Non-alcoholic fatty liver disease (NAFLD) encompasses a broad spectrum of liver diseases ranging from simple steatosis to non-alcoholic steatohepatitis (NASH) with or without fibrosis (1). NAFLD is the most common cause of chronic liver disease in children and is believed to follow a more aggressive course compared to adult disease due to early-onset and distinct histological features. Yet, the prevalence of pediatric NAFLD has been difficult to assess clinically and there is no approved pharmacotherapy for its treatment.

The etiopathogenesis of NAFLD in children is complex. It is regarded as the hepatic manifestation of the metabolic syndrome given its strong association with insulin resistance (IR), type 2 diabetes (T2D), and dyslipidemia, but is also an independent risk factor for cardiovascular morbidity and mortality. Early diagnosis through screening followed by treating associated comorbidities, i.e., obesity and IR, is the standard of care. However, the commonly available screening tests have major limitations, for example serum ALT has low specificity and liver ultrasonography has low sensitivity, whereas reference standards such as liver biopsy and MRI are expensive and not readily accessible in most centers (2). Additional research is needed to identify molecular mechanisms and/or novel biomarkers to improve diagnostic accuracy and provide potential targets for pharmacotherapy. Recent research has highlighted the role of epigenetic factors on NAFLD development. MicroRNAs (miRNAs), a type of short non-coding RNAs in the length of 19-28 nucleotides, are implicated in the epigenetic regulation of gene expressions involved in the pathogenesis of NAFLD (3). Dysregulation of hepatic miRNAs is associated with NAFLD (4–6). For instance, decreased hepatic miR-122 promotes hepatic de novo lipogenesis, which is implicated in the development of steatosis and progression to NASH (7); circulating miR-122 and miR-192 may distinguish NAFLD patients from healthy controls; and miRNA-34a may differentiate NASH from steatosis (8). Interestingly, one clinical cohort demonstrated that the classification performance of validated miRNAs (or their ratios) for NASH is better than that reached by ALT or aspartate aminotransferase (AST) (9). In addition, cross-validated models combining both clinical and miRNA variables showed an enhanced prediction of NAFLD. Taken together these findings show that circulating miRNAs correlate to the molecular events contributing to the pathogenesis of NAFLD and monitoring miRNAs may improve the accuracy of diagnostic screening tools.

Pediatric studies investigating the relationship between miRNA expressions and clinical and metabolic markers in children at-risk for NAFLD are limited. Most studies utilized healthy children without obesity as controls (6, 10–12) and one study (5) compared miRNA profile among pre-pubertal children. Considering the puberty-associated IR and strong association between onset of puberty and NAFLD, a more appropriate control group, one with similar risk factors is needed to decipher the effect of obesity or associated metabolic complications such as IR on miRNA profile in patients with NAFLD. In the present study, we aim to determine the associations between circulating miRNAs and metabolic and hepatic features of NAFLD and serum levels of insulin, adiponectin, and fibroblast growth factor (FGF)-21 in children with obesity and varying degrees of IR and intrahepatic triglyceride (IHTG).

Hepatic miRNAs are involved in lipid and glucose metabolic pathways; however, little is known about associations of miRNA expression in pediatric NAFLD. In this secondary analysis, we investigated the relationships between serum miRNA profile, metabolic biomarkers (insulin, lipid profile, adipokines, and liver enzymes), and IHTG percent in a well-described cohort of pubertal children with obesity and varying degrees of IR. We provided new evidence that serum concentrations of miR-21-5p, -22-3p, -150-5p, and -155-5p were higher in patients with NAFLD, and the metabolic profile of adolescents with IR were comparable among those with and without NAFLD. We also demonstrated that miR-27-3a expression was higher in adolescents without IR compared to those with IR regardless of NAFLD status.

A growing body of evidence suggests that the expression pattern of several miRNAs, including miR-21 and -22, are associated with the severity of NAFLD. miR-21 and -22 are two of the most abundantly expressed liver miRNAs. miR-21 is hypothesized to regulate the genes in hepatic cholesterol and triglyceride metabolisms (17). Furthermore, ablation of hepatic miR-21 was shown to decrease hepatic inflammation and improve fibrosis (18). In our study, we showed that serum miR-21 and -22 were both positively correlated with IHTG, TG, and LDL-C only in the NAFLD group. No correlation was observed between these miRNAs and liver enzymes in any of the groups. Based on these results it is tempting to speculate that in children with obesity and IR, increased expression of miR-21 and -22 in the circulation could be a harbinger of impending NAFLD. However, a limited number of studies in adults have reported contradictory results about miR-21 expression pattern. Sun et al. demonstrated decreased concentration of miR-21 in the circulation in patients with NAFLD (19), while Becker and colleagues did not find any difference between the control and NAFLD groups, but higher levels in patients with NASH (7). Moreover, while the role of miR-22 in the progression of steatosis to advanced stages such as fibrosis and cirrhosis is recognized (20), its role in the development of steatosis is less understood. Further validation studies are needed to determine if miR-21 and -22 are predictive markers for NAFLD progression in pediatric populations.

In the current study, we also showed that miR-22 expression negatively correlated with FGF21 and adiponectin levels in adolescents with IR regardless of NAFLD status. There were no differences in FGF21 levels among the groups with or without IR or NAFLD. We speculate that increased miR-22 blunts hepatic FGF21 synthesis and secretion in children with NAFLD, and therefore promotes worsening of steatosis. In a recent study, Hu et al. (21) utilizing a human liver cell line and fatty liver specimens demonstrated that increased hepatic miR-22 expression inhibited FGF21 expression and promoted lipogenesis through directly suppressing fibroblast growth factor receptor 1 (FGFR1) while reducing PPARα and PPARγ coactivator 1α (PGC1α), two transcriptional factors regulating FGF21 expression. Moreover, they also showed inhibition of miR-22 eliminates alcohol-induced steatosis in murine models, possibly due to restoring FGF21 expression. Considering the beneficial properties of FGF21 in hepatic lipid metabolism (22), these findings provide insight on the mechanistic role of miR-22 in NAFLD development.

Altered hepatic and serum levels of miR-150 have been reported in animal models and patients with insulin resistance and liver diseases. However, whether miR-150 exerts a protective role in NAFLD or promotes development and progression of the steatosis into more advanced stages is yet to be determined. Zhuge et al. (23) demonstrated elevated levels of miR-150 in the serum of adult patients with NAFLD compared to healthy controls, and in the liver of mice fed with high-fat diet compared to chow-fed mice. They also reported that miR-150 knock-out mice were protected from developing hepatic steatosis and insulin resistance even when fed high fat diet. In line with these findings, Huang et al. showed that increased miR-150 expression promotes steatosis in human fetal hepatocyte line (24). On the contrary, Ying et al., also utilizing miR-150 deficient mice, showed that miR-150 plays an important regulatory role in adipose tissue inflammation and that deficiency of miR-150 in mice is associated with severe systemic inflammation and insulin resistance (25). Our findings did not support any relationship between miR-150 and markers of insulin resistance such as insulin, adiponectin, or FGF21, but showed that miR-150 might be an important regulator in the cholesterol and triglyceride metabolisms.

The mechanistic role of miR-155 in hepatic lipid metabolism is less understood and debated. Earlier studies suggested a protective role for miR-155, through its downstream effector liver X receptor, for the development of steatosis (26). In line with these reports, Johnson et al. (27) suggested that miR-155 expression in white adipose tissue is pivotal to prevent progression of obesity-associated inflammatory response. However, Ying et al. (28) reported that macrophage-derived exosomal miR-155 obtained from the adipose tissue of obese mice could induce IR in lean mice via its target PPARγ. More recently, Bala et al. (29) showed that miR-155 knock-out mice displayed less steatosis and fibrosis compared to wild type mice. Studies in humans are scarce and contradictory. While Wang et al. (30) demonstrated decreased liver and serum miR-155 levels in adult patients with NAFLD, Zhou et al. (31) showed increased serum levels in children with NAFLD. In the current study, we found positive correlations between serum miR-155 level and insulin and adiponectin concentrations in children with IR regardless of NAFLD status, and IHTG and liver enzymes in children with NAFLD only. Taken together, these findings may suggest that elevated miR-155 may play a role in the development and progression of NAFLD through induction or exacerbation of IR.

miR-27 is another understudied and potentially overlooked miRNA in the pathophysiology of pediatric NAFLD. In an in vitro study, Ji and colleagues (32) showed that overexpression of miR-27a and -27b were influential in fat accumulation in hepatic stellate cells. This was supported by the findings of Singaravelu et al. (33) who demonstrated that overexpression of miR-27b results in larger and more abundant lipid droplets in hepatocytes infected with hepatitis C virus, likely due to inhibition of PPARα. Increased expression levels of miR-27b were also shown in humans with NAFLD compared to healthy controls (4). On the contrary, Zhang et al. (34) showed that miR-27a, through the inhibition of two important regulatory genes in the fatty acid synthesis and thereby decreasing the rate of de novo lipogenesis, decreases lipid accumulation in the hepatocytes. In line with this report, we found increased serum miR-27 levels in adolescents without IR regardless of the NAFLD status. We observed negative correlations between miR-27 and TG for all groups, miR-27 and insulin for groups with IR, and finally miR-27 and IHTG for groups with NAFLD. In contrast to adult studies, our comparison group was composed of children with similar degrees of obesity and body fat percent. Since IR is considered the first-hit in the development of NAFLD, we speculate miR-27 plays a protective role and prevents excessive hepatic lipid deposition via regulation of hepatic insulin signaling pathways.

In conclusion, our results add to the growing body of literature showing miRNAs are important regulators of systemic glucose and hepatic lipid metabolism in children with obesity and varying degrees of IR. These findings may have clinical implications. Particularly miR-21-5p, -22-3p, -150-5p, and -155-5p may be used to identify patients with IR at-risk for developing NAFLD, while miRNA-27-3a could be used to identify those who have a low risk of developing IR and hence NAFLD. We acknowledge that the small cohort may not equally distribute potential confounders such as race/ethnicity or pubertal developmental stages among the groups being compared. In addition, given the cross-sectional design, a cause-and-effect relationship cannot be established in this association study. However, our findings do address the current need for more accurate diagnostic tools for determining patients with IR at-risk for NAFLD. Further characterization of the mechanical roles of these miRNAs in the pathogenesis of IR and NAFLD may help develop targeted pharmacotherapies.

The data analyzed in this study is subject to the following licenses/restrictions: The data that support the findings of this study (all of the individual participant data collected during the study - after deidentification, statistical analysis plan, and analytic code) are available from the corresponding author immediately following publication with the researchers who provide a methodologically sound proposal. Requests to access these datasets should be directed to ET, etas@uams.edu.

The studies involving human participants were reviewed and approved by Institutional Review Board of the University of Arkansas for Medical Sciences. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

HL and ET designed the research. ET conducted the study. HL, KEM, XO, KM, RB, EB, and ET processed and interpreted the clinical and imaging data. HL and ET performed the statistical analyses. HL and ET wrote the manuscript. ET had primary responsibility for final content and edits. All authors read and approve the final manuscript.

Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number 5P20GM109096. HL, EB, KEM, and ET were also supported by the United States Department of Agriculture/Agricultural Research Service (USDA-ARS Project 6026-51000-012-06S).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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