Identification of Autophagy-Related Genes and Their Regulatory miRNAs Associated with Celiac Disease in Children

Abstract

Celiac disease (CD) is a severe genetic autoimmune disorder, affecting about one in 100 people, where the ingestion of gluten leads to damage in the small intestine. Diagnosing CD is quite complex and requires blood tests and intestinal biopsy examinations. Controversy exists regarding making the diagnosis without biopsy, due to the large spectrum of manifesting symptoms; furthermore, small-intestinal gastroscopy examinations have a relatively complex management in the pediatric population. To identify novel molecular markers useful to increase the sensitivity and specificity in the diagnosis of pediatric CD patients, the expression levels of two key autophagy executor genes (ATG7 and BECN1) and their regulatory validated miRNAs (miR-17 and miR-30a, respectively) were analyzed by relative quantitative real-time-PCR on a cohort of confirmed CD patients compared to age-related controls. Among the investigated targets, the non-parametric Mann–Whitney U test and ROC analysis indicated the highest significant association of BECN1 with CD status in the blood, while in intestinal biopsies, all of the investigated sequences were positively associated with CD diagnosis. Nomogram-based analysis showed nearly opposite expression trends in blood compared to intestine tissue, while hierarchical clustering dendrograms enabled identifying CD and control subgroups based on specific genes and miRNA expression signatures. Next, using an established in vitro approach, through digested gliadin administration in Caco-2 cells, we also highlighted that the modulation of miR-17 endogenous levels using enriched exosomes increased the intracellular autophagosome content, thereby altering the autophagic status. Altogether, these results highlighted novel molecular markers that might be useful to increase the accuracy in CD diagnosis and in molecular-based stratification of the patients, further reinforcing the functional involvement of the regulation of the autophagy process within a digestive and autoimmune-related disorder as CD.

Autophagy balances inflammation in innate immunity

“…10 Other connections have been reported between several autophagy loci and genetic predispositions for chronic inflammatory disorders and autoimmune diseases. These include systemic lupus erythematosus (IRGM, 11 ATG5, 12 PRDM1-ATG5 13 ; and DRAM1 14 ), asthma (ATG5 15 ), rheumatoid arthritis (albeit somewhat marginally significant ATG5 rs6568431 association 16 ), Vici syndrome (EPG5 17 ), celiac disease 18 and other conditions (CLEC16A, ULK3, and MIR's that target autophagy genes), [18][19][20][21] multiple sclerosis (CLEC16A 21 ), and other neurological disorders, including amyotrophic lateral sclerosis and frontotemporal dementia, especially in patients with stronger neuroinflammatory components (where connections to autophagy have been made through mutations in TBK1 22 ). CLEC16A is also genetically linked to type 1 diabetes mellitus, a condition associated with immune infiltration, and plays a role in mitophagy and autolysosome function.…”

Intestinal and Circulating MicroRNAs in Coeliac Disease

“…In the small intestine of children with CD, an increased expression of β-catenin has been observed, which suggests an enhanced cellular proliferation [ 14 ]. In digestive and autoimmune-related disorders such as CD, the regulation of the autophagy process might be also involved, as indicated by the decreased expression of miR-17 and miR-30a [ 18 ]. Accordingly, bioinformatics analyses show an involvement of miRNAs both in the transcription of Wnt target genes and in the positive regulation of cell proliferation ( Table 3 ).…”

“…In the last few years many studies focused the attention on the expression profiles of miRNAs in human small intestine of patients affected by CD, in the attempt to understand the mechanisms implicated in CD through the investigation of their mRNA targets [ 14 , 15 , 16 , 17 , 18 ]. The expression patterns of miRNAs in the small intestine of a cohort ( n = 40) of 20 children with active CD, nine on a gluten-free diet (GFD), and 11 controls have been investigated [ 14 ].…”

“…Quantitative PCR was performed on blood samples and intestinal biopsies derived from pediatric CD patients, and results compared with controls. The analysis highlighted the association of autophagy-related genes and miRNAs with CD condition [ 18 ] outlining the importance and involvement of autophagy processes in the CD pathogenesis.…”

The Emerging Role of the Autophagy Process in Children with Celiac Disease: Current Status and Research Perspectives

“…Comincini and collaborators [35] have recently tackled a different question regarding celiac disease, i.e., the possibility to identify novel molecular markers in order to increase the sensitivity and specificity in the diagnosis of pediatric celiac disease patients. To this end, the expression levels of two key autophagy executor genes (ATG7 and BECN1) and their regulatory validated miRNAs (miR-17 and miR-30a, respectively) were analyzed by relative quantitative real-time PCR on a cohort of confirmed celiac patients compared to age-related controls, analyzing peripheral blood, and corresponding duodenal specimens.…”