therapy over the past two decades, and the prophylactic anti-HBV vaccine, available since 1981, prenatal maternal HBeAg seropositivity in endemic countries remains significantly connected to HCC and elimination of cccDNA remains the major challenge in HBV-related treatment strategies [1,12,13].

Hepatitis C virus
Hepatitis C virus has a positive-sense single-stranded RNA genome of approximate length of 9.6 kb and is classified in the family Flaviviridae. The HCV genome encodes a polyprotein that is cleaved into three structural and seven nonstructural proteins. Based on HCV genomic sequence diversity, HCV is classified into seven genotypes and more than 60 subtypes are identified [2,14]. Genotypes 1, 2, and 3 are globally distributed and cause most of HCV infections in North America and Europe. In Middle East and North and Central Africa, genotype 4 prevails generally, in South Africa genotype 5, in Asia genotype 6, while a recently discovered genotype 7 originates from Central Africa [2,15].
Currently, no vaccine is available for the prevention of HCV infection. Most HCV transmissions occur by intravenous drug abuse, sexual transmission, and occupational exposure to HCV-infected blood [2]. Potential long-term outcomes in chronically HCV-infected people are liver cirrhosis and HCC, which remain the leading cause for liver transplantation. HCV is globally infecting over 150 million individuals [16][17][18]. Recent estimate on global anti-HCV prevalence is 115 million-infected individuals, of whom 80 million are actively viremic [19].
A recent study by Sibley et al. [20] forecasted changes in HCV-related disease up to 2030 and concluded that HCV-related morbidity and mortality are estimated to increase due to an aging of the HCV-infected population and currently available treatment will be inadequate if reductions in HCV-related disease of this magnitude are to be achieved [20].

MicroRNAs
Micro-ribonucleic acids (microRNAs/miRNAs) are noncoding RNAs of 18-25 nucleotides in length that complementarily target the 3′-untranslated regions (3′ UTRs), or less commonly 5′-untranslated regions (5′ UTRs) of messenger RNAs (mRNAs) [3]. Genes encoding miRNAs are located in intragenic regions or introns of mRNAs or noncoding RNAs [21]. MiRNAs are transcribed from the genome by the RNA polymerase II into primary-miRNA (pri-miRNA) hairpins, which are processed by Drosha (class III RNase) into pre-miRNAs. Pre-miRNAs are exported from the nucleus to the cytoplasm, where they are processed by a second RNaseIII Dicer into short double-stranded mature miRNAs, consisting of 5' and 3' arms. Finally, singlestranded miRNAs are assembled with specific proteins and form a RNA-induced-silencing complex (RISC). At least two to seven nucleotide complementarities with the target sequence are required for RISC-mediated target silencing [22][23][24].
The binding of miRNAs in posttranscriptional or translational level provides a rapid and sensitive mechanism of gene expression regulation, either by suppressing the translation of mRNA or by promoting mRNA degradation [25]. Gene silencing by a full complementary miRNA sequence directs cleavage of the target mRNA, while partial complementary miRNA sequence suppresses mRNA translation [26,27]. Currently, more than 2,588 mature miRNAs are reported in a human genome [28,29] and due to sufficient partial complementarity to the target sequence it has been shown that one type of miRNA could affect up to 200 genes, and over a 100 different targets can be involved in approximately 100 different biochemical pathways [30,31].

MiRNAs mechanism
MiRNAs are participating in various cellular processes such as cell development, differentiation, proliferation, metabolism, immune responses, apoptosis, and oncogenesis [32,33]. Estimates in humans suggest that 60-70% of all genes are regulated by miRNAs [4]. Being involved in numerous biological pathways, their expression and regulation reflect in various diseases, stages of the particular disease, especially in cancer development [34,35].
MiRNAs are cell-free-circulating molecules that can be detected in almost every body fluid. Their high stability and accessibility make them ideal noninvasive markers for the early diagnosis of different pathophysiological processes. Indeed, a large amount of evidence suggests that miRNA profiles could provide a classification system for various tumors, as well as an important tool for the diagnosis and treatment of cancer and viral diseases [36][37][38][39][40][41]. Accordingly, cellular miRNAs have an ability to regulate pathogenesis of viral infections, and at the same time viruses manipulate with host cellular machinery, including miRNAs [42,43].
Increased interest in hepatitis B and hepatitis C disease pathogenesis and diagnostics has lead to the emergence of various studies over the last 15 years that have tried to evaluate plasma and tissue levels of miRNAs in order to provide or improve the diagnosis of HBV and HCV infections as well as HBV-and HCV-related HCC [5].

MiRNAs in normal liver
Liver consists of various cell types, mainly divided in the parenchymal cells (hepatocytes) and non-parenchymal cells (biliary epithelial cells and lymphoid cells, etc.). Each cell type expresses its unique miRNA profile. While miRNAs are up-or downregulated in almost every stage of hepatic development, they accelerate or inhibit liver proliferation and play the major role in regulation of diverse liver functions [44]. It has been shown that a total of 277 miRNAs are expressed in the liver, with miR-122 being one of the most abundant and liverspecific miRNAs [45,46]. Besides miR-192, miR-199a/b-3p, miR-101, miR-99a, and let-7a/b/c/f (let-7 family), which account for 80% of the total miRNA in liver, miR-122 accounts for 70% of the total liver miRNAs [45,47]. Expression of miRNAs in the normal liver has been established by microarray systems and library sequencing [47][48][49].

Function of miRNAs in normal liver
The function of miR-122 has been explored in a variety of in vivo studies, including the miR-122 gene knockdown or silencing of miR-122 with antagomirs [50][51][52]. In the miR-122 gene knockdown mice, it has been shown that the deletion of the miR-122 gene resulted in hepatosteatosis, hepatitis, and the development of liver tumors [52]. Beyond that, studies implicate miR-122 as a key regulator of cholesterol and fatty-acid metabolism [50]. However, results of studies evaluating up-and downregulated miRNA profiles in differentiating liver cells are not consistent. Besides various technical issues, including differences in clinical samples and different miRNA matrices in miRNA assays, different degrees of miRNA expression among studies suggest that the miRNA profile is also influenced by the origin of the progenitor cell and that it is difficult to compare miRNA profiles in different cellular developmental stages [53].
However, it has been shown in two studies by Liu et al. [54] and Tzur et al. [55] that embryonic liver mainly contains miRNAs- 122, -192, -194, -451, and -483-3p, whereas miR-122 can be detected in embryonic stem cells as well as in hepatocytes and continues to be expressed in the adulthood. Studies of hepatic malignancy pathologies have shown that miRNAs have specific targets in specific disease states [44,53,56]. Interestingly, a biphasic pattern of miRNA expression was observed in rats after liver surgical resection [57]. In the first 18 h after hepatectomy, about 40% of miRNAs were upregulated, whereas by 24 h there was a negative feedback mechanism which downregulated about 70% of all miRNAs [57]. These negative feedback loops are postulated to play an important role in liver regeneration processes, required for recovery of liver cells after injury; however, the abundance of miRNAs does not directly correlate with the predictable role of specific cells.
Since essential knowledge on liver regeneration processes has been delivered from rodent model studies, further studies are warranted to confirm post-hepatectomy miRNA level changes in humans [4,57]. Investigation of miRNA levels in specific stages of liver organogenesis may reveal potential biomarkers for liver disease states.

miRNA and HBV-and HCV-related liver disease
HCC is the fifth most common cancer worldwide. Deregulation of miRNA expression could be one of the key factors in the development of liver pathology, including viral hepatitis and HCC [11]. Evidence is rapidly growing that specific miRNAs could be used as potential biomarkers for HCC, tumor progression, and response to therapeutic targets [4].
Infection with HBV and HCV can lead to chronic hepatitis, liver cirrhosis, or even HCC. Approximately 50-80% of HCC cases are associated with chronic HBV or HCV infection [10]. In the past several years, the involvement of miRNA in the pathogenesis of HBV-/HCVrelated liver diseases has been well documented [53]. Since miRNAs can be directly involved in antiviral immune-pathological events, it is inevitable that miRNA target sequences in viral populations remained conserved, providing relevant evidence of the biological significance of potential miRNA-based antiviral interventions [58]. For the time being, no HBV-or HCVencoded miRNAs have been reported. Using computational approaches, one candidate HBV miRNA has been found but its function remains undetermined [59].
It should be noted that miRNA dysregulation has been studied in various experimental settings, mainly involving in vitro systems, HBV-/HCV-replication-supporting cell lines, transgenic mice, cultured hepatocytes (mouse/rat/human), circulating blood cells or serum of HBV-/HCV-infected individuals, and liver tissue samples. MiRNA expression in model systems has been measured mainly with qualitative real time-polymerase chain reactions (RT-PCRs) and/or miRNA microarrays and less frequently with next-generation sequencing (NGS) (for review, see [23,60]).

MiRNAs and HBV infection
It is well known that numerous cellular miRNAs are able to promote or repress the HBV lifecycle, either by directly targeting HBV transcripts or by indirectly targeting cellular mediators, involved in the HBV pathogenesis. Alternatively, HBV infection dysregulates cellular miRNAs and in this manner controls the host gene expression to promote its replication [60].
A variety of miRNAs have been reported in regulation of HBV replication namely miR-122, let-7 family, miR-199 family, miR-15 family, miR-125 family, and miR-17-92 cluster (extensively reviewed in [60]). Reported effects of the most abundant liver miRNA, miR-122, on HBV lifecycle remain mixed. Whereas some studies reported miRNAs as inhibitors of HBV replication [61][62][63][64], others have failed to even identify miR-122 as a regulator of the HBV lifecycle [65]. For example, in a study by Qiu et al. [63], in comparison to the control system, cotransfection of Huh7 cells with miR-122 inhibitor, and a plasmid encoding the HBV genome, the production of HBsAg and HBeAg increased, suggesting a negative regulatory effect of miR-122 on HBV lifecycle.
The study of Wang et al. [64] reported downregulation of miR-122 expression in liver of patients with HBV infection, in comparison to healthy controls, and showed that the miR-122 levels were negatively correlated with intrahepatic viral load and hepatic inflammation. Researchers concluded that HBV-induced miR-122 downregulation enhances HBV replication through cyclin G1-modulated P53 activity and that HBV mRNAs harboring complementary sites to miR-122 sequester miR-122 and contribute to viral persistence and carcinogenesis [61,64].
Guo et al. [66] showed that members of the miRs-371-372-373 (miRs-371-3) gene cluster were co-upregulated in HBV-producing HepG2.2.15 cells and revealed that miRs-372/373 promotes HBV expression by targeting the transcription factor NFIB. Furthermore, Zhang et al. [65] reported that miR-1 promotes HBV replication, transcription, and antigen expression by indirect modulation of host genes expression in HCC cell line. In addition, they have shown that miR-1 arrested cell cycle, inhibited proliferation, and therefore reversed the cancer cell phenotype, which is in contradiction with HBV-induced carcinogenesis, since HBV infection promotes hepatocellular proliferation [65].
Jin et al. [67] have shown that downregulation of miR-501 in HepG2.2.15 cells could significantly inhibit HBV replication, thus representing a potential therapeutic target. They suggested that miR-501 promotes HBV replication through inhibition of the HBXIP, which interacts with HBx protein and normally represses HBV replication [67]. In a mouse model, Dai et al. [68] reported that miR-15b promoted HBV replication by direct inhibition of hepatocyte nuclear factor HNF1α.
On the other hand, miR-125 family members, miR-125a and miR-125b, have been reported to suppress the HBV lifecycle. In the PLC/PRF/5 cell line, miR-125a-5p was identified as a downregulator of HBsAg expression by directly targeting HBV RNAs [69], while miR-125b inhibited HBV in HepG2.2.15 cells [70]. The miR-22 has been reported as a regulatory molecule which inhibits HBV infection [71]. Furthermore, miR-199a-3p and miR-210 were shown to repress the HBV replication in HepG2.2.15 cells by directly targeting the HBV S protein-coding region [72], while inhibition of miR-20 and miR-92a-1 increased levels of HBV RNAs in HepAD38 hepatoma cells [73]. Hu et al. [74] demonstrated that miR-141 suppresses HBV replication by reducing HBV promoter activities and two separate studies suggested upregulation of miR-181a in HBV-infected hepatoma cells, implying an important role in the development of HCC [75,76].

MiRNAs and HCV infection
HCV lifecycle is influenced by host miRNAs in all stages: entry, translation, replication, and assembly [43]. As the HCV genome is single-stranded RNA, it serves as a template for its replication and direct binding site for host miRNAs. Among high number of miRNAs reported to be involved in the regulation of HCV infection and replication, most miRNAs have been documented to directly target the HCV genome: miR-1, miR-30, miR-122, miR-128, miR-196, miR-296, miR-351, miR-431, and miR-448 [77,78].
Microarray analysis on human hepatoma cells has revealed changed expression profiles of 108 human miRNAs after HCV infection [79]. Furthermore, Liu et al. [79] showed that after acute HCV infection, miR-122 was downregulated, whereas miR-296 and miR-351 were significantly upregulated. In addition, it has been shown that HCV infection upregulated the expression of miR-192, miR-194, and miR-215, whereas the expression of miR-320 and miR-491 was downregulated [80]. It was reported that miR-192/miR-215 and miR-491 could enhance HCV replication [80].
For the most abundant miRNA in the liver, miR-122, it has been demonstrated that it promotes HCV replication by direct binding to the less commonly used UTR-binding site, the 5′ UTR site of the HCV RNA, which leads to Argonaute (Ago) protein complex recruitment, stabilization of the viral RNA, and activation of the RNA translation [77,81,82]. In vitro studies have shown that miR-122 is essential for HCV replication [81].
On the other hand, it has been shown that miR-122 exhibits anti-inflammatory and antitumorigenic properties in mice knockdown studies [52]. Mixed results exist on expression levels of miR-122 and development of HCC-or HCV-induced HCC. Coulouarn et al. [83] have shown that the loss of miR-122 expression in liver cancer correlated with HCC progression, whereas in another study, the upregulation of miR-122 promoted the HCV-related HCC [84].
The increased expression of miR-155 in HCV-infected patients promotes hepatocarcinogenesis and inhibits apoptosis of hepatocytes [85]. Furthermore, the direct effect on HCV replication cycle has been determined in the cell culture system for the miR-196b, which is complementary to the NS5A region of the HCV genome and is downregulated in HCV-infected patients. MiR-196b inhibits HCV replication directly by targeting HCV RNA or indirectly by increasing the expression of HMOX1. It has anti-inflammatory, antioxygenic, and hepatoprotective properties [86]. Some miRNAs can facilitate HCV lifecycle by targeting host proteins involved in innate immunity-signaling pathways. For example, HCV induced upregulation of miR-130 blocks expression of interferon stimulatory gene IFITM1, which promotes HCV entry into host cells [87]. Furthermore, miR-491 promotes HCV replication through inhibition of the PI3 kinase/ Akt pathway, one of the pathways leading to cancerous properties [80]. Studies analyzing circulating miRNA profiles in serum provide novel insights on miRNA expression in HCV pathogenesis [88,89]. In a study by Shwetha et al. [89], it has been shown that the expression of miR-134, miR-198, miR-320c, and miR-483-5p was upregulated in patients infected with HCV 1 and HCV 3 genotypes.
Complex correlation between hepatic expression of abundant liver miRNAs, miR-122, miR-126, miR-136, and miR-181a, and histopathological and clinical characteristic of HCVinfected patients has been reported by Boštjančič et al. [31]. The study included liver biopsies of patients infected with different genotypes (1, 1a, 1b, 3, and 4) and provided an important insight into miRNA expression patterns in different stages and grades of liver disease and revealed association among specific miRNA deregulation and patient gender, serum HCV viral load, presence of steatosis, and mode of HCV transmission [31].
By contrast, another study by Elhelw et al. [90] has demonstrated upregulation of miR-181a in serum samples of HCV genotype 4-infected individuals and downregulation in HCV-infected Huh7 cells. In addition, the inverse correlation between miR-181a serum levels, viral load, and liver enzymes was observed. Due to complexity of viral and host factors involved in HCV infection and progression to HCV-induced HCC, multiple clinical parameters should be considered and controlled for in the future studies. A systematic approach was recently reported by Oliveira et al. [91]. The study evaluated liver and serum expression of miR-122 in patients infected with HCV genotypes 1 and 3 to identify possible associations between miR-122 expression and lipid profiles, HCV viral load, apolipoproteins, and liver enzymes [91].

MiRNAs as biomarkers in diagnostics and treatment of HBV and HCV
Early diagnosis and treatment of HCC remain challenging due to the lack of early detection methods, limited access to diagnosis, expensive medications and the presence of comorbidities, coinfections, and contraindications due to different host and viral factors. Most of HBV-/HCV-infected individuals remain undiagnosed before they seek medical help due to advanced HCC [10,92].
The gold standard for the etiology of liver diseases is liver biopsy, an invasive method being replaced recently by serological methods and imaging technologies. Current diagnostic techniques for HCC, which are generally divided into radiological (first-line diagnostic method is ultrasound) and serological methods (serological marker alpha-fetoprotein, AFP, and desgamma-carboxy prothrombin), provide limited reliability [93,94].

MiRNAs as biomarkers in body fluids
MiRNAs can be detected in various body fluids, such as plasma, serum, urine, and infected or diseased tissue and may exhibit host responses to the pathogen or other inflammatory processes; however, miRNA levels in body fluids may not necessarily reflect the miRNA level in the infected/diseased tissue, and second, the same miRNA may be upregulated in one state of disease and downregulated in another. To provide optimal management of HBVand HCV-related diseases, novel surrogate miRNA biomarkers should be considered [41]. A great amount of studies provide promising information on miRNAs as potential diagnostic biomarkers of HBV or HCV infection as well as potential diagnostic and treatment tool in HBV-/HCV-related HCC. According to specific miRNA targets and up-or downregulation of miRNA expression, potential imitating or antagonistic characteristics of miRNAs could be used in HBV-/HCV-related therapy.

HBV miRNA biomarkers
Currently used markers in the diagnostics of HBV can serve as indicators of specific HBV infection phases; however, none of them can be used to predict the HBV infection outcome [41].
In order to reliably differentiate HCC from chronic HBV infection, cirrhosis, and healthy subjects, plasma panel of seven miRNAs (miR-122, miR-192, miR-21, miR-223, miR-26a, miR-27a, and miR-801) has been investigated by Zhou et al. [104]. Mizuguchi et al. [107] employed sequencing-based miRNA clustering and showed that the panel of miRNAs was more effective for the detection of high-risk patients for HBV-related HCC recurrence after liver surgery, in comparison to investigation of a single miRNA. MiR-122, miR-21, and miR-34a were identified as potential biomarkers for the prediction of HBV-related HCC.
The miR-34a is a direct target of the P53 and it has been shown that the expression of miR-34a is downregulated in several human cancers, and when overexpressed, miR-34a can repress several oncogenes and induces apoptosis and arrest of the cell cycle. Deletion of gene region encoding miR-34a has been well detected in breast, lung, cervical, and prostate cancers (reviewed by Agostini and Knight [111]). A mimic miR-34a (MRX34) became a promising therapeutic tool for HCC and has reached a clinical trial, phase 1 in 2013 [111].
Individual miRNAs or combination of miRNA and serological markers for HCC have been examined and proposed. While an upregulated oncogenic miRNA-27a has been suggested as a therapeutic target in HBV-related HCC patients [108] and miR-101 as a potential noninvasive biomarker to differentiate HBV-related HCC from HBV liver cirrhosis [97,112], a combination of circulating miR-126 and AFP has been proposed as a promising noninvasive-specific diagnostic biomarker for HBV-related HCC. Furthermore, combinations of miR-126/AFP AFP and miR-142-3p/AFP showed higher efficiency rather than AFP alone in discriminating HCC from non-HCC patients [99]. Abbreviations: HCC, hepatocellular carcinoma; NGS, next-generation sequencing; ND, no data available; qRT-PCR, quantitative real-time polymerase chain reaction. Serum miRNAs could serve as biomarkers for the detection of liver pathologies [102]. Serum HBV-related miRNAs for HBV-related HCC diagnosis have been investigated by Tan et al. [101]. The study identified eight miRNAs (miR-206, miR-141-3p, miR-433-3p, miR-1228-5p, miR-199a-5p, miR-122-5p, miR-192-5p, and miR-26a-5p) and constructed a miRNA set that provided high diagnostic accuracy for HBV-related HCC [101]. The study published by Winther et al. [113] presented a panel of circulating plasma miRNAs that are differentially expressed in immunological phases of chronically HBV-infected children and positively correlated with the quantity of HBsAg.
Hou et al. [47] performed an extensive study of miRNomes in human normal liver, hepatitis liver, and HCC. Researchers presented 15 deregulated miRNAs in 40 HBV-related HCC samples in comparison to healthy controls. Additionally, the consistent decline of miR-199a/b-3p in HCC and its significant correlation with poor prognosis of HCC patients has been elucidated, suggesting miR-199a/b-3p as a potential HBV therapeutic target. Gao et al. Several studies have suggested the role of HBx protein in miRNA expression during HBV infection [67,96,98,106]. For example, the recent study, conducted on 120 patients with HBV-related HCC, has shown that the expression of miR-106b was significantly higher in HBV-related HCC patients in comparison to non-HBV/non-HCV-related HCC patients and suggested that HBx enhances miR-106b transcription and therefore promotes tumor progression in HBV-related HCC [98]. Transfection with the HBx expression plasmid has been recently used in an additional study by Yu et al. [114] to investigate HBx-related regulation of miR-19a, miR-122, and miR-223 in malignant hepatocytes. The study has shown that the expression of miRNAs was regulated by HBx protein, which enhanced the proliferation of HBx-transfected HepG2 cells.

HCV miRNA biomarkers
Apart from a variety of published studies focusing on the identification of HCV infection miRNA biomarkers [88], our review focused more on difference in miRNAs profiles between HCV-infected cancerous liver cells and HCV-infected cells without progression to HCC. Despite the fact that treatment for most common HCV genotype 1 has been evolving rapidly in the past 10 years, no effective and safe anti-HCV vaccine is available and only approximately 50% of patients, infected with HCV genotype 1 (the more common genotype in USA and Western Europe), reach sustained viral response (SVR) while treated with most accessible treatment, the interferon. Therefore, the management of HCV-induced liver disease remains problematic [115].
Antagonism of miR-122 by locked nucleic acid is a promising tool for the treatment of HCV.
The current most advanced research on miR-122 antagonist Miravirsen is discussed in the following section. Likewise, the therapeutic potential of mimic miR-196b is presented by Kaluzna [30], reviewing studies which confirmed the ability of miR-196b to inhibit HCV replication and revealed that interferon-induced overexpression of miR-196b decreases inflammation and leads to a better response in interferon-based HCV therapy.
Circulating serum levels of miR-122 and miR-222 have been shown to be useful potential diagnostic biomarkers for chronic HCV infection in Egyptian patients [116][117][118], whereas in another study miR-122, miR-199a, and miR-16 have been implicated as potential early diagnostic biomarkers for HCC in Egyptian patients, chronically infected with HCV [118] ( Table 2).
Of note, miR-222 and miR-224 have been reported to be upregulated in both, HBV and HCV infections [95,105,116,121]. However, Bandopadhyay et al. [106] and Zhang et al. [95] reported downregulation of miR-222 and miR-224 in HBV-infected patients, respectively. As expression profiles of circulating serum biomarkers became a subject of interest in different disease studies, serum miRNAs possibly involved in HCV-related HCC have been investigated in several studies. Oksuz et al. [119] examined HCV-infected patients with chronic infection, cirrhosis, and HCC and compared them with control group samples. When all groups of samples were compared, the study revealed deregulation of miR-30c-5p, miR-223-3p, miR-302c-3p, and miR-17-5p in cirrhosis and HCC, suggesting possible novel noninvasive biomarkers for HCC.
Increased expression of miR-155 in hepatocytes of patients infected with HCV has been confirmed in vitro and in vivo by Zhang et al. [85]. In addition, the study revealed that overexpression of miR-155 inhibits apoptosis, and promotes hepatocyte proliferation and tumorigenesis, which suggested miR-155 could be a negative prognostic biomarker for HCC (reviewed by Kaluzna [30]). In addition, miR-155 has been shown to be upregulated in HBV-infected human hepatoma cells, where it inhibits HBV infection [103] (Figure 1). Abbreviations: HCC, hepatocellular carcinoma; NGS, next-generation sequencing; ND, no data available; qRT-PCR, quantitative real-time polymerase chain reaction.
* Different mature miRNA from the same stem loop. Despite the fact that several studies examined the expression of miRNAs in HCC, discrepancies exist among published data. Diaz et al. [121] assumed that non-concordance may be the result of differences in selection of noncancerous control samples or commonly included HCC samples without prior confirmation of possible HBV or HCV infection. In some previous studies, Diaz et al. [121] investigated the expression of miRNAs in HCV-induced HCC, in comparison to a wide range of liver samples and identified 18 miRNAs exclusively expressed in HCV-induced HCC. Several other studies have as well examined subsets of miRNAs potentially involved in hepatocellular changes, advancing to HCC [80,84,122].
According to an increasing amount of miRNAs identified and examined throughout various stages Of HBV/HCV infection and HCC development (Tables 1 and 2), miRNAs not specifically presented in this review should be as well included as a subject of interest in future studies.

MiRNA-122 in HBV and HCV
The levels of liver-specific miRNA-122 (miR-122) are down-and upregulated in HBV and HCV, respectively. The miR-122 promotes the replication of HCV and blocks the replication of HBV. It has been shown recently that HBV inhibits the miR-122 expression, suggesting a possibility of miR-122 replacement therapy in HBV-infected individuals [61,64]. The study by Fan et al. [62] showed that miR-122 inhibited the expression of the NDRG3 protein, which subsequently inhibited malignant cell transformation and presented the miR-122 and its target NDRG3 as key diagnostic markers and potential therapeutic targets in HBV-related HCC.
On the other hand, in HCV infections, it has been shown in vitro and in vivo that antagonistic utility of miR-122 inhibits HCV replication cycle and reduces viral load, and thus represents an effective treatment of HCV infection. A model of Miravirsen interaction with miR-122 is shown in Figure 2 [77,123].
Recently, the safety and efficacy of the Miravirsen, a locked nucleic acid form of antisense-miR-122 that sequesters miR-122, has been evaluated in a phase 2a clinical study, which included 36 patients with chronic HCV genotype 1 infection from seven international sites [40]. The study observed that treatment with Miravirsen prolonged dose-dependent reductions in HCV RNA, without evidence of viral resistance [40]. Moreover, a recent study, conducted on 51 HCV-infected Japanese patients, treated with interferon, presented miR-122 as an independent predictor of SVR [124]. Unfortunately, no single miRNA representing a promising treatment option in HBV infections has been pointed out as yet.
Nevertheless, therapies that silence HBV RNAs are emerging. Multiple cell-line-based studies and in vivo studies on mouse models have evaluated synthetically engineered or chemically modified small RNAs that complementarily target HBV transcripts and lead to RNA degradation and thus to the inhibition of HBV replication. However, due to short duration of their activity, the lack of applicable animal model for testing in clinical trials, and subsequent Importance of MicroRNAs in Hepatitis B and C Diagnostics and Treatment http://dx.doi.org/10.5772/66498 pharmacokinetic difficulties, further investigations are warranted to evaluate RNA-interference-based approaches to clinical practice (reviewed by Ivacik et al. [125]).
HBV/HCV dual infection is not an uncommon event, occurring in approximately 2-10% of chronically infected HCV patients and in 5-20% of chronically infected HBV patients [126]. Dual HBV/HCV infection has prognosis of a more aggressive clinical course of liver disease than either mono-infection. Despite the fact that compiling evidence exists on reciprocal inhibition between HBV and HCV and that miR-122 represents a crucial host gene involved in pathogenesis of both viruses, the role of miR-122 in HBV/HCV dual infection has not been defined so far [127].

Conclusion
Cellular miRNAs contribute to HBV and HCV pathogenesis by direct or indirect interactions with viral genome or proteins and molecules critical for regulation of the cell cycle. Regulation of miRNAs expression upon HBV and HCV infection significantly differs between both viruses. Reports summarized in this chapter indicate that miRNAs represent an effective, noninvasive biomarker tools for early diagnosis of HBV and HCV infection, early diagnosis of liver disease and its progressive stages, particularly HCC. Mimic and antagonistic effects of cellular miRNAs have been considered in diagnostic and treatment of HBV/HCV-related liver disease, with miR-122 representing a promising treatment option for chronic infection with HCV genotype 1. Because most studies identified and validated miRNAs in heterogenic tumors and because miRNA targets were validated mostly in the already-transformed cell culture systems, transfected with plasmids encoding HBV or HCV genome or parts of their genome, discrepancies exist in candidate biomarker miRNAs across published studies. Due to an extensive number of miRNA targets and other clinical factors considered in significant number of studies published in the last 10 years, efforts should be made to establish a specific, repetitive, and easy-to-operate method to identify reliable panels of miRNA biomarkers for early diagnosis and treatment of HBV-HCV-related diseases. Suitable reference miRNA targets and positive and negative controls should be included in such profiling applications. The application of novel techniques such as next-generation sequencing, development of synthetic small RNAs, and hepatoma cell lines will impact the subsequent advances in miRNA studies related to HBV and HCV pathogenesis as well as miRNA deregulation in other pathological conditions.