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MicroRNAs as regulators, biomarkers and therapeutic targets in liver diseases
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  1. Xiaolin Wang,
  2. Yong He,
  3. Bryan Mackowiak,
  4. Bin Gao
  1. Laboratory of Liver Diseases, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland, USA
  1. Correspondence to Dr Bin Gao, Laboratory of Liver Diseases, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20892-000, USA; bgao{at}mail.nih.gov

Abstract

MicroRNAs (miRNAs) are small, non-coding RNAs that post-transcriptionally regulate gene expression by binding to specific mRNA targets and promoting their degradation and/or translational inhibition. miRNAs regulate both physiological and pathological liver functions. Altered expression of miRNAs is associated with liver metabolism dysregulation, liver injury, liver fibrosis and tumour development, making miRNAs attractive therapeutic strategies for the diagnosis and treatment of liver diseases. Here, we review recent advances regarding the regulation and function of miRNAs in liver diseases with a major focus on miRNAs that are specifically expressed or enriched in hepatocytes (miR-122, miR-194/192), neutrophils (miR-223), hepatic stellate cells (miR-29), immune cells (miR-155) and in circulation (miR-21). The functions and target genes of these miRNAs are emphasised in alcohol-associated liver disease, non-alcoholic fatty liver disease, drug-induced liver injury, viral hepatitis and hepatocellular carcinoma, as well liver fibrosis and liver failure. We touch on the roles of miRNAs in intercellular communication between hepatocytes and other types of cells via extracellular vesicles in the pathogenesis of liver diseases. We provide perspective on the application of miRNAs as biomarkers for early diagnosis, prognosis and assessment of liver diseases and discuss the challenges in miRNA-based therapy for liver diseases. Further investigation of miRNAs in the liver will help us better understand the pathogeneses of liver diseases and may identify biomarkers and therapeutic targets for liver diseases in the future.

  • cancer
  • fibrosis
  • fatty liver
  • hepatitis

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Key messages

  • MicroRNAs (miRNAs) are small, non-coding RNAs that are critical regulators for liver physiological processes by negatively regulating target mRNA expression.

  • miR-122, miR-194/192, miR-223, miR-21, miR-155 and miR-29 are specifically expressed or enriched in several types of hepatic cells or in circulation, playing important roles in the pathogeneses of liver disease.

  • miRNAs can be packaged and released in extracellular vesicles, serving as messengers for intercellular communication in liver disease.

  • miRNAs appear to have great potential as novel, non-invasive biomarkers for early diagnosis, prognosis and assessment of liver disease.

  • miRNAs have potential to be used as therapeutic targets; however, miRNA-based therapy is challenging and is still in the early stages of development.

Introduction

MicroRNAs (miRNAs) are small (20–24 nucleotide) non-coding RNAs that regulate gene expression and cellular processes by targeting messenger RNA (mRNA) transcripts. MiRNA biogenesis takes place in multiple steps, including transcription, cleavage and final maturation.1 miRNAs are transcribed by RNA polymerase II, which generates the long primary transcripts characterised by hairpin structures (primary-miRNAs, pri-miRNAs) where miRNA sequences are embedded. Then, pri-miRNAs bind to the RNase III protein Drosha, which cuts the 3ʹ and 5ʹ strands of pri-miRNAs and generates precursor-miRNAs (pre-miRNAs). Next, pre-miRNAs are exported from the nucleus into the cytosol, where they are cleaved by Dicer to yield a double-stranded RNA duplex that is made up of the mature miRNA guide strand and the complementary passenger strand. The passenger strand is usually subject to degradation, but some studies suggest that it also represents a functional strand and plays biological roles. In the last step, the mature single stranded miRNA is incorporated into the RNA-induced silencing complex, leading to its binding to the complementary sequences in the 3’-untranslated regions (UTRs) of target mRNAs, and resulting in mRNA translational inhibition or degradation.

miRNA profiles are unique to different cell-types and generate different functions. Hepatic miRNA profiles play an important role in the pathogenesis of liver diseases by regulating liver metabolism, injury, fibrosis and tumour development.2 3 miRNAs can also be packaged and released in extracellular vesicles (EVs), serving as a messenger for communication between hepatic cells and other tissues. Importantly, some miRNAs are superior biomarkers compared with other known biomarkers, and some miRNA mimics/antimiRs have shown beneficial effects for the treatment of liver diseases in preclinical studies. In this review, we summarise recent studies on the roles of miRNAs in hepatic functions as well as in liver diseases and discuss the utility and promise of miRNAs as diagnostic, prognostic and predictive biomarkers and therapeutic targets for liver diseases, with a focus on those miRNAs which are specifically expressed or enriched in several types of liver cells, immune cells, or in circulation.

miRNAs in the Liver

The expression of miRNAs is under the tight and dynamic control of many regulatory factors and dysregulation of miRNAs is associated with numerous pathologies.4 Systematic analysis of the distribution of miRNAs has shown that many miRNAs are expressed in a tissue or cell-specific manner. For example, miR-122 is a highly abundant, liver-specific miRNA that accounts for approximately 70% and 52% of the whole hepatic miRNome in adult mouse and human, respectively, with negligible expression in other cell types; therefore, it is considered as a hepatocyte specific miRNA.5 Other miRNAs such as miR-194/192, are enriched in hepatocytes but are also expressed in other tissues. miR-29 is highly expressed in hepatic stellate cells (HSCs),6 whereas miR-21 is the strongest elevated miRNA in activated HSCs.7 miR-30a and miR-30c have been described as biliary-specific miRNAs in zebrafish.8 However, cholangiocyte-specific miRNAs in humans or mice have not been reported.

The broad functions of miRNAs in the liver have been investigated by using hepatocyte-specific Dicer knockout (KO) mice, in which miRNA processing is blocked in hepatocytes, resulting in loss of all miRNAs in hepatocytes. The pathophysiology of hepatocyte-specific Dicer1 KO mice bears some resemblance to that of miR-122 KO mice, such as liver steatosis, inflammation and hepatocellular carcinoma (HCC),9 10 which is probably because miR-122 constitutes 70% of the entire miRNA pool in the liver. Surprisingly, HSC-specific Dicer KO, which deletes all miRNAs in HSCs, does not significantly affect HSC activation and liver fibrosis,7 suggesting that either miRNAs do not play a role in HSC activation or miRNAs regulate both profibrotic and antifibrotic genes. Indeed, several miRNAs have been reported to regulate liver fibrosis (see discussion later). While the deletion of all miRNAs has provided insight into the overall functions of miRNAs in the liver, the downstream effects of specific miRNAs produced by the liver or present at high levels in circulation are much more nuanced. Therefore, the following sections will focus on several cell-specific miRNAs and how each one affects liver function and disease.

Hepatocyte-specific miR-122

miR-122 expression is transcriptionally controlled by liver-enriched transcription factors including CCAAT/enhancer-binding protein (C/EBP)α, hepatocyte nuclear factor (HNF)1α, HNF3β and HNF4α, which explains its specific expression in hepatocytes.11 miR-122 in the liver starts to express around embryonic day 12.5 (E12.5) in mice,8 and subsequently controls a group of genes (table 1) involved in hepatocyte proliferation, differentiation, maturation and polyploidy, thereby playing an important role in controlling liver development.11 12

Table 1

Regulation and partial target genes of microRNAs (miRNAs) in liver diseases

miR-122 is expressed at high levels in mature liver but is markedly downregulated in HCC. Such downregulation is closely correlated with hepatocarcinogenesis, poor prognosis and metastasis in HCC while restoration of miR-122 suppresses HCC growth and renders HCC sensitive to chemotherapeutic agents (see reviews2 13). The tumour-suppressive effect of miR-122 is believed to be mediated via the inhibition of a variety of tumourigenic genes (table 1) (see reviews2 13). Hepatic miR-122 levels are also decreased in patients with non-alcoholic steatohepatitis (NASH) compared with healthy controls,14 while serum miR-122 levels are elevated in patients with non-alcoholic fatty liver disease (NAFLD) and its serum concentration is even higher in patients with NASH.15 16 These opposing changes suggest the dynamic regulation of both the liver expression and release of miR-122 into circulation, demonstrating the complexity surrounding the functions of miR-122. The important anti-NASH functions of miR-122 are supported by the data from miR-122 KO mice that develop the full spectrum of NAFLD disorders (figure 1), which is partially due to the upregulation of miR-122 target genes (table 1).17 18 Consistently, miR-122 inhibition by antagomiR-122 worsens fatty liver in high-fat diet (HFD)-fed mice by reducing β-oxidation.19 In addition, hepatocytes can release hepatic miR-122 to attenuate triglyceride synthesis in skeletal muscle and adipose tissues, generating a crosstalk between the liver and remote tissues (figure 2).20 Similar to NAFLD, alcohol-associated liver disease (ALD) is also accompanied by decreased hepatic levels of miR-122, which is likely due to the elevation of transcription factor grainyhead like transcription factor 2 that attenuates miR-122 expression in the liver.21 Restoration of miR-122 in hepatocytes ameliorated ALD by targeting Hif1a, suggesting a protective function of miR-122 in ALD (figure 3).21

Figure 1

Roles of microRNAs (miRNAs) in the progression of non-alcoholic fatty liver disease (NAFLD). miR-21 accelerates NAFLD progression, whereas miR-122 and miR-223 ameliorate it. miR-21 and miR-122 control liver steatosis by regulating genes involved in triglyceride synthesis and secretion. miR-21 promotes liver inflammation and fibrosis via the inhibition of Ppara in inflammatory cells. Lipotoxic hepatocyte-derived exosomal miR-192 activates macrophages and, thus leads to non-alcoholic steatohepatitis (NASH) progression. miR-122 attenuates liver inflammation and fibrosis by reducing CD11bhiGr1+ inflammatory cell infiltration and by targeting Ccl2 and Klf6 in hepatocytes. miR-223 inhibits NASH progression by targeting Cxcl10 and Taz in hepatocytes and Nlrp3 in macrophages. miR-21 and miR-155 act as onco-miRs in NASH-related hepatocellular carcinoma (HCC), whereas miR-122 and miR-223 protect against HCC development.

Figure 2

Hepatocytes take up and release exosomal microRNAs (miRNAs). (I: take up) Extracellular vesicles (EVs) derived from adipose tissue macrophage containing miR-155 are taken up by hepatocytes, leading to increased insulin resistance by suppressing Pparg. miR-214 in hepatic stellate cells (HSCs) is shuttled by EVs to hepatocytes, resulting in inhibition on CCN2/Ccn2. Under high-fat diet (HFD), miR-223 is elevated in hepatocytes and attenuates non-alcoholic steatohepatitis (NASH) progression by targeting Cxcl10 and Taz. The elevated miR-223 in hepatocytes is likely due to its transfer from neutrophils by EVs. (II: Release) Hepatocytes can also release EVs that contain some miRNAs including miR-122, miR-192 and miR-1. Free fatty acids (FFAs) can stimulate hepatocytes to produce miR-122, which enters muscle and adipose tissues probably by EVs, and inhibits their triglyceride (TG) synthesis. Alcohol or TLR4/9 ligand can stimulate hepatocytes to release exosomal miR-122. Lipotoxic hepatocyte derived EVs can also transfer miR-192 into macrophages where it induces macrophage M1 polarisation. Besides, miR-1 is enriched in steatotic hepatocyte derived EVs, which promotes endothelial inflammation.

Figure 3

Roles of microRNAs (miRNAs) in the progression of alcohol-associated liver disease (ALD). miR-155 is increased in hepatocytes and macrophages in ALD, and facilitates alcohol-induced steatosis by inhibiting Ppara in hepatocytes and promotes M1 macrophage polarisation by targeting Cebpb. Upregulated miR-155 in macrophages contributes to alcohol-induced tumour necrosis factor α (TNF-α) production. Meanwhile, miR-155 inhibits autophagy and promotes miR-155-enriched extracellular vesicle (EV) release in both hepatocytes and macrophages. miR-122 expression is decreased in hepatocytes in ALD, there it protects against ethanol-induced damage by targeting Hif1a. miR-21 expression is upregulated in ALD and inhibits alcohol-induced apoptosis in both hepatocytes and hepatic stellate cells (HSCs) but promotes cytokine production in HSCs. miR-223 inhibits liver inflammation by suppressing the IL-6-p47phox-ROS pathway in neutrophils. Finally, increased circulating exosomal miR-155 may present as biomarkers for ALD. Il6, interleukin 6.

Another interesting function of miR-122 is to directly pair with distinct regions at the 5′-UTR of the HCV RNA genome and promote the replication of HCV RNA22 in contrast to the canonical miRNA mode of action to suppress their targets through binding the 3′ UTR of target mRNAs. Diverging from its role as a host factor for HCV replication, miR-122 suppresses HBV replication by downregulating a cyclin G1-p53 complex and blocking the specific binding of p53 to HBV enhancers.23 Conversely, HBV can modulate miR-122 levels that may facilitate its persistent infection and ultimately contribute to oncogenesis.24 A fusion of the HBV-encoded X gene and the human long interspersed nuclear elements can serve as a molecular sponge to absorb and deplete cellular miR-122, thereby promoting HCC by inducing hepatocyte β-catenin signalling activation, E-cadherin reduction and cell migration.24

Hepatocyte-enriched miR-194/192

The miR-194 and miR-192 (miR-194/192) gene cluster is controlled by the same promoter, and their expressions are controlled by liver-enriched transcription factors including HNF1α and HNF4α, thereby contributing to liver-enriched miR-194/192 expression.25 26 A variety of potential mRNAs targeted by miR-194/192 have been identified (table 1),25 26 and the proteins encoded by these genes are involved in glucose metabolism, cell adhesion and migration, and tumourigenesis25 26; however, the exact functions of miR-192 and 194 in the liver still remain obscure.

In patients with NAFLD, miR-192 in circulation is elevated in both simple steatosis and NASH compared with healthy controls while its liver expression is elevated in simple steatosis but in NASH.16 miR-192 is one of several obesity-associated exosomal miRNAs that play a role in the pathology of glucose intolerance and dyslipidaemia.27 A recent study indicates that miR-192 may regulate liver inflammation in NAFLD by promoting macrophage activation and proinflammatory functions via the inhibition of the rapamycin-insensitive companion of mammalian target of rapamycin (Rictor)/Akt/FoxO1 signalling pathway (figure 2).28 Hepatic cancer stem cells (CSC) are implicated in HCC initiation, metastasis and recurrence; interestingly, miR-192 is the most significantly downregulated miRNA in CSC+HCC cells.29 Such downregulation likely contributes to CSC+HCC activation because miR-192 suppresses the CSC-related features of HCC cells, which is partially mediated by targeting poly(A) binding protein cytoplasmic 4.29 MiR-194 is also involved in HCC development and metastasis by targeting several genes (table 1).25 26 30 Owing to the antitumourigenic effects of miR-192/194 and its low expression levels in CSC+ HCCs, delivering miR-192/194 to HCC may be a potent strategy for HCC therapy.

MiR-223: neutrophil/myeloid-specific miRNA

X-chromosome linked miR-223 is expressed at the highest level in neutrophils and is considered a neutrophil-specific miRNA, playing a critical role in attenuating neutrophil maturation and activation.31 MiR-223 is also expressed in macrophages at ~10-fold lower compared with that in neutrophils and regulates macrophage polarisation, but its expression in other organs is relatively low or undetectable.32 Since hepatic neutrophil infiltration is a hallmark of ALD and NAFLD, it is plausible to speculate that this neutrophil-specific miR-223 plays an important role in the pathogenesis of these two diseases. Indeed, serum and/or hepatic levels of miR-223 are elevated in patients or mouse models with ALD33 or NAFLD.16 34 Genetic deletion of the miR-223 gene exacerbated ALD33 and NAFLD34 in mice. Mechanistically, miR-223 inhibits Il6 expression and subsequently attenuates the IL-6-p47phox-ROS pathway in neutrophils, thereby protecting against ALD (figure 3).33 In NAFLD model, miR-223 directly inhibits several inflammatory genes and oncogenes including (C-X-C motif) chemokine 10 (Cxcl10) and transcriptional coactivator with PDZ-binding motif (Taz) in hepatocytes, thus ameliorating NAFLD (figure 1).34

Elevation of serum miR-223 was also reported in other types of liver diseases including acute liver failure (ALF) in patients,35 and mouse models of liver injury induced by CCl4 injection35 and acetaminophen (APAP).36 In APAP-induced liver injury, miR-223 seems to act as a negative feedback loop to control APAP-induced liver inflammation by targeting IκB kinase α expression in neutrophils.36 Interestingly, liver fibrosis development is associated with elevation of hepatic miR-223 whereas liver fibrosis resolution is associated with downregulation of miR-223.35 Serum miR-223 tends to increase in fibrotic mice, while it is decreased in patients with liver fibrosis/cirrhosis.35 These opposing changes may be due to the reduced number of circulating neutrophils in the late stage of cirrhosis. The role of miR-223 in regulating liver fibrosis was demonstrated by a recent study showing that miR-223 can be transmitted via the EVs from neutrophils to hepatic macrophages, where miR-223 inhibits proinflammatory sensor NOD-, LRR- and pyrin domain-containing protein 3 and converts proinflammatory macrophages into a restorative phenotype that mitigates fibrogenesis (figure 4).37

Figure 4

Roles of microRNAs (miRNAs) in the progression of liver fibrosis. miR-29, miR-214 and miR-223 play protective roles in liver fibrosis through different pathways. miR-214 is present in exosomes from hepatic stellate cells (HSCs) and can be taken up by recipient hepatocytes and HSCs, resulting in suppression of its target CCN2/Ccn2 and CCN2 downstream genes, including alpha smooth muscle and collagens. Downregulation of CCN2 leads to reduced expression of fibrogenic genes. miR-29 directly suppresses the expression of Col1a1,Col1a2, Col4a5 and Col5a3 in HSCs. miR-223 is transmitted from neutrophils to macrophages by extracellular vesicles (EVs) and converts proinflammatory macrophages to restorative macrophages by inhibiting proinflammatory sensor NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3). Then, restorative macrophages release interleukin (IL)-10, thus indirectly attenuates fibrogenesis by impairing HSC activation and collagen synthesis. miR-21 represses its target Smad7, which is an inhibitor of TGFβ signalling, eventually promoting HSC activation and liver fibrosis.

miR-223 is commonly repressed in human HCC,38 which is partly due to the epigenetic regulation by sulfatide.39 Microarray analyses of fatty livers have revealed that the top dysregulated genes are cancer-related genes in 3-month HFD-fed miR-223 KO mice, which likely contributes to the increased susceptibility of miR-223 KO mice to NASH-associated liver cancer.34 Several potential carcinogenesis targets that are inhibited by miR­223 have been identified (table 1), contributing to anti-HCC effect of miR-223.34 38 In addition, miR-223 also regulates cholesterol homeostasis by suppressing cholesterol biosynthesis, promoting cholesterol efflux, and reducing selective high-density lipoprotein-cholesterol uptake via the inhibition of a variety of genes in hepatocytes (table 1).40

MiR-21: an enriched miRNA in the circulation

miR-21 is one of the most abundant miRNAs detected in the circulation41 and is widely expressed in various types of human tissues, including the liver.42 Upregulation of serum and hepatic miR-21 has been reported in several types of liver diseases, and thus, has attracted intense interest in the field.43Hepatic miR-21 expression is significantly increased in mice and patients with NAFLD/NASH.14 44–46 Emerging evidence suggests that miR-21 promotes steatosis by inhibiting multiple transcription factors (table 1) that promote very low density lipoprotein (VLDL) secretion and inhibit lipogenesis,47 by activating lipogenic protein SREBP1c via the regulation of the HMG-box transcription factor 1-p53-SREBP1c pathway,44 and inhibiting phosphatase and tensin homolog (PTEN) protein, a regulator of the phosphatidylinositol-3-kinase pathway.46 miR-21 also suppresses hepatic peroxisome proliferator-activated receptor alpha (PPARα) expression, thereby promoting NASH progression (figure 1).45 Similar to NAFLD, hepatic miR-21 expression is also upregulated patients with ALD48 and in mouse ALD models,49 but the function of miR-21 in ALD seems complicated (figure 3). KO of miR-21 attenuated liver inflammation,48 while anti-miR-21 treatment promoted liver fibrosis in ethanol-fed mice.49 These opposite effects may be due to the differences between long-term miR-21 KO and short-term anti-miR-21 treatment.48 49

Upregulation of miR-21 is also observed in HSCs in both human cirrhotic liver and in mouse fibrotic livers induced by CCl4, bile duct ligation (BDL), or thioacetamide (TAA).7 50 Inhibition of miR-21 by antagomir-21 or antisense oligonucleotides ameliorated liver fibrosis in TAA- or CCl4-treated mice50 or in hepatocyte-specific Pten KO mice.51 Mechanistically, miR-21 represses the expression of its target mothers against decapentaplegic homolog 7 (SMAD7), which is an inhibitory regulator of SMAD signalling, resulting in enhanced SMAD signalling and liver fibrosis (figure 4).50 However, the profibrotic role of miR-21 was challenged by a recent study, which reported that genetic deletion of miR-21 neither attenuated liver fibrosis in mouse models nor prevented HSC activation in culture.7 Further studies are needed to clarify the effect of miR-21 in hepatic fibrogenesis under different conditions.

Patients with ALF are associated with elevated serum miR-21 levels, which are even higher in patients with spontaneous recovery compared with non-recovered patients.52 Hepatic miR-21 expression is also upregulated after partial hepatectomy in mice,53 and knockdown of miR-21 delays liver regeneration.54 MiR-21 promotes hepatocyte proliferation by inhibiting expression of its target genes such as B-cell translocation gene 2 (Btg2), which is a cell cycle inhibitor,53 and Ras homolog family member B (Rhob).54 Moreover, miR-21 promotes HCC proliferation and is considered an onco-miR in liver cancer.55 56 MiR-21 is one of the most overexpressed miRNAs in HCC57 and its levels in the serum are significantly elevated in patients with HCC.41 58 In the Pten-null mouse HCC model, anti-miR-21 treatment induced CD24+ liver progenitor cell apoptosis, leading to reduced liver tumour development.51 Mechanistically, miR-21 promotes HCC proliferation and invasion through the regulation of its multiple target genes (table 1). Additionally, miR-21 can also modulate the interactions of HCC cells with the stromal microenvironment, leading to enhanced HCC cell migration and invasion.59

MiR-155: highly expressed in immune cells

MiR-155 is highly expressed in immune cells, including macrophages, and plays a key role in controlling their functions. Although it is detected at low levels in the liver, miR-155 has been reported to regulate liver injury, inflammation, fibrosis, and carcinogenesis. Patients with autoimmune hepatitis show increased hepatic miR-155 expression but a decrease in miR-155 expression in peripheral mononuclear cells compared with healthy controls.60 Moreover, increased circulating or hepatic miR-155 was observed in mice with liver injury induced by APAP-, TLR9/TLR4 ligand, or concanavalin-A (Con A).60 61 Deletion of miR-155 worsens Con A-induced liver injury, accompanied by defective recruitment of regulatory T cells to the liver and decreased cytokine production by T-helper cells.60 The target of miR-155 in immune-mediated liver injury was identified as SH2 domain–containing inositol 5-phosphatase (SHIP1), a regulator of Treg cell development.60 In contrast to its protective role in Con A-induced hepatitis, miR-155 promotes alcohol-induced steatohepatitis and liver fibrosis in ALD by inhibiting hepatic Ppara in hepatocytes62 and by targeting negative regulators of the LPS/TLR4 pathway, such as SHIP1, IL-1 receptor–associated kinase-M, and suppressor of cytokine signalling 1 in macrophages.63 In addition, miR-155 promotes alcohol-induced autophagy impairment and exosome release in both hepatocytes and macrophages by targeting mTOR, Ras homolog enriched in brain, lysosomal-associated membrane protein (LAMP1), and LAMP2 (figure 3).64

MiR-155 is considered an oncogenic miRNA that links inflammation with tumourigenesis.65 66 Hepatic miR-155 is up-regulated in human HCC samples,66 and in the choline deficient, defined amino acid (CDAA) diet-induced HCC mouse model.65 Activation of NF-κB signalling seems to upregulate miR-155 expression in hepatocytes and liver cancer associated with CDAA diet feeding in mice65 or HCV infection in patients.66 This upregulated miR-155 promotes hepatocyte proliferation and tumourigenesis by repressing CEBPβ and adenomatous polyposis coli.65 66 Finally, miR-155 is highly expressed in epithelial cell adhesion molecule+ HCC cells, which promotes malignant features, such as colony formation, cell migration and invasion by suppressing its potential targets, including CEBPβ, SMAD1 and myosin light chain kinase.67

MiR-29: widely expressed with relatively high levels in HSCs

The miR-29 family consists of miR-29a, miR-29b and miR-29c, which are widely expressed.6 In the liver, miR-29b is expressed at the highest levels in HSCs, which is ~7000-fold,~250-fold and ~150 fold higher than that in sinusoidal endothelial cells, hepatocytes and Kupffer cells, respectively.6 Hepatic miR-29a/b/c expression is downregulated in patients with advanced fibrosis and in mice with fibrosis induced by CCl4 or BDL.6 Treatment with miR-29a dramatically improved liver fibrosis in CCl4-induced and TAA-induced fibrosis models, suggesting miR-29 inhibits liver fibrosis.68 Mechanistically, miR-29 downregulates the expression of extracellular matrix genes, such as Col1a1, Col4a5 and Col5a3 in HSCs (figure 4).6 Thus, miR-29 may represent a novel therapeutic target for liver fibrosis.

Although it is expressed at relatively low levels in hepatocytes,6 miR-29 is also involved in hepatic glucose and lipid metabolism. Overexpression of miR-29a-c in the liver alleviated hyperglycaemic and insulin resistance in both db/db diabetic and HFD-induced obese mice,69 whereas inhibition of miR-29a enhanced triglyceride accumulation in the liver due to the increased hepatic lipid uptake via the upregulation of lipoprotein lipase.10 Mechanistically, miR-29a-c dampens hepatic glucose production by targeting two key gluconeogenic factor genes, PPAR-γ co-activator 1α and glucose-6-phosphatase.69

MiR-29 also exerts a tumour-suppressive effect in HCC.70 71 miR-29a/b/c is downregulated in human HCC tissues, and low expression of miR-29a is associated with poor survival in patients with HCC.70 71 Transfection of miR-29b in HCC cells delayed tumour formation and reduced tumour size in nude mice.71 miR-29a expression is transcriptionally suppressed by alpha-fetoprotein (AFP); conversely, miR-29a attenuates the protumour effects of AFP by targeting DNA methyltransferase 3A, leading to decreased DNA methylation in HCC.70 In addition, miR-29a/b/c promotes apoptosis of HCC cells by suppressing two cell survival genes, MCL-1 and BCL2.71 These important functions of miR-29 uncover its potential as prognosis marker and therapeutic target for HCC.

MiRNAs in EVs and intercellular communication

Most studies on miRNAs focus on the intracellular functions and regulation of target genes, while the roles of miRNAs in intercellular communication in liver diseases have been just begun to be explored. MiRNAs can be secreted into the extracellular space within nanoparticles termed EVs, particularly exosomes. These miRNA-containing EVs can be taken up by neighbouring cells or enter the circulation, enabling miRNAs to modify targets in recipient cells both locally and distally.

In the liver, EVs can be released by various types of liver cells such as hepatocytes, HCC cells, HSCs and immune cells, leading to the transfer of EV cargo, including miRNAs, to target cells (figure 2). However, the studies regarding EV-mediated miRNA transfer and their functions in liver diseases are relatively limited. Hepatocytes can release EVs containing miRNAs into circulation, such as miR-122,20 61 miR-19228 and miR-155,61 64 and this EV release can be stimulated by alcohol, TLR9 ligand, or FFA. In ALD, alcohol-exposed monocytes can release EVs carrying miR-27a, which stimulates naïve monocytes to polarise into M2 macrophages.72 In NAFLD, lipotoxic hepatocyte-derived exosomal miR-192 and miR-1 promote macrophage activation and endothelial inflammation, respectively.28 73 In addition, miR-223 is highly elevated in hepatocytes in HFD-fed mice, and it is not clear whether such elevation of miR-223 in hepatocytes is transferred from neutrophils,34 but a recent study revealed that neutrophils can transmit miR-223 via the EVs to macrophages, thereby promoting liver fibrosis resolution.37 Besides, hepatocytes and HSCs can accept miR-214-containing exosomes released by HSCs, resulting in suppressed connective tissue growth factor-dependent fibrogenesis.74 75

Emerging evidence suggests that exosomal miRNAs released by HCC cells play an important role in regulating the tumour microenvironment (figure 5). For instance, HCC cell-secreted exosomal miR-103 can be delivered into endothelial cells, and then destroy endothelial junction integrity and facilitate tumour metastasis by targeting VE-cadherin, p120-catenin and zonula occludens 1.76 Under ER stress, HCC cells release miR-23a-3p-enriched exosomes which upregulate programmed death-ligand 1 expression in macrophages, attenuating antitumour immunity in HCC.77 HCC cells can also receive EVs from other cells like HSCs. HSC-derived EVs can deliver miR-335 to HCC cells, where it suppresses its target genes involved in HCC proliferation and invasion.78

Figure 5

Functions of microRNAs (miRNAs) in hepatocellular carcinoma (HCC) cells. miRNAs affect HCC development through different pathways. miR-122, miR-223, miR-29 and miR-192 function as tumour suppressers, while miR-21 and miR-155 are onco-miRs. miR-122 prevents the development of HCC by inhibiting multiple targets involved in Wnt/IGF/VEGF signalling pathways. miR-223 attenuates HCC by targeting cancer-associated genes including Taz. miR-29 sensitises HCC cells to apoptosis, while miR-192 suppresses cancer stem cell development. miR-21 and miR-155 act as oncomirs that regulate multiple pathways in HCC cells including phosphatidylinositol 3-kinase (PI3K) and Wnt signalling pathways. Besides, HSC-derived extracellular vesicles (EVs) deliver miR-335 to HCC cells, inhibiting cell proliferation and invasion. miR-103 is enriched in high metastatic HCC-derived EVs and can be delivered into endothelial cells where it abrogates endothelial junction integrity by targeting junction proteins and ultimately promotes tumour metastasis. Endoplasmic reticulum stress (ER stress) causes HCC cells to release exosomal miR-23a-3p, which upregulates programmed death-ligand 1 expression in macrophages and subsequently suppresses antitumour immunity.

Circulating exosomal miRNAs are considered new messengers for crosstalk between distant organs. Exosomes secreted by adipose tissue macrophages in obese mice transfer miR-155 to insulin target cells, including hepatocytes, leading to glucose intolerance and insulin resistance by targeting peroxisome proliferator-activated receptor gamma (Pparg) (figure 2).79 This is an exciting new area of research that can connect distant tissues and cell types to the development of liver disease via exosomal miRNAs.

MiRNAs as biomarkers: promise and potential

MiRNAs have the potential to become novel, non-invasive biomarkers because they are highly stable and easily detected in circulation. Indeed, numerous studies have demonstrated the superiority of several miRNAs as biomarkers for early diagnosis, prognosis and assessment of liver diseases compared with the traditional biomarkers. Many of them are registered in the clinicaltrials.gov database to evaluate their potential as diagnostic, prognostic and treatment-response biomarkers for liver diseases.

As a hepatocyte-specific miRNA, miR-122 has been extensively investigated as a serum biomarker for the severity of hepatocyte damage which causes release of miR-122 into the circulation. Serum miR-122 is elevated in patients with various types of liver diseases as discussed above. Interestingly, elevation of serum miR-122 was even observed prior to serum ALT elevation in drug-induced liver injury (DILI), suggesting that miR-122 is a more sensitive and early biomarker for DILI.80 However, a recent study challenged this notion because there are the large interindividual and intraindividual variability of circulating miR-122 among healthy volunteers.81 Further studies are needed to clarify whether miR-122 is a good marker for DILI. In addition, serum miR-122 is also elevated in cystic fibrosis with liver disease compared with both cystic fibrosis without liver disease and healthy controls, suggesting the potential use of miR-122 as a biomarker for liver damage in cystic fibrosis.82 Similar to miR-122, serum miR-192, another liver-enriched miRNA, is also a sensitive marker to predict early signs of DILI.83 In addition, the potential application of serum exosomal miR-192 as a biomarker for the diagnosis of NAFLD progression was suggested by a recent study.28 While the potential of miR-192 as a biomarker is promising, more rigorous studies are needed for its validation and to explore its potential as a biomarker for a broad spectrum of liver injury.

Circulating miR-155 was found in different fractions of serum depending on the type of liver injury.61 In alcohol or TLR4/TLR9-mediated liver injury, circulating miR­155 is predominantly associated with the EV­-enriched serum fraction, whereas in APAP-induced DILI, miR-155 is mainly present in the protein-rich fraction.61 Therefore, miR-155 in different fractions of serum may be a potential biomarker for identifying the aetiology of liver injury, and it will be interesting to further explore its contribution to communication between different types of cells in the liver and immune system.

Identification of miRNAs as biomarkers for HCC diagnosis and prognosis has been received great attention. For example, a miRNA classifier (Cmi) containing seven miRNAs has been built to identify small-size, early-stage and AFP-negative HCC.84 The miR-151a-5p/miR-192-5 p and miR-122-5 p/miR-151a-5p ratios were identified as reliable markers to predict postoperative liver dysfunction in patients with liver malignancies undergoing liver resection.85 Patients with HCC with low miR-26 expression in their tumours have poor prognoses but respond favourably to interferon alpha therapy.86 A recent study identified nine plasma miRNAs including miR-122 as biomarkers that can predict regorafenib response in patients with HCC.87 Another study showed that higher miR-541 expression in HCC tissues indicated a better response to sorafenib treatment.88 Given the important role of miR-21 in HCC progression, plasma miR-21 has been explored as a biochemical marker for HCC, and found to be superior to α-AFP in differentiating HCC from chronic hepatitis and healthy volunteers and the combination of miR-21 and AFP showed improved differentiating power.58 In addition, a plasma miRNA panel containing seven miRNAs including miR-21 was able to accurately diagnose HBV-related HCC.41 Taken together, targeting miR-21 might be a promising strategy for the diagnosis and treatment of HCC in the future.

One challenge for miRNA biomarker development is accounting for comorbidities that modify miRNA profiles in patients with liver disease. With the development of high throughput detection methods for miRNAs such as microarray and next-generation sequencing, it is feasible to get a global profile of circulating miRNA, which may identify a panel of miRNAs as biomarkers with improved diagnostic sensitivity and/or specificity compared with currently used biomarkers. For instance, a set of 11 miRNAs were identified that not only can discriminate APAP hepatotoxicity from another common hepatotoxic condition, ischaemic hepatitis, but also respond more rapidly than ALT during N-acetylcysteine treatment.89 miR-34a, miR-122 and miR-192 are potential biomarkers to distinguish NAFLD and NASH severity.15 Furthermore, the combination of miRNAs and traditional biomarkers may be useful in clinical practice for earlier diagnosis and expediting medical intervention decisions.

Recently, exosomal miRNAs, which are enriched and well-protected in EVs, have attracted great attention as a new class of biomarkers. Clinical studies have showed the promise of exosomal miRNAs, such as miR-122 and miR-21, for the early detection and prediction of HCC,90 and let-7s for detection of liver fibrosis in patients with chronic hepatitis C infection.91 Exosomal miR-122, miR-155 and miR-192 have also been suggested as biomarkers for liver injury in ALD and NAFLD (figures 2 and 3).28 61 64 Since exosomal miRNAs derived from HCC cells are involved in intercellular communication, tumour microenvironment and tumour metastasis, they may be good candidates for early-stage HCC and minimal metastatic HCC diagnosis (figure 5). Currently, advanced sequencing technologies, such as small RNA sequencing, are starting to be applied to EV-related miRNA profiling in the context of liver diseases. However, more studies are needed to further explore the significance of circulating exosomal miRNAs as biomarkers in liver diseases.

MiRNA-based therapies: conceptual validation and challenges

MiRNAs regulate diverse biological functions in the pathogeneses of liver diseases, which makes them become attractive therapeutic targets for various types of liver diseases and have been tested in preclinical models. For example, injection of miR-223 3p is an effective therapy in mouse models of both acute hepatitis and NASH.92 In addition, inhibition of miR-221-3 p ameliorated liver fibrosis in chronic CCl4 treated mice.93 MiRNAs also influence response to therapy of liver diseases. For instance, the ATP-binding cassette (ABC) transporters, ABCA1, ABCC1, ABCC5, ABCC10 and ABCE1, which are responsible for chemotherapy resistance in HCC, have been identified as the targets of multiple miRNAs. Thus, it is possible that miRNA-based therapy may help to overcome clinical drug resistance.94 The first miRNA-targeting drug that entered clinical trials was anti-miR-122 miravirsen for the treatment of HCV infection,22 but this anti-miR-122 therapy has received less attention after the discovery of effective direct-acting antiviral drugs. In addition, miR-34a is considered a tumour suppressor in various types of cancers including HCC95 and cholangiocarcinoma96 by downregulating multiple oncogenes; but this concept was challenged by other studies which showed the oncogenic role of miR-34a in HCC with β-catenin mutations97 and in cholangiocarcinoma by targeting tumour-suppressor gene PER1.98 The clinical trial of MRX34, a liposomal miR-34a mimic in solid tumour therapy including HCC was terminated due to immune-related serious adverse events.99 To our knowledge, many miRNAs listed in table 1 may have therapeutic potential for various types of liver diseases.

MiRNA-based therapeutics are still at an early stage of development and there were miRNA-based clinical trials for HCV and HCC therapy as mentioned above but no miRNA-based therapies have been proved for clinical treatment of liver diseases to date. One major obstacle is the delivery system for miRNA mimics (to overexpress the transcript) as well as anti-miRNAs (to suppress the transcript function). Various strategies have been proposed to increase the stability and efficiency of miRNAs for delivery. Notably, EVs are less toxic, highly stable and are preferentially taken up by the liver, making EVs as attractive delivery vehicles for miRNA-­based therapies in the context of liver diseases. Some studies have explored the application of EVs in miRNA-based therapy for liver diseases. For example, HSC-derived EVs have been used as vehicles to deliver miR-335 to HCC cells and eventually inhibit HCC growth.78 In a cholangiocarcinoma rat model, miR-195-loaded EVs shrank tumour size and improved survival of treated rats.100 However, the safety and efficacy of EV-mediated therapy requires further confirmation. Apart from being used as delivery vehicles, EVs may also present as the targets for therapy. Some studies have shown the roles of exosomal miRNAs in facilitating liver disease progression. For example, hepatocyte-derived exosomal miR-192 promotes liver inflammation in NASH28 and HCC cell-derived exosomal miR-103 promotes metastasis.76 Therefore, the interruption of specific EV-mediated miRNA trafficking may provide a novel therapeutic approach for liver diseases. Nevertheless, the factors that mediate EV packaging, release, and uptake remain obscure and further studies are needed.

Another challenge of miRNA-based therapy is specificity, given that one particular miRNA can simultaneously control multiple target genes and function differentially in various types of cells. Therefore, unexpected off-target effects of miRNA-based therapy may be generated and should be considered in therapeutic development. Chemical modifications of miRNA mimics/antimiRs and new technology (eg, TargomiRs or nanoparticles) may be helpful to improve the targeting specificity.

Conclusions and future perspective

Searches of PubMed with the keywords miRNAs and liver resulted in over 8000 publications, and therefore, our review primarily focuses on recent advances regarding the functions of miR-122, miR-194/192, miR-223, miR-21, miR-155 and miR-29 in liver biology and liver disease pathogeneses. While we were not able to discuss other miRNAs related to pathogeneses of liver diseases in depth due to space constraints, we would like to summarise some important findings and refer the readers to more targeted reviews on these topics. For instance, several recent studies suggest that miR-378 plays an important role in controlling steatosis, liver inflammation and fibrosis.101 102 Notably, most previous studies have described only a one-to-one, one-to-multiple, or multiple-to-one relationship between miRNA and its target genes in liver diseases as discussed above. Few studies investigated the effects of a combination of miRNAs on multiple targets in the liver. One example is that 13 miRNAs synergistically downregulated the ABC transporter genes in HCC,94 resulting in alterations of multidrug resistance phenotype and chemotherapeutic treatment failure.94 In ALD, miR-21 and let-7 have been suggested to regulate HSC survival, activation, and cytokine production through multiple targets.48 49 103 In addition, different miRNAs may have synergistic/additive functions or opposing functions. It has been reported that miR-21 promotes hepatocyte proliferation by repressing the cell cycle inhibitor Btg2 and this effect is further enhanced by a decreased expression of miR-378.53 Furthermore, several miRNAs, such as miR-21,50 miR-29,6 miR-21474 and miR-22337 have been reported to exert profibrotic or antifibrotic functions while deletion of all miRNAs in HSC-specific Dicer KO mice revealed no significant effects on HSC activation and liver fibrosis.7 This is probably because the profibrotic and antifibrotic miRNAs counteract each other, so the deletion of all miRNAs in HSCs did not show significant changes.7 Thus, future studies should focus on the combination effect of various miRNAs on multiple targets in the liver which may help fill in the whole picture for the roles of miRNAs in the pathogeneses of liver diseases.

Dysregulation of miRNAs have been reported in liver diseases which can be modified by miRNA mimics or anti-miRs. As such, miRNAs have potential to act as promising biomarkers and therapeutic strategies for the treatment of liver diseases. Importantly, some dysregulated miRNAs promote liver disease progression, but some act as defensive responses. For example, miR-122 has been shown to exert anti-NASH functions,17 18 and therefore, the upregulated expression of hepatic miR-122 during liver steatosis16 may be a defensive response to reduce steatosis, while the downregulated miR-122 in NASH14 may be a causal factor for NASH progression. MiR-223 is a well-documented anti-inflammatory miRNA, and thus, elevation of hepatic miR-223 likely compensatively protects against NASH34 and ALD33 while downregulation of miR-223 in HCC likely acts as a causal factor to accelerate HCC progression.38 Another important notion is that the same miRNA may play different or even opposite roles in different liver diseases. For instance, miR-192 promotes liver inflammation in NAFLD by targeting RICTOR in macrophages28 while it suppresses CSC-related malignant features of HCC by targeting poly(A) binding protein cytoplasmic 4 in HCC cells.29 These disease-specific functions of miR-192 may be due to targeting cell-specific genes in macrophages and HCCs, leading to different functions in each disease pathology. Therefore, future studies should focus on cell-specific and disease-specific functions of miRNAs in the liver, which will likely help us better identify therapeutic targets for the treatment of liver diseases and avoid off-target effects.

MiRNAs can also affect liver diseases by targeting non-liver tissues. One example is that miR-223 protects against obesity-associated adipose tissue inflammation and systemic insulin resistance by regulating macrophage polarisation, ameliorating NAFLD progression.32 MiRNA-containing exosomes isolated from obese mice induce glucose intolerance, insulin resistance and promote liver steatosis in lean mice by inhibiting Ppara expression in white adipose tissue.27 Moreover, adipose tissue macrophage derived exosomes transfer miR-155 into insulin target tissues, including the liver, adipose tissue and muscle, thereby promoting insulin resistance.79 Therefore, miRNAs in non-liver tissue, such as adipose tissue, may be potential targets for the treatment of NAFLD.

Many miRNAs have been or are currently being evaluated in preclinical studies or clinical trials for their potential as diagnostic, prognostic and treatment-response biomarkers for liver diseases. Large cohort clinical studies should be performed to further explore and validate the superiority and utility of miRNAs in the prediction, early detection and monitoring of disease progression and/or treatment response in liver diseases. Lastly, although many challenges remain, a deeper and comprehensive understanding of miRNAs and their activities in liver disease will help design feasible miRNA-based therapies. Advances in technologies for studying miRNAs, such as ‘–omics’, sequencing technologies and miRNA mimic104 or inhibition screening,105 may help identify novel targets for diagnostic and therapeutic development in liver disease.

Acknowledgments

The current review covers a very broad topic, and the authors apologise to the colleagues whose work was not mentioned or cited in this paper because of space constraints and limitation of a number of references that are allowed to be cited.

References

Footnotes

  • Contributors XW searched the data for the article and wrote the manuscript. YH and BM made substantial contributions to the discussion of content and edited the manuscript. BG supervised the whole project and edited the manuscript.

  • Funding This work described from BG lab in this review article was supported by the intramural program of NIAAA, NIH (BG). XW is a visiting scholar from Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.

  • Competing interests None declared.

  • Patient consent for publication Not required.

  • Provenance and peer review Commissioned; externally peer reviewed.