MicroRNAs as controlled systems and controllers in non-alcoholic fatty liver disease

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a multi-faceted condition including simple steatosis alone or associated with inflammation and ballooning (non-alcoholic steatohepatitis) and eventually fibrosis. The NAFLD incidence has increased over the last twenty years becoming the most frequent chronic liver disease in industrialized countries. Obesity, visceral adiposity, insulin resistance, and many other disorders that characterize metabolic syndrome are the major predisposing risk factors for NAFLD. Furthermore, different factors, including genetic background, epigenetic mechanisms and environmental factors, such as diet and physical exercise, contribute to NAFLD development and progression. Several lines of evidence demonstrate that specific microRNAs expression profiles are strongly associated with several pathological conditions including NAFLD. In NAFLD, microRNA deregulation in response to intrinsic genetic or epigenetic factors or environmental factors contributes to metabolic dysfunction. In this review we focused on microRNAs role both as controlled and controllers molecules in NAFLD development and/or their eventual value as non-invasive biomarkers of disease.

Key Words: Liver steatosis, Fibrosis, Non-alcoholic fatty liver disease, Non-alcoholic steatohepatitis, MicroRNAs

Core tip: Genetic background, epigenetic mechanisms and environmental factors contribute to Non-alcoholic fatty liver disease (NAFLD) development and progression. Among epigenetic mechanisms microRNAs are the most studied in NAFLD, contributing to metabolic dysfunction and liver damage. As during the last five years several authors investigated the role of microRNAs as potential therapeutic targets and/or non-invasive biomarkers we believe that this mini-review could be a good tool for researchers that critically approach this topic.

INTRODUCTION

Non-alcoholic fatty liver disease (NAFLD) is a multi-factorial condition encompassing a wide spectrum of liver damage, ranging from simple steatosis non-alcoholic fatty liver (NAFL) to steatohepatitis non-alcoholic steatohepatitis (NASH). While NAFL is considered a relatively benign condition with little risk of progression, NASH, described as the inflammatory phase of steatosis, is sometimes associated with fibrosis and may potentially progress to irreversible cirrhosis and in some cases to hepatocellular carcinoma (HCC)[1]. The interesting particularity of this pathology is that, even though the histo-pathological pattern of NAFLD/NASH resembles that observed during alcohol-induced liver damage, the disease occurs in subjects with no significant or even absent alcohol consumption. Furthermore, it also occurs in the absence of other underlying aetiology such as virus infection, autoimmune hepatitis, drug- induced and inherited metabolic disorders[2].

Alarming epidemiological data shows that the incidence of NAFLD has increased over the last twenty years becoming the most common cause of chronic liver disease worldwide[3-5]. Noteworthy, several studies now reveal the NAFLD prevalence in Western nations fluctuates between 20% and 30%, going up to 90% in obese individuals, while NASH affects approximately 2%-3% of the general population, rising up to 37% in the obese individuals[6,7].

It is now widely accepted that the major predisposing risk factors of NAFLD are represented by obesity, visceral adiposity, insulin resistance (IR) and many other disorders that define the metabolic syndrome[7]. Moreover, several clinical and experimental lines of evidence report a reciprocal relationship between NAFLD and diabetes onset[8].

On the other hand, different factors including genetic background, epigenetic mechanisms and environmental factors, as diet and physical exercise, participate in NAFLD development. Additionally, adipose tissue and gut may intervene in establishing a complicated cross-talk causing the production and the release of circulating endotoxins, adipokines and pro-inflammatory cytokines, which in turn contribute to IR and free fatty acids (FFAs) accumulation bursting oxidative stress and hepatocellular damage[9,10]. To further complicate this scenario, NAFLD is still a condition under-diagnosed and there is currently no specific drug therapy approved. In fact, the mainstay of NAFLD treatment is lifestyle modification in term of weight loss and diet changes. However, the majority of paediatric and adult patients have difficulty to achieve and to maintain weight loss, thus reducing the beneficial long-term effects of lifestyle intervention[11,12]. In the latest years, it has been widely documented that anomalous microRNA (miRNA or miR) profiles are strongly associated with several pathological conditions including some kind of cancers and liver, heart, kidney and autoimmune diseases[12]. Furthemore, the deregulation of microRNA expression in response to intrinsic factors (genetic or epigenetic) or extrinsic factors (environmental challenge or stress conditions) may also contribute to metabolic dysfunction[13]. Therefore, it is not surprising that numerous recent evidence reports altered expression patterns of microRNAs in NAFLD, suggesting their role in the pathogenesis of the disease.

Given its importance, in this review we will discuss the recent emerging implication of microRNAs both as controlled and controllers in NAFLD development and/or their eventual prognostic value of disease progression.

MICRORNA CONTROLLED SYSTEMS AND FUNCTION

In the world of non-coding RNAs (ncRNAs), microRNAs are small endogenous single-stranded RNA molecules acting in transcriptional and post-transcriptional regulation of gene expression. Conserved across diverse species including animals, plants and even viruses, microRNAs exert their function of base-pairing with complementary sequences within mRNA targets, usually resulting in gene silencing through translational repression or mRNA stability alteration[14]. By the way, a positive regulation (transcriptional and translational activation) mediated by microRNAs is also possible[15-19]. Since the discovery of microRNAs in C. elegans over ten years ago[20], much attention has been paid to deeply understand microRNAs biology and regulatory function. At present, the latest versions of the miR dedicated Database (http://www.mirbase.org) list over 24521 entries and the number of publications dealing with microRNAs is growing day by day[21].

To date, it is estimated that these small molecules are likely to regulate approximately 30% of the human protein-coding genome[22] and are now recognized as key regulator of almost every cellular process including proliferation, apoptosis, differentiation and cellular growth[23,24]. Furthermore, in the latest years, mis-expression of microRNAs has been linked to numerous disease states and for this reason, microRNA-based therapies are under investigation[13].

The human genome encodes hundreds of microRNA genes classified as intergenic, intronic or exonic. Independently of their genomic location, microRNA genes can be found either as single genes or as clusters of genes under the control of their own promoter or the promoter of protein-coding genes[14].

The majority of microRNAs are transcribed by RNA polymerase II (Pol II) or occasionally RNA polymerase III to generate a long hairpin precursor called pri-miRNA[25,26].

Pri-miRNAs are both capped at the 5’ end and poly-adenylated at the 3’ end like other protein coding primary transcripts[27-29] but they have an unique hairpin structure that allow the microRNA biogenesis machinery to distinguish them from the various RNA stem-loop-like structures present in the nucleus[26].

In the canonical pathway, the precursor pri-miRNA is processed through a two-step mechanism by the ribonuclease (RNase) III-family proteins Drosha and Dicer[30,31]. Actually, once the pri-miRNA is synthesized, it is cleaved by the nuclear Drosha, that together with the cofactor DGCR8 composes the microprocessor complex[32,33].

Many other proteins, such as helicases and heterogeneous nuclear ribonucleoproteins (hnRNPs) may take part to help, thus creating a complex regulatory network[34].

The cleavage gives rise to a hairpin structure of 60-110 nt, termed pre-miRNA. Pre-miRNA is then exported out from the nucleus to the cytoplasm by the cargo transporter Exportin-5/Ran-GTP complex, which also protects microRNAs from degradation[35,36]. However, some exceptions exist. In fact, it has been shown that intronic microRNAs (mirtrons) can bypass the Drosha cleavage step, undergoing splicing and debranching instead[37].

In either case, once in the cytosol, the RNaseIII enzyme Dicer-1 further processes pre-miRNA, producing an imperfect about 20-bp double-stranded miRNA duplex (miRNA/*miRNA)[31,38-41]. The duplex is composed by the guide strand and the passenger (or miRNA*) strand. The guide strand, generally the one with the lowest thermodynamic stability at its 5’ end, is preferentially incorporated into the miRNA-induced silencing complex (miRISC)[33]. On the contrary, the passenger is excluded from the RISC and it is degraded[33,42,43]. Anyway, it should be kept in mind that sometimes miRNA* strands can be loaded into miRISC to function as microRNAs and that each strand of the duplex can be tissue-specific[44].

In mammals, the miRISC is mainly composed by the Argonaute (AGO) proteins, as core components, by the GW182 family proteins and by other accessory proteins. The formation and activity of the miRISC implies many additional factors, some of which play an important regulatory function[45]. As part of miRISC, the miRNA can induce translational repression or deadenylation and degradation of target mRNA usually through antisense base-pairing in the 3’-UTR of target mRNAs[46].

Whereas the full mechanism of microRNA-mediated translational repression is still poorly understood, more details are known about mRNA deadenylation[45-47]. In mammals and Drosophila the carboxy-terminal domain of GW182 proteins plays a central role interacting with the poly(A) binding protein (PABP) and recruiting the deadenylases CCR4 and CAF1 indeed[47,48].

In addition, in D. melanogaster, the C-terminal and the Ago-interacting N-terminal GW182 domains, contain other regions that are active in translational repression, opening numerous regulatory mechanisms[49].

Interestingly, in plants a typical miRISC-mediated gene regulation is guided by a perfect match between microRNAs and target RNAs that lead to an endonucleolytic cleavage and then to degradation. Even if rare, this mechanism can be found in animals too. However, microRNAs in plants may imperfectly base-pair to mRNAs, as occurs in mammals, and repress translation in this way[50].

MICRORNA AS REGULATORS AND CONTROLLERS IN NAFLD

Experimental evidence

Recently, it has been demonstrated that variations in the expression levels of some micro-RNAs are related to the pathogenesis of NAFLD and its progression to NASH. In fact, some of them are key regulators of glucose, cholesterol and lipid metabolism and their deregulation may result in intra-hepatic excessive accumulation of triglycerides and fatty acids[51].

One of the first microRNAs correlating with lipid metabolism and homeostasis is miR-122, the most abundant in the liver[52]. In fact, an important murine study showed that inhibition of miR-122 using antagomirs results in a 25%-30% reduction of plasma cholesterol levels and in an altered expression of several hepatic genes involved in the biosynthesis of cholesterol, as the 3-hydroxy-3-methylglutaryl-Coa synthase 1 (Hmgcs1), 3-hydroxy-3-methylglutaryl-Coa reductase (Hmgcr) and the 7-dehydrocholesterol reductase (Dhcr7)[53]. Furthermore, Esau et al[54] highlighted how the antisense-based silencing of miR-122 was able to induce a significant decrease in fatty acids and cholesterol biosynthesis and an increase in the fatty acids β-oxidation pathway in mice fed a high fat diet. Interestingly, targeting miR-122 by antisense inhibitors with “locked nucleic acid” (LNA) technique correlated with decreased circulating cholesterol levels in monkeys[55].

Recently, it has been found that miR-122 is involved also in the propagation of hepatitis C virus (HCV) suggesting that the silencing of this microRNA may represent a safe therapeutic approach for the treatment of both chronic HCV infection and diseases related to the alteration of cholesterol and lipid metabolism[56].

Further, the hepatic expression pattern of some crucial microRNAs has been analyzed in two interesting different animal models of NASH. In C57BL/6J and DBA/2J mice fed with methyl-deficient diet, the hepatic over-expression of miR-34a, miR-155, miR-200b and miR-221 and a concomitant down-regulation of miR-29c, miR-122, miR-192 and miR-203 was reported[57]. The microRNAs analysis on rats fed with hypercaloric diet enriched in fat (HFD) and fructose (HFD-HFr), highlighted a down-regulation of miR-27a, miR-122, miR-451 and an up-regulation of miR-200a, miR-200b and miR-429[58].

Moreover, in a recent paper of our group, mir-200b, together with miR-155, have been also linked with enhancer of zeste homolog 2 (EZH2) expression in both in vivo and in vitro models of NAFLD[59].

An additional microRNA involved in the regulation of lipid metabolism is miR-15b. Indeed, its potential contribution has been revealed through two studies: the first conducted on HFD fed rats and the second on the L02 hepatocyte cell line treated with palmitate. The data obtained by real-time PCR showed increases of miR-15b hepatic expression in both in vivo and in vitro NAFLD models compared to the controls. To further investigate miR-15b roles, miR-15b was over-expressed in QSG7701 hepatic cells, causing an increase in triglyceride levels and a decrease in glucose consumption. Therefore, these data collectively suggest that augmented expression of miR-15b can lead to an alteration in glucose and lipid metabolism, possibly increasing the risk of NAFLD onset[60]. An in vitro study conducted by Zheng et al[61] highlighted the miR-10b involvement in NAFLD pathogenesis. In fact, miR-10b was found altered in steatotic hepatic cells. More precisely, when over-expressed, miR-10b induces an increase in the cellular lipid content and in the triglycerides levels acting on peroxisome proliferator-activated receptor-α (PPARα), a nuclear receptor involved in the catabolism of fatty acids. Other post-transcriptional regulators of lipid metabolism seem to be miR-33a and miR-33b, intronic microRNAs, located within SREBF2 and SREBF1 genes, respectively, both implicated in the positive expression of cholesterogenic and lipogenic genes[62].

Notably, several studies reported a tight co-expression of miR-33a/b with their host genes, with whom they act in concert to promote hepatic lipogenesis[63]. However, other microRNAs, such as miR-103 and miR-107, which are up-regulated in diet-induced obese rats, have been associated with metabolic disorders related to IR development. Interestingly, given that the antisense-mediated silencing of miR-103 and 107 improved insulin sensitivity, these two microRNAs might be used as therapeutic targets to improve obesity IR-related[64]. Moreover, miR-33a/b play also a key role in post-transcriptional repression of ATP-binding cassette transporter sub-family A member 1 (ABCA1), which is essential for the binding of cholesterol to apolipoprotein A1 (APOA1) during HDL formation. The mechanisms involves the direct binding of miR-33 to the 3’ UTR of ABCA1 gene[65]. Interestingly, it has been reported that miR-33a/b are involved in lipid and glucose homeostasis also interacting with the deacetylases NAD+ dependent sirtuin 6 (SIRT6), which in turn regulates the expression of Sterol Regulatory Element-Binding Protein (SREBP) target genes[66]. In fact, it has been demonstrated that SIRT6 knockout is associated with the development of liver steatosis in mice[67]. In line with previous published papers, among SIRT6 regulators, the NAD+-dependent deacetylase sirtuin (SIRT1) seem to play an important function in mice. SIRT1 complexes with several factors such as FOXO3a and NRF1 on the SIRT6 promoter regulates positively SIRT6 expression, which, in turn, negatively regulates glycolysis, triglyceride synthesis, and fat metabolism acting on the transcription of many genes involved in these processes[67]. According to the literature, SIRT1 is further regulated at post-transcription level by miR-34a. Cantó et al[68], reviewed that miR-34a inhibits the expression of SIRT1 and prevents the activation of PPARα and of liver X receptor (LXR), a key regulator of energy homeostasis. In addition, the repression of SIRT1 results in inhibition of both SREBP and nuclear factor-κB (NF-κB), a critical mediator of inflammation. Moreover, recent research has shown that miR-34a reduces levels of NAD+ and SIRT1 activity by acting directly on the nicotinamidide phosphoribosyl transferase (NAMPT) required for NAD+ biosynthesis, resulting in a decrease in β-oxidation and in an increase in lipogenesis. Besides, miR-34a silencing in obese mice leads to the restoration of NAMPT/NAD+ levels relieving hepatic steatosis and inflammation[69]. Notably, miR-34a also acts on the repression of β-Klotho, a coreceptor of the fibroblast growth factor 19 (FGF19), implicated in the regulation of glucose metabolism in the post-prandial response under physiological conditions. This evidence comes from a paper showing that antisense inhibition of miR-34a in obese mice is able to restore both β-Klotho levels and FGF19 target gene expression improving obesity[70]. Studies reporting experimental evidence of microRNA deregulation during NAFLD are summarized in Table 1.

Table 1 miRs deregulated in experimental models of non-alcoholic fatty liver disease.

Ref.	miR	miR regulation	Experimental model
[55]	miR-122	Up-regulation	In vivo: Silencing by antisense-base technology in a diet induced obesity mouse model
[56]	miR-122	Up-regulation	In vivo: Inhibition by antisense oligonucleotides “locked nucleic acid” (LNA) in monkeys
[59]	miR-34a miR155 miR-200b miR-221	Up-regulation	In vivo: C57BL/6J and DBA/2J methyl-deficient diet mice fed
	miR-29c miR-122 miR-192 miR-203	Down-regulation
[60]	miR-200a miR-200b miR-429	Up-regulation	In vivo: HFD/HFr fed rats
	miR-27 miR-122 miR-451	Down-regulation
[61]	miR 200b miR 155	Up-regulation	In vivo: HFD/HFr fed rats In vitro: HepG2 hepatoma cell line upon FFA stimulation
[62]	miR-15b	Up-regulation	In vivo: HFD fed rats In vitro: L02 human hepatocyte cell line treated with palmitate
[63]	miR-10b	Up-regulation	In vitro: Steatotic model of human L02 hepatocytes
[66]	miR-103 mi-R107	Up-regulation	In vivo: Dietary obese rats
[71,72]	miR-34a	Up-regulation	In vivo: Dietary obese mice

HFD/HFr: Hypercaloric diet enriched in fat/fructose.

microRNA as biomarkers in NAFLD

Since serum miRNAs have been recognized as diagnostic and prognostic markers for lung, prostate, breast, ovarian, and liver cancers, the overall idea is to identify the altered microRNA expression profiles potentially associated with the pathogenesis of NAFLD and eventually to evaluate serum levels of microRNAs as potential non-invasive biomarkers for the diagnosis and monitoring of disease[71]. The microRNAs suggested as potential biomarkers for human NAFLD are reported in Table 2.

Table 2 miRs deregulated in human non-alcoholic fatty liver disease.

Ref.	miR	miR regulation	Source	Phenotype
[74]	miR-34a miR-122 miR-21 miR-451	Up-regulation	Blood	Only miR-122 is correlated with the severity of liver steatosis
[75]	miR-122 miR-34a miR-16	Up-regulation	Blood	Positive correlation with fibrosis, inflammation and hepatic steatosis
[77]	miR-122 miR-122	Up-regulation Down-regulation	Blood/liver	From mild to severe hepatosteatosis End-stage liver fibrosis
[79]	miR-15b	Up-regulation	Blood	Positive correlation with NAFLD parameters
[80]	miR-34a miR-146 miR-122	Up-regulation Down-regulation	Liver Liver	Association between NASH and altered hepatic miRNA expression

NAFLD: Non-alcoholic fatty liver disease; NASH: Non-alcoholic steatohepatitis.

A recent study conducted by Yamada et al[72] on Japanese subjects, adds evidence on the possibility to use circulating microRNAs as biomarkers for NAFLD. In this study, serum levels of five selected miRNAs (miR-145, miR-451, miR-122, miR-34a and miR-21), well known to be implicated in cholesterol and fatty acid homeostasis in liver tissue, were evaluated in individuals with hepatic steatosis assessed by ultrasound scan. Here the authors found increased levels of miR-34a, miR-122, miR-21 and miR-451 in NAFLD patients compared to non-NAFLD participants. These findings are in line with other previous human studies showing that altered expression of circulating levels of these and other microRNAs are related to NAFLD pathogenesis. Indeed, in a human study by Cermelli et al^[73] serum levels of miR-122 and miR-34a were found significantly increased in patients with NASH, showing a positive correlation with fibrosis and inflammation stage. The same study also found a positive correlation between increased circulating levels of miR-16 and simple hepatic steatosis. The possible explanation of miR-34 mis-expression lies on the fact that miR-34a acts by inducing pro-apoptotic genes and p53 transcription, thus sustaining the liver damage occurring during NAFLD[74]. In a more recent human study in NAFLD patients, serum and hepatic miR-122 levels were found increased moving from mild to severe steatosis. In contrast, a significant inverse correlation with progression of fibrotic liver damage was observed[75]. This particular expression trend of miR-122 could be explained by molecular mechanisms occurring during the progression of the disease. Persistent liver injury results in loss of hepatocytes, the main miR-122 producing cells, and thus may determine an inadequate miR-122 expression[73]. Another mechanism supporting the link between miR-122 and fibrotic liver damage is the inhibitory effect of this miR on hepatic stellate cell activation and collagen deposition[76]. Taken together, this evidence suggests that the measure of serum miR-122 levels can provide a useful predictive marker of liver fibrosis in patients with NAFLD.

In a recent cohort study of patients with NAFLD, the increase of body mass index, blood glucose or alanine transferases levels, were interestingly associated with a parallel increase in miR-15b serum levels, suggesting a potential role also for miR-15b as a diagnostic candidate[62]. However, comparing studies on circulating levels and hepatic expression of microRNAs in NAFLD it is possible to find interesting differences. Indeed, in a study of hepatic microRNA expression patterns, miR-34a and miR-146b were significantly increased in livers of NASH subjects while decreased expression of miR-122 was found in the majority of patients[77]. In Table 3 the most important NAFLD-related miRNAs with known and validated target genes are reported.

Table 3 Key miRs involved in non-alcoholic fatty liver disease and their target genes.

Ref.	miR	Target genes
[55,56]	miR-122	Hmgcs1, Hmgcr, Dhcr7
[70-72]	miR-34a	SIRT1, NAMPT, β-Klotho coreceptor
[63] [66]	miR-10b miR-33a/b	PPAR-α SIRT6 ABCA1

CONCLUSION

Despite numerous research efforts, NAFLD molecular pathogenesis is only partially understood. Recent evidence indicates, that during NAFLD development and progression, some tissue-specific or circulating microRNAs may play crucial roles in the post-transcriptional regulation of genes that regulate critical process in liver damage associated with this disease. In this picture, therefore, the study of microRNA could assume a dual significance. On one hand, the comprehension of regulatory upstream/downstream factors associated with these microRNAs might provide a source of novel potential therapeutic targets to block NAFLD progression. On the other hand, the analysis of the microRNA expression pattern in NAFLD might highlight which of them have a strong correlation with liver damage and possess adequate characteristics as non-invasive biomarkers.