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Item Type: Tesi di dottorato
Uncontrolled Keywords: celiac disease, microRNAs, Notch pathway
Date Deposited: 03 Dec 2010 10:35
Last Modified: 30 Apr 2014 19:43


Celiac disease (CD) is a chronic inflammatory disease characterized by small intestinal mucosal injury and nutrient malabsorption in genetically susceptible individuals following the dietary ingestion of gluten1. The disease is characterized by villous atrophy, intraepithelial lymphocyte infiltration, chronic inflammation and activation of lamina propria T cells. The common genetic background in CD is the presence of heterodimeric HLA class II molecules DQ2 or DQ8 that account for ~40% of the genetic predisposition in CD2; several susceptibility loci not related HLA have been identified, but their contribution is only 3–4%3. Further, in addition to DNA variations, also gene transcription regulation, such as microRNAs (miRNAs) mechanism, could be involved in CD pathogenesis. MiRNAs are small non coding RNAs regulating basic cellular functions including proliferation, differentiation and death4. Indeed, it is easy to conceive that a defective miRNA-based mRNA regulation may compromise normal cell function and cause genetic diseases. The aim of this project is to perform an extensive study of the different miRNAs expression pattern in intestinal mucosa from CD patients at different stages of the disease [17 with active CD and 9 at gluten free diet (GFD)] and from 11 controls, to investigate the role of these molecules in transcriptional regulation in CD. We detect and quantify the expression profiling of 365 mature miRNAs using TaqMan Low Density Array methodology and Comparative CT method. Expression profiling revealed that the 25% of miRNAs tested are not expressed in jejunal intestine. Among the large set of expressed microRNAs over 50% of miRNAs are expressed at similar levels in CD and in control patients whereas respectively, the 23% and 19% of miRNAs were differently expressed in active CD and in GFD patients vs controls. Among these miRNAs upregulated, miR-449a shows very high levels: 55.18±16.45 and 15.43±7.69 (RQ±SEM) in active CD and in GFD children respectively. The quantitative RT-PCR confirmed the high level of miR-449a in active CD patients vs controls (mean RQ±SEM: 2.8±0.9). The bioinformatic analysis identified several target genes of miR-449a, belonging to Notch pathway such as NOTCH1, KLF4, DLL, LEF1 and NUMBL. Notch signaling is a gatekeeper of the progenitor/stem cell compartment of the intestine5. The luciferase reporter assay confirmed that the effective, specific and functional interaction between miR-449a and NOTCH1. The quantitative RT-PCR confirmed the expression of NOTCH1 and HES1 (a target gene of NOTCH1) mRNAs in active CD patients (RQ±SEM: 3.4±1.3 and 2.2±0.6, respectively) and in GFD patients (RQ±SEM: 6.5±4.7 and 4.2±2.9, respectively). In addition, immunohistochemistry showed that NOTCH1- and HES1- positive cells were significantly fewer in CD patients vs controls. Because NOTCH1 signals interact with the WNT pathway to influence the intestinal stem cell fate, we investigated both the WNT pathway and secretory goblet cells. We showed a similar β-catenin expression in CD and in control children, suggesting that the WNT pathway is not altered in CD, and a reduced number of goblet cells indicating an altered differentiation in celiac small intestine. In conclusion, we investigated miRNA expression in celiac small intestine and detected very high miR-449a expression levels in both active CD and GFD patients. These data demonstrate an altered miRNA gene regulation in CD patients than in controls, in association with a reduced NOTCH1 pathway and with a decrease differentiation of intestinal cells towards the secretory goblet cell lineage. Our findings suggest a novel pathogenic event in CD. References: 1Meresse B. et al. Celiac disease: from oral tolerance to intestinal inflammation, autoimmunity and Lymphomagenesis. Nature 2009(2):8-23. 2Di Sabatino A. et al. Coeliac disease. The Lancet 2009 (373)1480-1493. 3Schuppan D. et al. Celiac disease: from pathogenesis to novel therapies. Gastroenterology. 2009 137(6):1912-1933. 4Hwang H-W. et al. MicroRNAs in cell proliferation, cell death, and tumorigenesis. British Journal of Cancer 2006(94):776–780. 5Radtke F. et al. From Gut Homeostasis to Cancer. Current Molecular Medicine 2006(6):275-289.

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