MicroRNAs as the promising markers of comorbidities in childhood obesity—A systematic review

Summary Introduction Rising child obesity rate creates a need for tools quantifying changes in children suffering from obesity, for purposes of detection or prevention of comorbidities. A candidate for such a role seems to be microRNAs, which in vivo serve as the suppressing factors in gene expression. Objectives This study aimed at reviewing recent discoveries in this field and concluding directions of research or application of studied molecules. Methods Repeated browsing of databases and screening of results, led to final approval of 16 articles. Filtered studies examined differences in microRNA expression between subjects with obesity and children suffering from its comorbidities. Results Studies concerning endothelial dysfunction identified molecules miR‐320a and miR‐630 as a possible diagnosis and treatment option. Search for the alternative markers in diagnosis of non‐alcoholic fatty liver disease suggested value of molecules: miR‐199a‐5p and miR‐122. miR‐486, miR‐146b, and miR‐15b may serve in grading the development of type 2 diabetes in children, although further research raised doubts. Panel of molecules was indicated as useful in early detection of metabolic syndrome and insulin resistance associated alterations. No valid link between studied microRNAs and atherosclerosis was found. Conclusions MicroRNAs seem to be promising prognostic markers for diagnosis of endothelial dysfunction, non‐alcoholic fatty liver disease, type 2 diabetes, metabolic syndrome and insulin resistance in children.


| INTRODUCTION
Obesity in children is one of the most demanding challenges emerging in the modern world of paediatric medicine. It is described in relation to patient's body-mass index (BMI). In children a threshold of obesity is BMI above 95th percentile in a group of the same age and sex. Despite various prevention programs, analyses, and research, its occurrence is still high, 1 which exposes youth to complications of obesity, such as endothelial dysfunction (ED), 2 non-alcoholic fatty liver disease (NAFLD), 3 type 2 diabetes mellitus (T2DM), 4 atherosclerosis (AS), 5 and obstructive sleep apnea. 6 The possibility to quantify the progression of these diseases is necessary in order to overcome their development. As the standard markers and tests in many cases are insufficient measures for this challenge, the novel markers such as microRNA (miRNAs; miRs) may prove to be a sound solution. MiRNAs are small, non-coding molecules that play a suppressive role in expression of genes by binding to 3 0 UTR end of their target messenger ribonucleic acid (mRNA). They can be found in body tissues or fluids, like saliva, plasma, serum, or whole blood; encapsulated into extracellular vesicles (EVs) or as circulating miRNA. 7,8 Changes in a profile of the circulating and tissue miRNAs directly influence the physiology of tissues and cells involved in glucose and lipid metabolismpancreatic β-cells, hepatocytes, skeletal muscle tissue, and adipose tissueby modulating mRNA translation. 9,10 Taking into consideration the easy accessibility and measurability of materials rich in miRNAs, such as blood, plasma, and saliva, [11][12][13] examining the associations between diseases and miRNA levels might lead to creation of diagnostic and prognostic markers of high sensitivity and specificity.
Research on miRNA is nowadays one of the more popular scopes of interest in numerous branches of medicine. Recently the differences in miRNA profiles between the children with and without obesity have been investigated. 14,15 Results of studies on this topic were analysed and summarized into the systematic review in 2019 by Oses et al. It also presented two miRNAs significantly overexpressed in children with NAFLD and T2DM. 16 Our study concentrated solely on reviewing studies that examined miRNAs as biomarkers of paediatric obesity comorbidities. The scope of interest was expanded to comorbidities other than NAFLD and T2DM. The set of included studies was updated. In accordance with 'participants, interventions, comparisons, outcomes, and study design (PICOS)' scheme, the following  Retrieved data underwent further screening on a basis of titles, abstracts, and keywords (performed by MH and JH) according to inclusion and exclusion criteria, which led to exclusion of reviews, systematic reviews, author manuscripts, personal communication, letters to editor, conference material, and case reports. Articles concerning the miRNA as the markers of obesity itself in children, as well as the studies of miRNA not obtained from blood or plasma were ruled outin order to be plausible for the role of markers, miRNAs must be easily accessible (blood or plasma) and correlate with comorbidities of obesity (rather than the obesity itself ). Inadequate matching of experimental and control groups in the matter of age or exceeding upper age threshold were also the discriminating factor.
At the stage of data synthesis the retrieved studies were assessed by two independent researchers (AZ and PM) in terms of methodology quality, as well as relevance of the results. The principal measures of differentiating potential were area under the receiver-operator curve (ROC) (AUC) score, fold change, and p-values (statistical significance threshold: p < 0.05).

| RESULTS
Multiple browsing of databases, such as PubMed, askMEDLINE, Wiley Online Library, and Elsevier, conducted in the span of January 2020 - In total 16 studies fully met the inclusion and exclusion criteria.
The process of data collection for this review is presented in Figure 1.
Main characteristics of selected articles can be found in Table 1.
Enrolled studies focused on associations between changes in miRNA profiles of patients with obesity and presence of 6 obesity comorbidities. Their methodology is discussed in the following sections separately for each complication.

| Type 2 diabetes mellitus
The goal of study 7 was the comparative analysis of miRNA levels in paediatric groups of children with (OB) or without obesity (CG) and were created in order to assess the potential of differentiating between the groups.
These two studies presented, though to a different extent, a profile of three miRNAs (namely miR-486-5p, miR-146a-5p, and miR-15b) potentially useful in detection of T2DM development in children.
Results of study 8 are less satisfactory than those of study 7, showing lower AUC score values. However, it should be pointed out, that in study 7 the cumulative effect of simultaneous measurement of two or three of aforementioned molecules was not examined.
Another two miRNAs (miR-29a, miR-122) were examined in study 9 in terms of differences of their levels in children without obesity, with obesity but without T2DM, and in those with obesity and T2DM. Two hundred and ninety eight patients, aged 9-15 years, were divided into above groups based on their BMI and WC (obesity vs. non-obesity) and glucose levels (fasting ≥126 mg/dl, random ≥200 mg/dl, OGTT

| Metabolic syndrome
Similarly to the obesity, we can observe the increasing metabolic syndrome (MetS) prevalence worldwide. This term represents the cluster of metabolic changes, which stem directly from long-term positive energy balance. These alterations include obesity, followed by hypertension, and atherogenic dyslipidemia. 40 Recently the study on hepatic transaminases, anthropometric, and blood metabolic parameters. Similar to study 10, the associations between the miRNA profile and adiponectin, as well as obesity-related inflammatory markers levels were investigated. The population of the study varied in age (9-18 years, mean 13.1 ± 2.7 years) with a disproportion for sex (37 male/72 female), therefore the results were adjusted for age and sex, which led to differences between the sexes in significance of correlation of some miRNAs with studied parameters.

| Insulin resistance
Three studies (studies 13, 14, and 15) concentrated on the correlation between miRNAs and insulin resistance (IR). In study 13, the children and adolescents aged 10-17 years were divided into two groups: children with obesity (OB; 9 patients) and children with obesity and IR Analysis of glucose metabolism parameters and their association with miRNA profile in a group of 58 children with overweight or obesity was conducted in study 15. There was no statistical difference in mean age or insulin levels and HOMA-IR between the sexes, though the latter parameters were insignificantly higher in girls. Two examined miRNAs: miR-191-3p and miR-375, were also further analysed in terms of interactions with their respective pathway targets.  Examined miRNAs, their level changes, as well as their significance and AUC score are presented in Table 3. Studies 12 and 15 were not applicable for the purpose of this table, and as such were not included.

| Endothelial dysfunction
Condition of endothelium as a whole can be inspected via levels of endothelial products, such as NO and ET-1, their ratio (NO/ET-1) or through functional test -RHI measurement, whose score is dependent on endothelial reaction engaging NO. 43,44 Some miRNAs play an established role in physiology of endothelium, angiogenesis, although they have not been yet applied clinically as the markers of endothelial dysfunction. 45,46 Physical activity is Method used for the measurement of RHIperipheral arterial tonometryis highly dependent on age and sex of subjects, 47 therefore the uniformity of groups in this regard is highly required. Unfortunately, in this study the patients' age parameter had a wide range (12-18 years) and the RHI results might be unreliable measure of endothelial condition in that case.
Similar to study 1, study 2 was also an interventional study, which The goal of study 3 was to establish the differences in expression of miRNAs specific for cardiovascular diseases between the groups of children with NEF and the ones with ED using microarray assay.
Results were further validated (using qRT-PCR) for three miRNAs whose expressions were found to be significantly different in groups.
The procedure was repeated for groups of children with ED and with NEF, but without obesity, in order to rule out the influence of BMI-z Gene mapping revealed putative targets of these three molecules

| Non-alcoholic fatty liver disease
Diagnosis of NAFLD is based on rating the function and condition of liver with liver-associated enzymes -ALT, AST, GGT, 48,49 or through biopsy, which provides information concerning steatosis and fibrosis of this organ. Alternative method is combining measurement of ALT/AST and elastography. 50 Adequate identification scheme for this disease is highly required, considering its rising occurrence. Recently many studies concentrated on said assessment methods 50,51 although the use of miRNA markers seems to be yet out of the scope of interest.
In the cohort of this study the greatest fold change between children with and without NAFLD occurred with miR-199a-5p (17,18), followed by third highest value of AUC parameter. There was however no significant correlation between this marker and BMI, ALT, and AST parameters. But bearing in mind its role in preadipocyte proliferation and differentiation, insulin signalling, and correlation with state of fibrosis in human liver, miR-199a-5p still poses as a strong candidate for the potential NAFLD development marker.
In the contrast, miR-122 scored the highest AUC and second highest fold-change (12.48) between the groups and was also positively related with both AST (r = 0.50) and ALT (r = 0.60). Previous studies in adults established its protective role in non-alcoholic steatohepatitis (NASH), 52 as well as in T2DM 53 and maturity onset diabetes of the young. 54 Limitation of this study is the lack of comparative analysis of the findings might be connected directly to NAFLD, the influence of obesity itself on the results cannot be ruled out.
MiR-122 was also solely investigated in study 6. in comparison to Obtaining the data from two different centres bears a risk of results being influenced by differences in methodology, used tools and kits, which must be considered a limitation of this study.
Further validation of these findings is needed to approve these markers for clinical use, which in paediatric population would be desirable for measurement of liver steatosis and fibrosis, due to its noninvasive character, as opposed to the liver biopsy. 3

| Type 2 diabetes mellitus
Obesity stimulates increase of IR, leading to the fully developed noninsulin dependent diabetes mellitus. 55 It does so by chronic low-grade inflammation of adipose tissue, thus the anti-inflammatory treatment is able to slow its progress. 56 Genetic background of this pathological process is not yet clear, and examining this field might enable prevention of the development of T2DM in children suffering from obesity.
In order to establish a profile of miRNAs involved in development of this disease, two comparative analysis of miRNA expression were performed in study 7one between children with or without obesity and second between patients with T2DM and adults with NGT. After serum miRNA sequencing, followed by RT-qPCR validation, expression levels of 8 miRNAs proved to be significantly different in children with obesity. Interestingly, the highest Pearson's r values were linked with miRNAs, whose expression increased the most -miR-486, miR-146b, and miR-15b.
In miRNA analysis in T2DM/NGT groups the same 3 miRNAs, which were most increased in children with obesity, turned out to be also the most overexpressed in patients with T2DM, when compared to the ones with NGT (p < 0.01). They were also positively correlated to FPG values (miR-486: r = 0.923; miR-146b: r = 0.882; miR-15b: r = 0.969). ROCs and AUC values were calculated for each miRNA, as well as for their combination (AUC scores given in Table 5).
These miRNAs were overexpressed in model mice with obesity and diabetes, in relation to development of these two conditions.
They were also found in heart, liver, spleen, pancreas, kidney, white fat, lung, and skeletal muscles of adult C57BL mice. MiR-486-5p added to preadipocyte culture increased their proliferation, while miR-146a-5p inhibited this process. MiR-15b had no significant effect.
In pancreatic beta-cell dysfunction assay, insulin synthesis, and secretion were impaired in groups treated with miR-146a-5p and miR-15b-    As the MetS is the early state leading to severe changes and comorbidities in organism, an adequate early detection of its development using studied molecules could enable prevention of health worsening at its very beginning.

| Insulin resistance
Despite the small size of groups in study 13 (9 patients
Interestingly, each of these molecules targets ABCA1 genes expression. After modulating macrophages with VAT-derived EVs, expression of cholesterol efflux-related genes did not differ between the cells modulated by EVs of subjects with and without obesity. Nonetheless, treatment with EVs itself led to differences in expression between experiment and control groups, as well as between the experiment groups treated with different EV concentration (3 and 1 μg/ml).
Although the study identified a panel of miRNAs significantly different levels between the subjects with high and low cholesterol efflux capacity, these results lack the correlation with BMI and obesity in subjects, which is essential in order to treat them as relevant in prediction of risk of developing atherosclerosis in children with obesity.

| CONCLUSIONS
Review of literature provided us with a good insight into the possibilities of applying miRNAs in diagnosis and prevention of obesity comorbidities.
• Results of reviewed studies indicated miR-320a as a potential marker useful in diagnosis of ED, whereas miR-630 was shown to influence the condition of endothelium, therefore posing as a strong candidate for future therapeutic strategy for ED.
• Two miRNAs: miR-199a-5p and miR-122, presented a good potential in diagnosis of NAFLD and grading its development, however further evaluations are recommended.
• Studies concerning type 2 diabetes mellitus suggested miR-486, miR-146b, and miR-15b as markers of disease progression, although the efficacy of these molecules differed between the studies (AUC: 0.88-0.98 in study 7 vs. 0.54-0.62 in study 8). MiR-122, associated with NAFLD, and miR-29a were correlated with levels of glucose metabolism parameters. They may also be involved in development of T2DM.
• MiRNAs examined in studies 10, 11, and 12 showed significant correlation with parameters of metabolic syndrome (BMI, diabetic, and lipid profile parameters), which might be used for early detection and planning adequate prevention of this disease.
• Studies 13, 14, and 15. concentrated on miRNAs significantly associated with the development of IR, suggesting the role of several miRNAs (miR-27a, À30d-5p, À122-5p, À221-3p, and À215-5p) in its pathogenesis • Research on a link between miRNAs and cholesterol efflux capacity, which is an important factor in AS development, returned no satisfying results.
Results of above studies indicate that miRNA might play an important role in diagnosis of obesity comorbidities in paediatric population, especially ED, NAFLD, and T2DM. Possibility of early detection of developing obesity-related pathologies is crucial for creation of efficient strategy aimed at slowing or even inhibiting their progression.