Nonalcoholic fatty liver disease (NAFLD) is currently the most common chronic liver disease worldwide in part due to the concomitant obesity pandemic and insulin resistance (IR). It is increasingly becoming evident that NAFLD is a disease affecting numerous extrahepatic vital organs and regulatory pathways. The molecular mechanisms underlying the nonalcoholic steatosis formation are poorly understood, and little information is available on the pathways that are responsible for the progressive hepatocellular damage that follows lipid accumulation. Recently, much research has focused on the identification of the epigenetic modifications that contribute to NAFLD pathogenesis. Noncoding RNAs (ncRNAs) are one of such epigenetic factors that could be implicated in the NAFLD development and progression. In this review, we summarize the current knowledge of the genetic and epigenetic factors potentially underlying the disease. Particular emphasis will be put on the contribution of microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs) to the pathophysiology of NAFLD as well as their potential use as therapeutic targets or as markers for the prediction and the progression of the disease.
Nonalcoholic fatty liver disease is the accumulation of lipids in the liver above 5% of the total liver weight, in the absence of other medical conditions. It is currently the most common chronic liver disease worldwide, with a global prevalence of around 25% [
Progression of NAFLD and ncRNAs associated with each stage. The first stage of NAFLD is simple steatosis (or nonalcoholic fatty liver (NAFL)) characterized by abnormal accumulation of fat in the liver. If not reversed, simple steatosis may transform into nonalcoholic steatohepatitis (NASH), which in turn can lead to liver cirrhosis and eventually to hepatocellular carcinoma. The miRNAs, lncRNA, and circRNAs known to be associated with each stage are shown in blue, green, and yellow boxes, respectively. Few interactions between the different classes of ncRNAs in NAFLD were reported and are indicated with the arrow-ended brackets.
The etiology of NAFLD is not well understood. However, it is widely recognized that it is closely associated with excess fat, mainly visceral adiposity, IR, T2D (type 2 diabetes), hypertension, and dyslipidemia [
As said above, NAFLD implicates intricate interactions between many risk factors and genetic predisposition [
Candidate genes and variant associated with NAFLD: function and phenotype.
Pathway | Genes | Variant/SNP | Phenotype/function | Stage/phase | Studies | References |
---|---|---|---|---|---|---|
Lipid metabolism | PNPLA3 | rs738409 C/G | Hepatic steatosis, histologic lobular inflammation, HCC fibrosis, lipolytic and lipogenic function in vitro | NAFLD/NASH/activated HSCs | GWAS, case-control | [ |
PPAR | rs1801282 G/C | Protection against liver injury | NAFLD | Case-control, GWAS, meta-analysis | [ | |
LPIN1 | rs13412852 C/T | Regulation of lipid metabolism, reduced lipolysis, decreased flux of FAs to the liver, decreased fibrosis | NAFLD | Case-control | [ | |
NCAN | rs2228603 C/T | Regulates cell adhesion and migration/hepatic steatosis | NAFLD | GWAS | [ | |
rs3745367 G/A | Involved in lipid metabolism, hepatic insulin resistance, inflammatory cascade reactions, and fibrogenesis | NAFLD | GWAS, case-control | [ | ||
Cholesterol biogenesis | SREBPF 1 | rs11868035 A/G | The severity of steatosis and necroinflammation/impaired glucose homeostasis and lipoprotein and adiponectin responses to fat ingestion | NAFLD | Case-control | [ |
SREBPF 2 | rs2228314 G>C | Histological characteristics and NASH diagnosis | NASH | Case-control | [ | |
Fatty acid uptake and transport | PPAR | rs1801282 | Protection against liver injury | NAFLD | Case-control, GWAS, meta-analysis | [ |
APOC3 | rs2854116 T/C rs2854117 C/T | Hepatic steatosis/inhibits lipoprotein lipase and triglyceride clearance | NAFLD | Case-control, meta-analysis | [ | |
FABP1 | rs2241883 T/C rs1545224G/A | Impact blood lipoprotein/lipid levels and responses to lipid-lowering therapy and glycogenolysis/fibrosis steatosis | NAFLD/NASH | Case-control | [ | |
MTTP | rs1800591 | Synthesis and secretion of VLDL in the liver, transfer protein involved in apoB-lipoprotein assembly/hypobetalipoproteinemia | NAFLD/NASH | GWAS, case-control | [ | |
Oxidative stress | PPAR | rs1800206 | Steatosis, inflammation, and fibrosis | NAFLD/NASH | GWAS, case-control | [ |
PNPLA3 | rs738409 C/G | Hepatic steatosis, histologic lobular inflammation, HCC development, fibrosis/lipolytic and lipogenic function in vitro | NAFLD/NASH/activated HSCs | GWAS, case-control | [ | |
TM6SF2 | rs58542926 | Hepatic steatosis, necroinflammation, ballooning, higher serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels/fibrosis, cirrhosis | NAFLD/NASH | GWAS | [ | |
GCKR | rs780094 A>G rs1260326 C>T | Steatosis/fibrosis, inability to regulate glucose influx into hepatocytes, increased de novo lipogenesis | NAFLD/NASH | GWAS, meta-analysis | [ | |
HSD17B13 | rs72613567 T>A | Localizes to hepatocyte lipid droplets/decreased HSD17B13 and PNPLA3 production | ↓NASH | Case-control | [ | |
Insulin resistance | ADIPOQ | rs2241766 rs1501299 | Insulin-sensitizing, anti-inflammatory adipokine/severity of liver disease and with an atherogenic postprandial lipoprotein profile in NASH | NAFLD | Case-control | [ |
IRS1 | rs1801278 | Downstream regulator of insulin action, deceased insulin signaling/fibrosis | NAFLD | Case-control | [ | |
PPAR | rs8192678 | Transcriptional coactivator that regulates genes involved in lipid and glucose metabolism | NAFLD | Case-control | [ | |
TCF7L2 | rs7903146C/T | Regulates gene expression in cellular metabolism and growth | NAFLD | Case-control | [ |
Epigenetics describes the changes in gene expression caused by mechanisms unrelated to modification in the DNA sequence [
Noncoding RNAs are RNAs that result mostly from alternative splicing of the more extensive transcripts, which become the precursors for smaller ncRNAs [
The human genome encodes about 2000 miRNAs, which target 30–60% of the genes [
Specific miRNAs with NAFLD development and progression in human patients.
Candidate biomarkers | Human | Stage/phase | Function | Approach | References | |
---|---|---|---|---|---|---|
Serum | Liver | |||||
miR-122 | Upregulated | Downregulated | NAFLD/NASH/activated HSCs | Lipid metabolism, intestinal permeability, inflammation, fibrogenesis, proliferation | RT-qPCR | [ |
miR-192 | Upregulated | Downregulated | NAFLD/NASH/activated HSCs/fibrosis | HSC activation | RT-qPCR | [ |
miR-34a | Upregulated | Upregulated | NAFLD/NASH/steatosis/activated HSCs/fibrosis | Inflammation | RT-qPCR | [ |
miR-1290 | Upregulated | Upregulated | NASH | Inflammation | RT-qPCR | [ |
miR-27b-3p | Upregulated | Upregulated | NASH | Inflammation | RT-qPCR | [ |
miR-192-5p | Upregulated | Upregulated | NASH | Inflammation | RT-qPCR | [ |
miR-34a-5p | Upregulated | Upregulated | NASH/NAFLD | Lipid metabolism, oxidative stress, apoptosis, useful miRNA biomarkers to discriminate between NAFLD and NASH patients | RT-qPCR | [ |
miR-375 | Upregulated | Upregulated | NAFLD/NASH/fibrosis | Glucose homeostasis, intestinal permeability modulation, inflammation | RT-qPCR | [ |
miR-155 | Upregulated | Upregulated | NASH | Master regulator of inflammation | RT-qPCR | [ |
miR-125b | Upregulated | Upregulated | Steatosis/NASH/fibrosis | Lipid and glucose homeostasis, adipocyte differentiation, fibrogenesis | RT-qPCR | [ |
miR-33a/b | Upregulated | Upregulated | NASH | Lipid and cholesterol metabolism, glucose homeostasis | RT-qPCR | [ |
miR-451 | Upregulated | Downregulated | NASH/NAFLD | Inflammation | RT-qPCR | [ |
miR-155 | Upregulated | Upregulated | NASH | Lipid metabolism, intestinal permeability modulation, inflammation | RT-qPCR | [ |
miR-221 | Upregulated | Upregulated | NASH/fibrosis/HCC | HSC activation, fibrogenesis | RT-qPCR | [ |
miR-222 | Upregulated | Upregulated | NASH/fibrosis/HCC | HSC activation, fibrogenesis | RT-qPCR | [ |
miR-15 | Upregulated | Downregulated | Fibrosis/HCC | HSC activation, proliferation, and metastasis | RT-qPCR | [ |
miR-16 | Upregulated | Downregulated | Fibrosis/HCC | HSC activation, proliferation, and metastasis | RT-qPCR | [ |
miR-21 | Upregulated | Upregulated | NASH/fibrosis/HCC | Gut microbiota modulation, inflammation, proliferation | RT-qPCR | [ |
miR-22 | Upregulated | Upregulated | NASH/fibrosis/HCC | Inflammation | RT-qPCR | [ |
miR-29 | Upregulated | Upregulated | NAFLD/NASH/activated HSCs | Inflammation | RT-qPCR | [ |
miR-122 is highly abundant in the liver [
miR-21 is upregulated miRNAs in serum and liver from patients with fibrosing NASH and HCC [
In high-fat diet-fed mice, the levels of miR-34a were upregulated significantly in liver tissues, resulting in the downregulation of its direct hepatic targets PPAR
miR-192 is a profibrogenic implicated in the development of fibrosis and activation of TGF
miR-370 is a potent posttranscriptional regulator of lipid metabolism. For instance, knockdown of miR-370 in HepG2 cells results in the upregulation of lipogenic genes such as
The miR-33 family consists of two members, miR-33a and miR-33, that locate in the introns of, respectively, SREBP-2 and SREBP-1 genes [
lncRNAs are transcripts that cannot translate into proteins and account for most of ncRNAs [
The biogenesis of lncRNAs is not fully unraveled. Its understanding is crucial not only for distinguishing lncRNAs from other types of RNAs but also to decipher its functional significance. It is cell type- and stage-specific and is under the control of cell type- and stage-specific stimuli. To date, the molecular mechanisms underlying the lncRNAs biogenesis are not fully resolved. The lncRNA can be transcribed by the RNA polymerase II from exonic, intergenic, or the distal protein-coding regions of the genome to produce the premature lncRNA. This later gets 3
Schematic presentation of the genomic loci of different long noncoding RNA (lncRNA). lncRNA classification depends on the genomic position: (i) intergenic RNAs are located between two protein coding genes, (ii) intronic lncRNAs are positioned within an intronic region of a protein coding-gene, (iii) and (v) sense and antisense lncRNAs are transcribed from complementary strands but in different direction, respectively, and (iv) bidirectional lncRNAs originate from the bidirectional transcription of protein-coding genes.
Numerous lncRNAs are implicated in liver disease and have potential diagnostic, prognostic, and therapeutic importance [
Confirmed lncRNAs in NAFLD.
Name of lncRNA | Type of lncRNA | Chromosome | Expression | Stage | Gene targets | References |
---|---|---|---|---|---|---|
MEG3 | Intergenic | 14q32 | Downregulated in human hepatic | NAFLD/NASH | [ | |
MALAT-1 | Intergenic | 11q13.1 | Upregulated | Fibrosis | [ | |
HOTAIR | Antisense | 12q13.13 | Upregulated/replicated in human | HCC | [ | |
APTR | Intergenic | 7q21 | Upregulated/involved in regulating cell cycle progression and cell proliferation in liver cirrhosis | HCC | [ | |
PVT1 | Intergenic | 8q24.21 | Upregulated/not replicate in human | HCC | [ | |
lnRNA-CoX2 | Antisense | 1p33 | Upregulated | Liver fibrosis | [ | |
NEAT1 | Intergenic | 11q13.1 | Upregulated | NAFLD/NASH | [ |
Overexpression of NEAT1 reduces the levels of miR-122, which mediates the effects of NEAT1 effects on HSC activation, by way of a mechanism ascribed to a Kruppel-like factor 6 (Klf6) [
circRNAs are a novel class of ncRNAs containing miRNA response elements (MREs). In humans, the first endogenous circRNA was reported in 1991 [
The biogenesis of circRNAs occurs during the transcription of most human genes due to a competition between the exonic linear splicing and an alternative splicing named back-splicing circularization [
Schematic presentation of the biogenesis of circRNAs. circRNAs are lncRNAs that undergo back splicing and can originate from transcripts containing only intronic, one or more exonic, or both intronic and exonic fragments.
Our knowledge about the role of circRNAs in NAFLD is scarce. Below, we review the current knowledge about the involvement of circRNAs in NAFLD progression, with a focus on the implication of the circRNA-miRNA-mRNA axis (Table
circRNA in NAFLD.
Name of circRNA | Target miRNA | Gene targets | Expression | Stage/phase | References |
---|---|---|---|---|---|
Upregulated | HCC | [ | |||
Downregulated | NAFLD | [ | |||
Upregulated | NAFLD | [ | |||
Upregulated | NAFLD | [ | |||
Upregulated | NAFLD | [ | |||
Upregulated | NAFLD | [ |
As discussed above, some of the reported miRNAs, lncRNAs, and circRNAs are strongly associated with NAFLD. To date, liver biopsy remains the gold standard for a firm diagnosis of NAFLD. However, the procedure remains invasive, difficult, prone to error sampling, and not practical for population screening of NAFLD. Moreover, different imaging methods have been applied to diagnose NAFLD but failed to distinguish the stages of the disease [
As we discussed above, miRNAs are becoming potential noninvasive biomarkers for the diagnosis, prognosis, and therapeutic targets of several diseases. Despite its many benefits, there are still obstacles and challenges to be surmounted before their adoption in clinical applications. First, the fundamental technical constraint to solve is the isolation and purification of samples, as the quality and purity of RNA is the basis of detection and quantification. Unlike intercellular miRNAs, circulating miRNAs are easily interfered by other serum components, and one need to be vigilant when purifying from serum [
Many lncRNAs play significant roles in multiple physiological processes involving gene regulation, as mentioned above, but it also opens the possibility of using these types of RNAs as diagnostic markers and therapeutic targets particularly when they can be readily detected in biological fluids [
Given the high stability of circRNAs in circulation, there is considerable interest in their potential use as biomarkers or therapeutic targets [
The development of several diseases, including NAFLD, involves numerous genetic and epigenetic factors. With the advance in high-throughput profiling methods, the coming years will undoubtedly see the discovery of new genetic determinants of NAFLD. Moreover, the interaction of epigenetic changes with inherited risk factors to determine an individual’s susceptibility to NAFLD will require more investigations to unravel the underlying mechanism fully. So far, no therapy exists for NAFLD, and the lifestyle modification aimed at weight loss remains the only therapy that gave relatively promising results. The evaluation of circulating ncRNA represents a promising strategy to assess and noninvasively monitor liver disease severity. Still, more investigations are required to identify and validate the efficiency and accuracy of these markers and to study their therapeutic potential. In a nutshell, studying the ncRNA in NAFLD will shed light on the pathophysiology of the disease. Still, it can also potentially help identify novel drug candidates as well as noninvasive and accurate predictive, diagnostic, and prognostic biomarkers.
Acyl-CoA-binding domain-containing 3
Acetyl-CoA carboxylase
Acyl-CoA synthetase short-chain family member 2
Actin alpha 2
Alu-mediated transcriptional regulator
Carbohydrate-responsive element-binding protein
Circular RNA
Chain of type 1 collagen
C-X-C motif chemokine ligand 5
Diacylglycerol O-acyltransferase 1
Diacylglycerol O-acyltransferase 2
ADN-methyltransferase 1
Farnesyl diphosphate farnesyl transferase-1 gene
Fatty free acids
Fibronectin domain-containing protein 5
Farnesoid X-activated receptor
Genome-wide association scan
Hepatocellular carcinoma
High-density lipoprotein
Hydroxy methylglutaryl-coenzyme A synthase 1
Homeobox transcript antisense RNA
Interleukin-6
Insulin resistance
lncRNA-cyclooxygenase 2
Noncoding RNAs
Lipin 1
Metastasis-associated lung adenocarcinoma transcript 1
Maternally expressed gene 3
MicroRNA
Matrix metalloproteinase 2
Matrix metalloproteinase 9
Messenger RNA
Microsomal TG transfer protein
Nonalcoholic fatty liver
Nonalcoholic fatty liver disease
Nonalcoholic steatosis
Nonalcoholic steatohepatitis
Noncoding RNAs
Nuclear-enriched abundant transcript 1
Patatin-like phospholipase domain-containing protein 3
Peroxisome proliferator-activated receptors
Tensin homolog expression
Plasmacytoma variant translocation 1
Single-nucleotide polymorphism
Sterol regulatory element-binding transcription factor 1
Transmembrane 6 superfamily members 2
Very-low-density lipoprotein.
The authors declare no conflict of interest for this article.
This work was supported by the Qatar Biomedical Research Institute. Open Access funding provided by the Qatar National Library.
Supplementary data 1: overview of the miRNA biogenesis pathway. miRNA genes are transcribed in the nucleus by RNA Pol II as long pri-miRNA transcripts that are 5