Circulating miRNAs Are Associated with Inflammation Biomarkers in Children with Overweight and Obesity: Results of the I.Family Study.

Increasing data suggest that overnutrition-induced obesity may trigger an inflammatory process in adipose tissue and upturn in the innate immune system. Numerous players have been involved in governing the inflammatory response, including epigenetics. Among epigenetic players, miRNAs are emerging as crucial regulators of immune cell development, immune responses, autoimmunity, and inflammation. In this study, we aimed at identifying the involvement of candidate miRNAs in relation to inflammation-associated biomarkers in a subsample of European children with overweight and obesity participating in the I.Family study. The study sample included individuals with increased adiposity since this condition contributes to the early occurrence of chronic low-grade inflammation. We focused on the acute-phase reagent C-reactive protein (CRP) as the primary outcome and selected cytokines as plausible biomarkers of inflammation. We found that chronic low-grade CRP elevation shows a highly significant association with miR-26b-3p and hsa-miR-576-5p in boys. Furthermore, the association of CRP with hsa-miR-10b-5p and hsa-miR-31-5p is highly significant in girls. We also observed major sex-related associations of candidate miRNAs with selected cytokines. Except for IL-6, a significant association of hsa-miR-26b-3p and hsa-miR-576-5p with TNF-α, IL1-Ra, IL-8, and IL-15 levels was found exclusively in boys. The findings of this exploratory study suggest sex differences in the association of circulating miRNAs with inflammatory response biomarkers, and indicate a possible role of miRNAs among the candidate epigenetic mechanisms related to the process of low-grade inflammation in childhood obesity.

The association of circulating miR-191 and miR-375 expression levels with markers of insulin resistance in overweight children: an exploratory analysis of the I.Family Study.

BACKGROUND: In recent years, the exciting emergence of circulating miRNAs as stable, reproducible, and consistent among individuals has opened a promising research opportunity for the detection of non-invasive biomarkers. A firm connection has been established between circulating miRNAs and glycaemic as well as metabolic homeostasis, showing that levels of specific miRNAs vary under different physio-pathological conditions. OBJECTIVE: In this pilot study, we investigated the expression of candidate miRNAs, hsa-miR-191-3p and hsa-miR-375, in relation to biomarkers associated with insulin sensitivity in a subgroup (n=58) of subjects participating to the European I.Family Study, a project aimed to assess the determinants of eating behaviour in children and adolescents and related health outcomes. The sample included overweight/obese children/adolescents since overweight/obesity is a known risk factor for impaired glucose homeostasis and metabolic disorders. Biological targets of candidate miRNAs were also explored in silico. RESULTS: We observed a significant association of the two miRNAs and early changes in glycaemic homeostasis, independent of covariates including country of origin, age, BMI z-score, puberty status, highest educational level of parents, total energy intake, energy from fats, energy from carbohydrates, and energy from proteins. CONCLUSION: Identification of circulating miRNAs associated with insulin impairment may offer novel approaches of assessing early variations in insulin sensitivity and provide evidence about the molecular mechanisms connected to early changes in glycaemic homeostasis. TRIAL REGISTRATION: ISRCTN, ISRCTN62310987. Retrospectively registered, http://isrctn.com/ISRCTN62310987.

Circulating miRNAs are associated with sleep duration in children/adolescents: Results of the I.Family Study.

NEW FINDINGS: What is the central question of this study? Are differential patterns of circulating miRNAs associated with sleep duration in normal-weight European children and adolescents? What is the main finding and its importance? Differences in the expression level of circulating miR-26b-3p and miR-485-5p are positively associated with total sleep duration in healthy normal-weight children and adolescents. ABSTRACT: It is commonly recognized that sleep is essential for children's health, and that insufficient sleep duration is associated with negative health outcomes. In humans, sleep duration and quality are influenced by genetic, environmental and social factors. Epigenetic mechanisms, likewise, regulate circadian rhythms and sleep patterns. In the present study, we aimed to identify circulating microRNAs associated with sleep duration in a subsample of normal-weight European children/adolescents (n = 111) participating in the I.Family Study. Subjects were divided into two groups based upon self-reported sleep duration, according to the recommended amount of sleep for paediatric populations. Sleep needs for children <13 years were at least 9 h per day, and for children >13 were at least 8 h per day. There were group differences (short sleepers versus normal sleepers) in circulating levels of miR-26b-3p (mean (95% CI) = 2.0 (1.3-2.7) versus 2.3 (1.9-2.7), P = 0.05) and miR-485-5p (mean (95% CI) = 0.6 (0.3-0.9) versus 0.9 (0.7 - 1.0), P < 0.001), adjusting for country of origin, age, sex, pubertal status, screen time and highest educational level of parents. Our findings show for the first time that sleep duration reflects the profile of specific circulating microRNAs in school-aged children and adolescents. It is conceivable that epigenetic modifications, mainly related to circadian rhythm control, may be modulated or interfere with sleep duration.

Circulating microRNAs are associated with early childhood obesity: results of the I.Family Study.

BACKGROUND: Nearly 10 years ago, the World Health Organization reported the increasing prevalence of overweight and obesity worldwide as a challenge for public health due to the associated adverse consequences. Epidemiological studies established a firm relationship between an elevated body mass index and chronic conditions such as diabetes, dyslipidemia, hypertension, heart disease, non-alcoholic fatty liver disease, and some types of cancer. Omic studies demonstrated that microRNA (miRNA) profile changes in tissues correlate with a number of diseases, including obesity. Recent studies showed a remarkable stability of miRNAs also in blood, emphasizing their potential as theranostic agents for a variety of disorders and conditions. A number of miRNAs enriched in homeostasis of obesity and metabolic disorders have been characterized in previous researches. AIM: This work was finalized to investigate the differential circulating miRNAs signature in early childhood obesity. Our cross-sectional study analyzed the signature of circulating miRNAs in plasma samples of normal weight (n = 159) and overweight/obese (n = 149) children and adolescents participating to the I.Family study, an EC-funded study finalized to investigate the etiology of overweight, obesity and related disorders and the determinants of food choice, lifestyle, and related health outcomes in children and adolescents of eight European countries (www.ifamilystudy.eu). RESULTS: Differences in miRNA signature with respect to anthropometric and biochemical variables were analyzed. A high degree of variability in levels of circulating miRNAs was identified among children from different countries, in line with recent reports supporting the hypothesis that these molecules are likewise affected by environmental and lifestyle factors. A panel of miRNAs differentially expressed in overweight/low-grade obesity children was characterized (miR-551a and miR-501-5p resulted upregulated; miR-10b-5p, miR-191-3p, miR-215-5p, and miR-874-3p resulted downregulated). ROC curves were also constructed for experimentally confirmed miRNAs. Single miRNAs generally exhibited low AUC values with the highest values for miR-874-3p and miR-501-5p which in combination provided an interesting value (AUC = 0.782). Pearson's analysis confirmed that miR-10b-5p, miR-215-5p, miR-501-5p, miR-551a, and miR-874-3p significantly correlated with BMI z-score. Molecular interactions of obesity-associated miRNAs were also predicted by bioinformatics tools. CONCLUSIONS: Our work showed that several circulating miRNAs are differentially represented in overweight/low-grade obesity children and adolescents. Although causal pathways cannot be firmly inferred, it is conceivable that circulating miRNAs may be new biomarkers of early childhood obesity. TRIAL REGISTRATION: ISRCTN, ISRCTN62310987. Registered 23/02/2018 - Retrospectively registered.

Laser Capture Microdissection as a Tool to Study the Mucosal Immune Response in Celiac Disease.

Laser capture microdissection (LCM) is a powerful tool for selection and isolation of single cells or compartments from complex primary tissues to perform molecular analyses. Celiac disease is a genetic autoimmune disorder where the ingestion of gluten leads to damage in the small intestine. Increased intraepithelial lymphocytes and the presence of the lamina propria inflammatory infiltrate of the duodenal mucosa is a common part of the disease. These cells promote inflammatory processes through the release of cytokines. Here, we describe the use of LCM and real-time quantitative PCR (RT-qPCR) to analyze cytokine profile information in distinct duodenal mucosa tissue compartments of celiac patients.

Urinary volatile organic compounds in overweight compared to normal-weight children: results from the Italian I.Family cohort.

Accumulating evidence shows that urinary volatile organic compounds (VOCs) could be perturbed in many physiological and pathological states, including several diseases and different dietary exposures. Few studies investigated the urinary metabolic signature associated to excess body weight and obesity in adult populations, while a different VOCs profile was found in exhaled breath in obese as compared to lean children. Aim of this study was to evaluate the VOCs profile in the urine of 21 overweight/obese (OW/Ob) and 28 normal-weight (NW) children belonging to the Italian cohort of the I. Family study. Urine samples were analysed by Solid Phase Micro-Extraction (SPME) GC-MS under both acidic and alkaline conditions, in order to profile a wider range of urinary volatiles with different physicochemical properties. Multivariate statistics techniques were applied to bioanalytical data to visualize clusters of cases and detect the VOCs able to differentiate OW/Ob from NW children. Under alkaline conditions, fourteen VOCs were identified, distinguishing OW/Ob from NW children. Our results suggest that VOCs signatures differ between OW/Ob and NW children. However, the biological and pathophysiological meaning of the observed differences needs to be elucidated, in order to better understand the potential of urinary VOCs as early metabolic biomarkers of obesity.

Circulating microRNAs are deregulated in overweight/obese children: preliminary results of the I.Family study.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs involved in the modulation of gene expression and in the control of numerous cell functions. Alterations of miRNA patterns frequently occur in cancer and metabolic disorders, including obesity. Recent studies showed remarkable stability of miRNAs in both plasma and serum making them suitable as potential circulating biomarkers for a variety of diseases and conditions. The aim of this study was to assess the profile of circulating miRNAs expressed in plasma samples of overweight or obese (OW/Ob) and normal weight (NW) prepubertal children from a European cohort (www.ifamilystudy.eu). The project, aimed to assess the determinants of eating behavior in children and adolescents of eight European countries, is built on the IDEFICS cohort (www.ideficsstudy.eu), established in 2006. Among the participants of the I.Family Italian Cohort, ten OW/Ob (age 10.7 ± 1.5 years, BMI 31.6 ± 4.3 kg/m(2)) and ten NW (age 10.5 ± 2.7 years, BMI 16.4 ± 1.7 kg/m(2)) children were selected for the study. Gene arrays were employed to differentially screen the expression of 372 miRNAs in pooled plasma samples. Deregulated miRNAs (p < 0.05) were further validated in the individual samples using a real-time PCR (RT-qPCR) approach. RESULTS: Using a significance threshold of p < 0.05 and a fold-change threshold of ± 4.0, we preliminarily identified in the pooled samples eight miRNAs that differed between the OW/Ob and NW groups. The validation by RT-qPCR in the individual plasma samples showed a twofold upregulation of miR-31-5p, a threefold upregulation of miR-2355-5p, and a 0.5-fold downregulation of miR-206 in OW/Ob as compared with NW. The molecular functions of these differentially expressed plasma miRNAs as well as their expected mRNA targets were predicted by bioinformatics tools. CONCLUSIONS: This pilot study shows that three circulating miRNAs are differentially regulated in OW/Ob as compared with NW children. Although causal pathways cannot be firmly inferred by these results, that deserve confirmation in larger samples, it is conceivable that circulating miRNAs may be novel biomarkers of obesity and related metabolic disturbances.