The Regulatory Role of MicroRNA in Hepatitis-B Virus-Associated Hepatocellular Carcinoma (HBV-HCC) Pathogenesis

The incidence and mortality of hepatitis B virus (HBV)-associated hepatocellular carcinoma (HBV-HCC) is an intractable public health problem in developing countries that is compounded by limited early detection and therapeutic options. Despite the early promise of utilizing the regulatory role of miRNA in liver cancer, this field remains largely in the work-in-progress phase. This exploratory review paper adopts a broad focus in order to collate evidence of the regulatory role of miRNA in each stage of the HBV-HCC continuum. This includes the regulatory role of miRNA in early HBV infection, chronic inflammation, fibrosis/cirrhosis, and the onset of HCC. The paper specifically investigates HBV dysregulated miRNA that influence the expression of the host/HBV genome in HBV-HCC pathogenesis and fully acknowledges that this does not cover the full spectrum of dysregulated miRNA. The sheer number of dysregulated miRNA in each phase support a hypothesis that future therapeutic interventions will need to consider incorporating multiple miRNA panels.

HBV and hepatitis C virus (HCV) infection is linked with more than 90% of all HCC incidence in developing countries compared to 40% in developed countries, with HBV being the major contributor in 66% of the cases [6]. According to the estimates of the global burden of disease study, deaths due to viral hepatitis increased by 63%, from 0.89 million to 1.45 million, between 1990 and 2013 [7,8]. HBV has been coined as "the second most dangerous carcinogen after tobacco" [8,9]. HBV, the prototype member of the family Hepadnaviridae, is hepatotropic and non-cytopathic. HBV enters the hepatocyte via the sodium-taurocholate cotransporting polypeptide (NTCP) receptor [10] and replicates by reverse transcription of a RNA intermediate, the pregenomic RNA (pgRNA). Although not required for replication of HBV, integration of HBV DNA can occur within the host genome and this has been implicated in hepatocarcinogenesis [11].

HBV-HCC Pathogenesis Continuum
HBV infection can result in a cascade of complex interactions between the host and the virus, which can lead to a spectrum of clinical manifestations including the asymptomatic carrier state, acute or fulminant hepatitis, and, ultimately, chronic hepatitis with progression to HCC with or without intermediary cirrhosis [12]. In 95% of infected adults and in only 10% of children, acute infection is self-limiting, with the virus persisting in 5% and 90% of the cases, respectively [12,13], with the risk of HCC increasing 100-fold if a patient is positive for both the hepatitis B surface antigen (HBsAg) and the hepatitis B e antigen (HBeAg). Even in the absence of HBsAg, there is a reduced and yet significant risk of HCC [14]. As viral load increases, the host immune response, triggered by HBV surface antigens (HBsAg), elicit T-cell responses accompanied by secondary inflammatory response [14], and an increase in free radicals, interferon, tumor necrosis factor (TNF), and hepatic injury. Moreover, if integration of HBV DNA into the host hepatocyte genome occurs, it results in the oncogenic disruption of cellular genes [15]. This disruption triggers apoptosis, regeneration, and early senescence [16], in addition to host cell deletions, cis/trans-activation, translocations, the production of fusion transcripts, aberrant epigenetic changes, and generalized genomic instability [17]. In parallel with these changes, the continuous destruction of well-differentiated hepatocytes and organized extracellular matrix results in their eventual replacement with undifferentiated liver stem cells and poorly organized fibrotic tissue [12].

Dysregulated MiRNA in the WNT/β-Catenin Pathway in HBV-HCC
The upregulation of the WNT/β-catenin pathway is a frequent event in early HCC [251]. It yields an aggressive phenotype that is implicated in the proliferation, migration, invasion, and survival of cancer cells [244]. Figure 2 illustrates some examples of HBx-dysregulated miRNAs and their target genes in this pathway. The HBx protein typically plays a role in enhancing WNT and β-catenin expression while suppressing the expression of E-Cadherin. WNT signaling, for instance, is not modulated as a result of HBx led downregulation of miR-122/148a/b [157,173,174] and Cadherin expression is suppressed because these miRNA fail to suppress cadherin suppressors like SNAIL 1 [174]. HBx-induced upregulation of mIR-21 also enhances WNT signaling because it reduces DCC6 suppression of WNT signaling [252] and contributes to the suppression of E-cadherin by suppressing PDCD4 expression [55]. HBx-upregulated miR-221/-222 also contributes to the suppression of cadherin by enhancing ZEB 1/2 via blocking one of its suppressors like TRPS-1 [54,157]. Cadherin expression is also suppressed as a result of HBx-induced downregulation of miR-200/-205/-101/34 that fails to regulate ZEB 1/2 [95,150,190]. HBx-induced downregulation of miR-122 reduces βcatenin-led transcription in the cytoplasm [156], while HBx upregulation of miR-155 blocks the APC tumor suppressor in the WNT pathway, as well as suppresses HBV replication by blocking enhancer 11 to potentially hide the virus from the host immune system [28,31].

Dysregulated MiRNAs in the JAK/STAT Pathway in HBV-HCC
The aberrant methylation in the CpG island of the SOCS-1 gene is a common feature in HBV-HCC and its silencing demonstrates its important tumor suppressor role in the JAK/STAT pathway [260]. Suppressor of cytokine signaling (SOCS-1) switches this signaling 'off' by means of its direct interaction with (JAK). The loss of function of SOCS-1 is a common feature in HCC and the HBxmediated upregulation of miR-155 is a contributing factor in HBV-HCC (see Figure 4) [261,262]. HBx upregulated miR-221/-203 contribute to a reduction in SOCS3 regulation of JAK/STAT signaling [263]. HBx also downregulates Let-7, which reduces its modulation of IL-6 induced activation of JAK/STAT signaling and mTOR mediated transcription of oncogenic proteins like C-MYC/MCL-1 [145,250,264]. However, HBx upregulation of miR-7 [241], has been demonstrated as a control by way of suppressing mTOR signaling in JAK/STAT pathway [250].

Discussion and Conclusions
This exploratory review deliberately adopts a broad focus to demonstrate the complex regulatory role of miRNAs in the HBV-HCC continuum. Multiple knowledge gaps, exposed in this paper, prompt further research, to clarify the complex regulatory roles of the same miRNA across the HBV-HCC continuum. In addition, numerous miRNA target the same genes and cascades of miRNA respond to injury and disease with differing levels of expression in tissue, serum and cell-lines. Circulating miRNA, for instance, may originate from different cells (e.g., blood-immune) rather than an HBV or cancer specific origin by way of secretion or cell death [265][266][267]. The broad focus of the paper is, therefore, specifically adopted to highlight this complexity rather than to explain it. The key focus of this exploratory review was, therefore, to illustrate the multiplicity of dysregulated miRNA in clearly defined stages of pathogenesis rather than to attempt to try and explain their role in mediating multiple targets or summarize their role across every stage of HCC pathogenesis (see miR-122/-21). A key limitation of the study, therefore, is that it conveys an overly simplistic role of specific miRNA in HBV-HCC pathogenesis.
The potential roles of HBx downregulated miR-124, illustrates this limitation (see Table 3). Its various roles could include interaction with lncRNA-MALAT1 to regulate HBx-induced cancer stem cell properties in HepG2 through PI3K/AKT signaling [268]. miR-124 also suppresses cell proliferation and tumor growth in HCC in vitro and in vivo models by direct targeting STAT3 and PIK3CA thereby repressing both JAK/STAT and PI3K/AKT pathways [164]. Other studies demonstrate that miR-124 can suppress cell proliferation in HCC by targeting PIK3CA [269], as well as extracellular matrix protein laminin gamma 1 (LAMC1), which is a key feature in HCC progression [270]. However, LAMC1 mRNA promote malignancy by competing with miR-124 by binding with CD151 [270] and the overexpression of LAMC1 promotes HCC progression pathway by interaction with integrin receptors on a cell surface to promote proliferation and metastasis in HCC [271]. This paper specifically adopts a broad, exploratory review of the regulatory role of HBV dysregulated miRNA in the HBV-HCC continuum and miRNA dysregulation may provide a means of screening HBV carriers and developing HCC biomarkers and alternate therapeutic options.

The Deregulation of MiRNA in HBV-HCC Continuum
MicroRNA (miRNA) act as post-transcriptional gene silencers that collectively reduce or inhibit their target mRNA expression, thereby playing a homeostatic role that fine tunes the translation of proteins. The ancillary role of miRNA, as mild suppressors, has been explained by the inherently stochastic nature of gene transcription and environmental fluctuations [20]. In the case of transient reaction to environmental conditions, miRNA quantity become temporarily dysregulated until homeostasis is restored [21]. In the case of the continuum from asymptomatic HBV infection leading to HCC, multiple miRNA become increasingly permanently dysregulated (Tables 1-4) as a result of HBV infection, epigenetic changes [22], inflammation [23], fibrosis [24], cirrhosis [16], and the onset of HCC. The increasing level of dysregulation in the HBV-HCC continuum is illustrated in a study that showed 37 miRNA deregulated in otherwise healthy controls (HC), 77 in asymptomatic HBV carriers (ASC), 101 in chronic hepatitis B infection (CHB) ( Table 1), and 135 in acute liver failure (ALF) [25]. In general, dysregulated miRNA in the HBV-HCC continuum fall into two major categories, namely, those that target HBV transcripts and those that target the host cell genome expression.

MiRNA Dysregulation in the HBV-HCC Continuum
This section illustrates the regulatory role of HBV dysregulated miRNA in the different stages of the HBV-HCC continuum (Tables 1-4). A brief description of each stage is provided before describing the regulatory role of miRNA.

Early/Chronic HBV Infection
In this stage the pivotal role of HBV, in cases where viral clearance does not occur, appears mainly to be due to the continuous destruction of hepatocytes by T-cells, which attempt to eliminate the infection in conditions of chronic inflammation and increasing oxidative stress [18]. If viral clearance does not occur, patients that progress to CHB will elicit a weak peripheral cytotoxic T-lymphocyte (CTL) response. Typically, an activated humoral response in CHB involves the production of interleukin-4 (IL-4), IL-5, and IL-10 secreted by type-2 helper T lymphocytes that promote antibody production rather than viral clearance. It is also hypothesized that low levels of intrahepatic HBV-specific CTLs are the cause of hepatic inflammation flares that occur in CHB patients without viral clearance [26]. Host cell miRNA modulate HBV expression by either targeting own cell transcription factors required for HBV genome transcription or by directly binding to HBV transcripts [13]. Many host cell miRNAs are also modulated by HBV proteins in order to ensure the persistence and replication of the virus.
MiRNA Regulating HBV Genome Expression HBV genome replication and survival is self-managed as a result of modulating (up/down) both host cell miRNA expression, as well as coding for its own miRNA [27]. The hepatitis B virus x protein (HBx), for example, blocks p53 stimulated miR-34 expression in hepatocytes leading to the upregulation of macrophage-derived chemokine (CCL22) that stimulates regulatory T-cells (Tregs) that, in turn, block effector T-cells allowing HBV expression to increase [28,29] HBV genome transcription or by directly binding to HBV transcripts [13] (see Scheme 1, pathway 1). Some miRNA, like miR-372, can influence HBV expression positively and negatively depending on their target pathways. HBx upregulated miR-372 [39,40] targets the c-AMP-response element binding protein (CREB) from binding to HBV enhancer 1/core promoter (Enh 1/ENI-Cp) to reduce HBV transcripts. It also targets the nuclear factor 1 B-type protein (NF1B) that fails to modulate HBV Enh 1 (ENI-Cp), thus promoting HBV expression [28,39] (see Schematic 1, pathway 7-8).

HBV-Induced Inflammation Pathways
In this stage, chronic inflammation, accompanied by T-cell response, causes tissue damage where the continuous replacement of hepatocytes is initially achieved by differentiated hepatocytes, which eventually become depleted and are replaced by the recruitment of non-epithelial cells in the liver [18]. While their activation and proliferation initially serves to support the immune response and the regeneration of the tissue, during chronic liver damage and with increasing inflammation, their expansion predominates and they gradually replace epithelial structures in the organ. Inflammation/injury induces pro-inflammatory cytokines like tumor-necrosis factor-alpha (TNFα) and interleukin 6 (IL-6) by activating Kupffer cells and liver-derived macrophages. The activation of pro-inflammatory cytokines like TNFα and Il-6 can induce both pro-apoptotic and anti-apoptotic effects in injured liver tissue [41,42]. A pro-apoptotic response would, typically, be triggered by inflammation/necrosis that triggers Toll-like receptor (TLR) signaling to induce Kupffer cells to synthesize pro-inflammatory cytokines like TNFα, IL-6, and interferon alpha (IFNα), that recruit natural killer cells (NKs) to promote TNF-related apoptosis-inducing ligand (TRAIL) [43][44][45] (see Schematic 2, pathway 9).
Alternatively, an anti-apoptotic response results from the activation of the canonical nuclear factor-κB (NFκB) inflammation pathway [46]. Injury triggers HSC/Kupffer cell stimulation of chemokines like TNFα and IL-6 that induce TLR signaling in hepatocytes. TLR signaling then activates the inhibitor of kappa kinase (IKK) complex to phosphorylate the IαKα/p65:p50 bound  Upregulation of miR-155 by HBx results in the downregulation of suppressor of cytokine signaling-1 (SOCSI) expression contributing to increased JAK/STAT signaling, leading to the suppression of HBV infection by augmenting interferon (IFN) signaling [30] (see Scheme 1, pathway 2). Alternatively, HBx upregulated miR-155 also subdues HBV replication by blocking the CCAAT/enhancer-binding protein (C/EBP) that binds and activates the HBV Enhancer (Enh) 11/core promoter [31] (see Scheme 1, pathway 3).
The liver miR-122 is widely reported as upregulated in serum following HBV infection (references in Table 1) and is regarded as a key suppressor of HBV expression [32][33][34]. However, it has been reported that HBx downregulates miR-122 and facilitates increases in HBV transcripts by failing to block Cyclin G1, which then blocks p53 from binding to HBV Enh1/core promoter thus facilitating an increase in HBV expression [35] (see Scheme 1,pathway 4). Other studies have demonstrated that miR-122 blocks HBV pgRNA that encodes the hepatitis B core antigen (HBcAg) and viral polymerase, and that HBV downregulated miR-122 blocks HBV replication by failing to modulate heme-oxygenase (HO-1), which blocks HBV covalently closed circular DNA (cccDNA) [36] (see Scheme 1, pathway 5).

HBV-Induced Inflammation Pathways
In this stage, chronic inflammation, accompanied by T-cell response, causes tissue damage where the continuous replacement of hepatocytes is initially achieved by differentiated hepatocytes, which eventually become depleted and are replaced by the recruitment of non-epithelial cells in the liver [18]. While their activation and proliferation initially serves to support the immune response and the regeneration of the tissue, during chronic liver damage and with increasing inflammation, their expansion predominates and they gradually replace epithelial structures in the organ. Inflammation/injury induces pro-inflammatory cytokines like tumor-necrosis factor-alpha (TNFα) and interleukin 6 (IL-6) by activating Kupffer cells and liver-derived macrophages. The activation of pro-inflammatory cytokines like TNFα and Il-6 can induce both pro-apoptotic and anti-apoptotic effects in injured liver tissue [41,42]. A pro-apoptotic response would, typically, be triggered by inflammation/necrosis that triggers Toll-like receptor (TLR) signaling to induce Kupffer cells to synthesize pro-inflammatory cytokines like TNFα, IL-6, and interferon alpha (IFNα), that recruit natural killer cells (NKs) to promote TNF-related apoptosis-inducing ligand (TRAIL) [43][44][45] (see Scheme 2, pathway 9).

MiRNA Regulating HBV-Induced Inflammation Pathways
At the commencement of the inflammation-fibrosis continuum in persistent HBV infection, a wide range of host miRNA are deregulated (see Table 1). Many of these miRNA subtly manipulate the host immune response to ensure both the survival and proliferation of the virus [28]. Complex interactions between HBV, the immune system, and miRNA trigger TLR pathways that interact with cytokines, interferons, and TNFs to modulate innate immune responses [51].
Examples of miRNA-influenced inflammation include miR-145/-148a/-200b/-200c/-455 and the Let-7 family [51]. Typically the Let-7 family, for instance, is downregulated in CHB by the HBx protein (or alternatively "mopped" up by HBV mRNA), thus preventing it from suppressing TLR4 signaling along the interleukin-1 receptor associated kinase (IRAK1)/TRAF6/NFKB pathway, to induce nuclear accumulation of p65:p50 [51] that promotes STAT3, resulting in cell proliferation and survival [48,52] (see Schematic 3, pathway 12). Alternatively, HBx downregulates Let-7 that can also activate STAT3 signaling by failing to modulate the expression of inflammatory cytokines, to influence miRNA-mediated suppression of key tumor suppressors [53] (see Schematic 3, pathway 13). HBx-induced upregulation of miR-21 by Il-6 mediated STAT3 signaling typically blocks tumor suppressors like PTEN and PDCD4 at an early stage in the inflammation-fibrosis axis and continues in the HBV-HCC stage [ Alternatively, an anti-apoptotic response results from the activation of the canonical nuclear factor-κB (NFκB) inflammation pathway [46]. Injury triggers HSC/Kupffer cell stimulation of chemokines like TNFα and IL-6 that induce TLR signaling in hepatocytes. TLR signaling then activates the inhibitor of kappa kinase (IKK) complex to phosphorylate the IαKα/p65:p50 bound complex in the cytoplasm that leads to the accumulation of transcription factors p65:p50 in the nucleus. This accumulation, in turn, promotes an anti-apoptotic response [41,47,48] (see Scheme 2 pathway 10).
In CHB, a deficiency in interferon can block the immune response by subduing TLR expression, and the HBx protein can stimulate TGF-β1 to induce T-cell regulators to subdue the immune response [49,50]. Persistent activation of the NFκB pathway in HBV infection is orchestrated by way of TLRs that bind to HBV proteins (e.g., HBx) and activate the NFκB pathway. Typically HBV proteins bind to TLR-4/7/9 and recruit the myeloid differentiation primary response adaptor protein (MYD88) to activate tumor necrosis factor receptor (TNF-R)-associated factor 6 (TRAF6) via transforming growth factor beta-activated kinase 1 (TAK1), to activate the inhibitor of nuclear factor kappa-B kinase (IKK complex), to induce nuclear translocation of p65:p50 accumulation in the nucleus, and this results in an anti-apoptotic effect [41,49,51] (see Scheme 2 pathway 11).

MiRNA Regulating HBV-Induced Inflammation Pathways
At the commencement of the inflammation-fibrosis continuum in persistent HBV infection, a wide range of host miRNA are deregulated (see Table 1). Many of these miRNA subtly manipulate the host immune response to ensure both the survival and proliferation of the virus [28]. Complex interactions between HBV, the immune system, and miRNA trigger TLR pathways that interact with cytokines, interferons, and TNFs to modulate innate immune responses [51].

MiRNA Regulating HBV-Induced Inflammation Pathways
At the commencement of the inflammation-fibrosis continuum in persistent HBV infection, a wide range of host miRNA are deregulated (see Table 1). Many of these miRNA subtly manipulate the host immune response to ensure both the survival and proliferation of the virus [28]. Complex interactions between HBV, the immune system, and miRNA trigger TLR pathways that interact with cytokines, interferons, and TNFs to modulate innate immune responses [51].

MiRNA Regulating HBV-Induced Fibrosis/Cirrhosis
In this stage, chronic tissue damage and inflammation are accompanied by the activation of non-epithelial cells in the liver, which proliferate. Initially this activation supports the immune response to regenerate damaged tissue, as these non-epithelial cells gradually replace epithelial structures in the liver. This process; however, eventually manifests as fibrosis/cirrhosis where well-organized parenchymal tissue is increasingly replaced by disorganized and dysfunctional fibrotic tissue [18]. In the inflammation-fibrosis axis, fibrogenesis is orchestrated by a complex network of common cytokine-mediated signaling pathways that regulate the activation of hepatic stellate cells (HSCs) and downstream extracellular matrix (ECM) proteins. These cytokines include TGF-β, platelet-derived growth factor (PDGF), TNF-α, interferons (IFNα/β), and interleukins (IL-1/6/17) [41,42]. Of all the different cytokine mediated pathways, upregulated TGF-B signaling is thought to be the principal fibrogenic pathway that activates HSCs to synthesize fibrogenic materials like collagen and alpha smooth muscle actin (α-SMA) [106][107][108][109]. In HBV-infected patients, the HBx protein has been identified as an activator of cytokine signaling [110], notwithstanding the synergistic effect induced by chronic inflammation, oxidative stress, and hepatocyte loss that triggers the activation of quiescent HSCs into myofibroblasts, which are the main source of ECM production (e.g., collagen 1/111, α-SMA) in the liver [106,107].
A hypothesized HBx-directed fibrogenic miRNA pathway involves miR-185 as follows: HBx promotes TGFβ signaling, that blocks miR-185 that, in turn, fails to modulate Rapamycin-insensitive companion of mammalian target of rapamycin (RICTOR/RHEB), resulting in the activation of HSCs, ECM proteins, and the development of fibrotic tissue [111,112] (see Scheme 4, pathway 15). Another important miRNA family that modulates fibrogenesis is the miR-200 cluster. In HBVrelated fibrogenesis, the HBx protein suppresses p53-led transcription of miR-192/-200, which then fails to modulate ZEB1/2, blocking the transcription of E-cadherin in the WNT/β-catenin pathway. A loss of E-cadherin is a key feature of fibrosis/EMT and occurs early in the HBV-HCC continuum [18,28,115] (see Schematic 4, pathway 17).
HBx-induced TGF signaling in the early phase of liver disease also promotes the increase in ECM, as a result of TGF-β1 upregulation of miR-33a to block inhibitory SMAD7, thus promoting Rsmad-induced TGF-β1, HSCs, ECM, and fibrosis [116] (see Schematic 4, pathway 18).
Another important miRNA family that modulates fibrogenesis is the miR-200 cluster. In HBV-related fibrogenesis, the HBx protein suppresses p53-led transcription of miR-192/-200, which then fails to modulate ZEB1/2, blocking the transcription of E-cadherin in the WNT/β-catenin pathway. A loss of E-cadherin is a key feature of fibrosis/EMT and occurs early in the HBV-HCC continuum [18,28,115] (see Scheme 4, pathway 17).   HBx-induced TGF signaling in the early phase of liver disease also promotes the increase in ECM, as a result of TGF-β1 upregulation of miR-33a to block inhibitory SMAD7, thus promoting Rsmad-induced TGF-β1, HSCs, ECM, and fibrosis [116] (see Scheme 4,pathway 18).

HBV Deregulated MiRNA in HBV-HCC
In this stage, persistent HBV infection, chronic inflammation, oxidative stress, and cirrhosis can dysregulate a wide range of host gene expression by initiating deletions, amplifications, mutations, epigenetic changes, or by targeting miRNA loci or their transcription factors [139]. In this disrupted tissue, HBV-HCC may develop from cells that are able to survive in cirrhotic livers and that are more resistant to adverse conditions, viral infection, and apoptosis [18]. In this regard, the HBx protein is thought to play a key role in the development of HCC because it can inhibit the TP53 function in early carcinogenesis, and may contribute to the accumulation of aberrant replacement cells by downregulating apoptosis [18,140]. The HBx transactivating protein promotes cell cycle progression, inactivates negative growth regulators, tumor suppressors, and senescence-related factors [141,142].

HBx-Upregulated MiRNA in HBV-HCC
In general, many HBx-upregulated miRNAs typically block tumor suppressor networks (see Table 4). For example, the phosphatase and tensin homologue (PTEN) tumor suppressor is downregulated by HBx upregulated miR-21, miR-29a, miR-221, and miR-222. Upregulated miR-21 is a key liver miRNA that has been consistently cited as a silencer of tumor suppressors like PTEN and PDCD4 (see references in Table 4).

HBV-HBx-Dysregulated MiRNA in the Principal HBV-HCC Cancer Pathways
Understanding of the molecular etiology of HCC remains incomplete [244]. Evidence to date shows that HCC generally involves a range of disruptions of the PI3K/MAPK pathways and the p53 network that includes cell cycle controls (RB1), as well as increased WNT signaling and the inactivation of key tumor suppressors (SOCSI) in the JAK/STAT network [18,19]. Alterations of RB1, p53, and WNT pathways in HCC are frequently associated with HCV, HBV, and alcoholic liver cirrhosis [245]. This section focuses on examples of HBx-induced dysregulation of miRNA in HBV-HCC pathways.

Dysregulated MiRNA in the WNT/β-Catenin Pathway in HBV-HCC
The upregulation of the WNT/β-catenin pathway is a frequent event in early HCC [251]. It yields an aggressive phenotype that is implicated in the proliferation, migration, invasion, and survival of cancer cells [244]. Figure 2 illustrates some examples of HBx-dysregulated miRNAs and their target genes in this pathway. The HBx protein typically plays a role in enhancing WNT and β-catenin expression while suppressing the expression of E-Cadherin. WNT signaling, for instance, is not modulated as a result of HBx led downregulation of miR-122/148a/b [157,173,174] and Cadherin expression is suppressed because these miRNA fail to suppress cadherin suppressors like SNAIL 1 [174]. HBx-induced upregulation of mIR-21 also enhances WNT signaling because it reduces DCC6 suppression of WNT signaling [252] and contributes to the suppression of E-cadherin by suppressing PDCD4 expression [55]. HBx-upregulated miR-221/-222 also contributes to the suppression of cadherin by enhancing ZEB 1/2 via blocking one of its suppressors like TRPS-1 [54,157]. Cadherin expression is also suppressed as a result of HBx-induced downregulation of miR-200/-205/-101/34 that fails to regulate ZEB 1/2 [95,150,190]. HBx-induced downregulation of miR-122 reduces β-catenin-led transcription in the cytoplasm [156], while HBx upregulation of miR-155 blocks the APC tumor suppressor in the WNT pathway, as well as suppresses HBV replication by blocking enhancer 11 to potentially hide the virus from the host immune system [28,31].

Dysregulated MiRNAs in the JAK/STAT Pathway in HBV-HCC
The aberrant methylation in the CpG island of the SOCS-1 gene is a common feature in HBV-HCC and its silencing demonstrates its important tumor suppressor role in the JAK/STAT pathway [260]. Suppressor of cytokine signaling (SOCS-1) switches this signaling 'off' by means of its direct interaction with (JAK). The loss of function of SOCS-1 is a common feature in HCC and the HBx-mediated upregulation of miR-155 is a contributing factor in HBV-HCC (see Figure 4) [261,262]. HBx upregulated miR-221/-203 contribute to a reduction in SOCS3 regulation of JAK/STAT signaling [263]. HBx also downregulates Let-7, which reduces its modulation of IL-6 induced activation of JAK/STAT signaling and mTOR mediated transcription of oncogenic proteins like C-MYC/MCL-1 [145,250,264]. However, HBx upregulation of miR-7 [241], has been demonstrated as a control by way of suppressing mTOR signaling in JAK/STAT pathway [250].

Discussion and Conclusions
This exploratory review deliberately adopts a broad focus to demonstrate the complex regulatory role of miRNAs in the HBV-HCC continuum. Multiple knowledge gaps, exposed in this paper, prompt further research, to clarify the complex regulatory roles of the same miRNA across the HBV-HCC continuum. In addition, numerous miRNA target the same genes and cascades of miRNA respond to injury and disease with differing levels of expression in tissue, serum and cell-lines. Circulating miRNA, for instance, may originate from different cells (e.g., blood-immune) rather than an HBV or cancer specific origin by way of secretion or cell death [265][266][267]. The broad focus of the paper is, therefore, specifically adopted to highlight this complexity rather than to explain it. The key focus of this exploratory review was, therefore, to illustrate the multiplicity of dysregulated miRNA in clearly defined stages of pathogenesis rather than to attempt to try and explain their role in mediating multiple targets or summarize their role across every stage of HCC pathogenesis (see miR-122/-21).
A key limitation of the study, therefore, is that it conveys an overly simplistic role of specific miRNA in HBV-HCC pathogenesis.
The potential roles of HBx downregulated miR-124, illustrates this limitation (see Table 3). Its various roles could include interaction with lncRNA-MALAT1 to regulate HBx-induced cancer stem cell properties in HepG2 through PI3K/AKT signaling [268]. miR-124 also suppresses cell proliferation and tumor growth in HCC in vitro and in vivo models by direct targeting STAT3 and PIK3CA thereby repressing both JAK/STAT and PI3K/AKT pathways [164]. Other studies demonstrate that miR-124 can suppress cell proliferation in HCC by targeting PIK3CA [269], as well as extracellular matrix protein laminin gamma 1 (LAMC1), which is a key feature in HCC progression [270]. However, LAMC1 mRNA promote malignancy by competing with miR-124 by binding with CD151 [270] and the overexpression of LAMC1 promotes HCC progression pathway by interaction with integrin receptors on a cell surface to promote proliferation and metastasis in HCC [271]. Elevated LAMC1 mRNA also acts as a sponge for miR-124 thus preventing its binding to another oncogenic membrane protein (CD151) that contributes to its elevated expression in HCC [270].
Similarly, the figures illustrating isolated roles of HBx-dysregulated miRNA in the main HBV-HCC pathways are clearly simplistic and only serve to demonstrate the interaction of the HBV dysregulated miRNA and their respective targets, thus ignoring the fact that they only represent a fraction of miRNA targeting these cancer pathways. The present review illustrates the complex range of miRNA regulatory roles in HBV-HCC pathogenesis. It also demonstrates how miRNA manipulation of HBV expression can be used as a tool to dissect HCC molecular pathways, and be harnessed to improve diagnosis, prognosis, anti-viral, and anti-tumor therapeutic modalities.

Conflicts of Interest:
The authors declare no conflicts of interest.