MicroRNAs (miRNAs) are small non-coding RNAs that modulate gene expression at the post-transcriptional level by affecting both the stability and translation of complementary mRNAs. Several studies have shown that miRNAs are important regulators in the conflicting efforts between the virus (to manipulate the host for its successful propagation) and the host (to inhibit the virus), culminating in either the elimination of the virus or its persistence. An increasing number of studies report a role of miRNAs in hepatitis B virus (HBV) replication and pathogenesis. In fact, HBV is able to modulate different host miRNAs, particularly through the transcriptional transactivator HBx protein and, conversely, different cellular miRNAs can regulate HBV gene expression and replication by a direct binding to HBV transcripts or indirectly targeting host factors. The present review will discuss the role of miRNAs in the pathogenesis of HBV-related diseases and their role as a biomarker in the management of patients with HBV-related disease and as therapeutic targets.

MicroRNAs (miRNAs) are small non-coding RNAs that modulate gene expression at the post-transcriptional level by affecting both the stability and translation of complementary mRNAs[1]. MiRNAs play crucial roles in a variety of physiological processes, such as cell development and differentiation[2,3]. miRNA mutations, dysregulation of their expression or dysfunction of miRNA biogenesis lead to an interference with biological pathways involved in the development and evolution of human diseases, including cancer, cardiovascular diseases and infectious diseases[4-8].

About the host-virus interplay, various studies have shown that miRNAs are important regulators in the conflicting efforts between the virus (to manipulate the host for its successful propagation) and the host (to inhibit the virus), culminating in either the elimination of the virus or its persistence. In fact, different viruses encode miRNAs that modulate not only the viral mRNA expression to regulate its own lifecycle, but also the host mRNA expression to establish a cellular environment resulting favorable to their replication; on the other hand, host cells encode miRNAs counteracting viral replication[8-10]. An increasing number of studies report a role of miRNAs in hepatitis B virus (HBV) replication and pathogenesis. HBV is a non-cytopathic virus belonging to the Hepadnaviridae family. It has a 3.2 kb partially double-stranded DNA, showing 4 known open reading frames (ORFs): The S region, which contains three in-frame initiator codons, codes for the small, medium and large surface antigen (HBsAg) proteins; the C region, with two initiator codons for the core and “e” antigen (HBcAg, HBeAg); the P frame, coding for an RNA-dependent DNA polymerase; and the X region, coding for a protein regulating the transcription of both viral and cellular genes[11].

The present review will discuss the emerging puzzle of HBV-hepatocyte interaction via miRNAs, indirectly or directly modulating HBV replication and pathogenesis, and thus will focus on the role of microRNAs in the natural history of HBV infection and on their possible future role as biomarkers in the management of patients with an HBV-related disease and as therapeutic targets.

Despite the universal vaccination campaigns against HBV infection undertaken in many countries over the last two decades, this infection remains a global health problem. In 2015, the WHO estimated that nearly 3.5% of the world population live with chronic HBV infection[12], with about 800000 deaths per year (460000 for complications of liver cirrhosis and 340000 for hepatocellular carcinoma-HCC)[13]. For non-immune/non-infected subjects any parenteral or mucosal exposure to blood, blood products or blood-contaminated material should be considered a risk for acquiring HBV infection[14]. In addition, being present in semen and cervical secretions at infectious concentrations, HBV is also transmitted by sexual and vertical routes[15]. The age at the time of infection strongly modulates the progression to chronicity, which occurs in around 90% of subjects infected at birth, a rate progressively decreasing with the increase in age at infection, up to 2%-5% in the adult population[16].

The geographical distribution of HBV chronic carriers is highly variable, ranging from 0.7%-1% in developed western countries to 8% or more in some countries in sub Saharan Africa and South-East Asia, depending on environmental factors, earning potential, educational levels and lifestyles. In countries with an intermediate-high level of endemicity, HBV infection is most frequently acquired at birth from an HBeAg-positive mother or through horizontal transmission in early childhood by household contacts (most frequently between siblings); in these cases the rate of progression to chronicity is high, which keeps the prevalence of infection in these geographical areas intermediate or high. Intravenous drug addiction is the major risk factors for acquiring HBV infection in countries with a low HBV endemicity like Western Europe[15] and North America[17], whereas promiscuous unprotected sexual activity is a main risk factor worldwide.

The clinical presentation of chronic HBV infection is variable, ranging from asymptomatic carriage of the virus to liver cirrhosis with or without HCC[18]. Patients with chronic hepatitis develop liver cirrhosis with an incidence of 1-5 per 100 persons/year, with a 5-year cumulative probability of progression ranging from 8% to 20%, depending on the degree of disease activity, HBeAg/anti-HBe status, the HBV load and co-morbidities[19]. The incidence of HCC in patients with HBV-related liver cirrhosis is estimated around 3.7 persons/year[12]. HCC may develop, but with a lower frequency also in patients with chronic HBV infection without cirrhosis, depending on demographic (male sex, older age), viral (higher levels of HBV replication; co-infections) and environmental (alcohol abuse) factors[20].

So far, there is no experimental evidence confirming the synthesis of miRNAs by HBV, though a computational analysis suggested one HBV pre-miRNA candidate[21]; however, HBV is able to modulate different host miRNAs, particularly through the transcriptional transactivator HBx protein. Conversely, different cellular miRNAs can regulate HBV gene expression and replication by a direct binding to HBV transcripts or indirectly targeting host factors, which in turn modulate viral replication.

An aberrant expression of miRNAs has a causative effect on several pathological conditions, including cancer[5]. In this field, several studies indicate that miRNAs can act as either tumor suppressors by downregulating the expression of oncogenes, or tumor promoters (oncomirs) by limiting the expression of oncosuppressor proteins[61-63]. Cancer cell downregulation of Dicer, an enzyme playing a critical role in the biosynthesis of miRNAs, or mutations in its structure suppress miRNA biogenesis, leading to increased tumor progression[64-67]. This implies that the oncosuppressive effect of miRNAs generally overcomes their oncogenic potential. It is also known that cell differentiation is often accompanied by increased Dicer expression[68-70].

In 2011, Hou et al[71] performed an extensive study of the miRNomes of healthy human liver and HCC. In this paragraph, their work will be discussed in some detail since it provides a good example of the experimental procedures currently employed to study the miRNAs with oncogenic or oncosuppressive roles. The authors used a next-generation sequencing (NGS) technique to analyze human liver miRNome and found that 9 miRNAs accounted for about 90% of the liver miRNA content, with miR-122 being the most represented (52%). Other highly expressed miRNAs included miR-192 (16.9%), miR-199a/b-3p (4.9%), miR-101 (3.7%), let-7a (3.3%), miR-99a (2.2%), let-7c (2.1%), let-7b (1.7%), and let-7f (1.5%). MicroRNAs -199a-3p and -199b-3p have an identical nucleotide sequence but are transcribed from three genes, a-1, a-2, and b, with a2 being the most expressed in the liver. The authors then analyzed liver biopsies from patients with hepatocellular carcinoma by comparing tumor samples with adjacent non-cancer tissues. In HBV-related HCCs, a remarkable decrease in 199a-3p was observed. This result was then validated by qRT-PCR in a cohort of 40 HBV-related HCCs. This analysis showed that the miRNA was downregulated in 100% of the patients, with a mean decrease of 8.3-fold. Other miRNAs markedly downregulated were miR-99a and miR-125b, both decreased by about 7-fold in the majority of the patients. MicroRNA-122 and miR-125a were also downregulated (Table 2). On the other hand, miR-21 was upregulated by 4.8-fold in half of the tumor samples. In the same study, genomic analyses of DNA samples from liver biopsies indicated that miR-199a-3p downregulation was not due to gene deletion or promoter DNA methylation but to histone modification and subsequent repression of transcription. Biological assays were then performed showing that transfection of a synthetic miR-199a-3p mimic in cultured HCC cell lines repressed cell proliferation and induced apoptosis, thus suggesting a tumor suppressive role in vitro. Experiments in vivo were then performed by monitoring human HCC cell growth in nude mice. Intra-tumoral injection of cholesterol-conjugated miR-199a-3p or its over-expression with a recombinant adeno-associated virus system markedly decreased tumor growth, further supporting its tumor suppressive role. The mechanism of action of miR-199a-3p was then studied. A computational analysis with TargetScan was used to identify the human genes whose mRNA sequence may allow binding of miR-199a-3p, possibly leading to gene silencing. Most of them were associated with the mitogen-activated protein kinase (MAPK) pathway, thus providing a possible explanation for the anti-proliferative activity of the miRNA. Among them, PAK4 was downregulated by miR-199a-3p transfection in HCC cells, and this effect was found to be due to a direct interaction with the gene transcript using a luciferase-based reporter assay. Finally, measurement of the miR-199a-3p content in the liver biopsies from two other cohorts of 142 and 152 patients showed that a markedly decreased HCC level of the miRNA correlated with poor survival. It should be noted that a downregulation of miR-199a-3p in HCC had already been observed in 2006 by Murakami et al[72] employing much less powerful miRNA detection techniques based on microarrays and Northern blotting analyses. The same study had also shown a tumor downregulation of miR-125a. Since then, other studies have confirmed the tumor-suppressive role of miR-199a-3p in HCC and identified CD44, CD51, c-MET, mTOR, YAP1, and ZHX1 as other direct miRNA targets contributing to its anti-proliferative and pro-apoptotic effects[73-78] (Table 2). The ability of miR-199a-3p to downregulate several target proteins should not surprise given the pleiotropic effects of microRNAs that are able to interact with several mRNAs provided with the same or similar binding sites located in their 3’-UTR. Therefore, a single microRNA often silences several genes with related functions, thus showing a marked effect on the cell physiology.

Other studies conducted with similar experimental approaches, profiling microRNAs by qPCR-arrays on multi-well plates, have confirmed the HCC downregulation of miR-99a, -122, -125a, -125b, and the upregulation of miR-21, also identifying their molecular targets (Table 2). Other miRNAs consistently downregulated in HCC included the let-7 family of microRNAs and miR-29; other examples of upregulated miRNAs were miR-155 and -221 (Table 2). These data have raised substantial interest in microRNAs in the field of molecular and cellular oncology, leading to the publication of several interesting reviews[79-84].

As regards the reasons for deregulated miRNA expression in HCC, it should be noted that the viral HBx protein plays a primary role in HBV cancerogenesis[85]. It is a transcriptional trans-activator lacking a DNA-binding domain but able to interact with several transcription factors[86,87]. Therefore, it is not surprising that HBx can modify the hepatic miRNA expression[88]. It is noteworthy that HBx downregulates the expression of oncosuppressive miRNAs let-7 and miR-122, whereas it upregulates oncomirs -21 and -221. MicroRNA-125a is induced by HBx[32,89] but is downregulated by its carboxyl-terminal truncated variant that is frequently found in HBV-related HCC[90]. These data indicate that the tumorigenic effect of HBx is partially mediated by microRNAs. Besides HBx, dysregulation of microRNAs in cancer cells may be determined by other genetic or epigenetic factors[91]. Chromosomal abnormalities, deletions, and mutations can downregulate cellular miRNA expression[92,93], and several miRNA genes associated with CpG islands are also transcriptionally repressed by promoter DNA methylation[94].

Some literature data suggest that microRNA-interference might be useful in the treatment of HBV-related chronic hepatitis and HCC. The RNA-interference directed to inhibit HBV replication has been investigated in several animal models and, more recently, in a clinical study[124]. In a phase 2 clinical trial enrolling entecavir- naïve or -exposed HBsAg-positive patients with chronic hepatitis[125], ARC-520, a mixture of small-interfering RNAs (siRNAs) targeting all viral transcripts, induced HBsAg reduction up to 1.5 log10 in HBeAg-positive and up to 0.5 log10 in HBeAg-negative subjects with a single intravenous administration of up to 4 mg/kg. The reasons for the limited efficacy in HBeAg-negative patients have been more recently investigated in a preclinical study on chimpanzees[126]. The authors demonstrated a lack of target sites for the siRNAs in the HBV DNA integrated in the host genome, which represents the dominant source of viral transcripts in HBeAg-negative patients. These findings highlight a novel issue that should be addressed by future research on HBV treatment. Other two RNAi-based therapies (TKM-HBV and ALN-HBV) are currently investigated in chimpanzees and mice with promising preliminary results[127].

There is some evidence of the efficacy of miRNAs mimics or inhibitors both in preclinical studies and in a recent phase I clinical trial regarding the treatment of hepatocellular cancer. In an HCC murine model, the systemic administration of miR-26a using an adeno-associated virus resulted in an inhibition of cancer cell proliferation, induction of tumor-specific apoptosis, and protection from disease progression[127-129]; a cholesterol-modified isoform of anti-miR-221 can reduce tumor cell proliferation and increase the tumor doubling time and the survival in mice with hepatocellular cancer. An miR-375 mimic delivered in gold nanoparticles has shown therapeutic efficacy without significant toxicity in primary and xenograft tumor mouse models[130]. Finally, MRX34, a liposomal miR-34a mimic, showed anti-tumor activity in a phase I clinical trial enrolling patients with refractory advanced primary liver cancers or other solid neoplasms[131], but this trial has been stopped because of serious adverse events.