Since we found miR b p to be

Since we found miR-15b-5p to be differentially expressed along the porcine intestine in 31 day old piglets in a former study [10], the entire miRNA family was analyzed in terms of interaction with potential target-genes. In silico analysis predicted miR-15 family members to target the hedgehog signaling peptide IHH revealing high conservation of binding within mammals. This fact increases the significance of the predicted interaction. In accordance with in silico prediction, performance of luciferase reporter gene assays, an established method to evaluate the miRNA target-sites [18], revealed downregulation of IHH by miRNAs miR-195, miR-15b, miR-424 and miR-497-5p. These data were supported by data obtained from mRNA degradation assays using the human intestinal cell line HT-29. Thus, data from in silico and in vitro experiments demonstrated a regulation of IHH through members of the miR-15 family. Data about rora and regulation of the hedgehog signaling pathway in the porcine gut is scarce. Therefore, tissue samples of 7–56 days old piglets were examined regarding expression and abundance of hedgehog molecules and corresponding miR-15 family members. Quantification of mRNA from the ileum and colon revealed a downregulation of IHH, SHH and DHH over the time. This trend was being observed for nearly all 11 analyzed hedgehog signaling factors. Expression of the miR-15 family in the porcine colon was analyzed in parallel to mRNA expression of hedgehog factors. MiRNAs miR-15a/b-5p and miR-195-5p exhibited matched and anticorrelated expression compared with the hedgehog ligands underlining the hypothesis, in vitro data and previous results that miR-15 family members regulate hedgehog signaling. Interestingly, miR-15a/b-5p was downregulated during the weaning period (day 28–31) and increased again between day 31–56. This is in accordance with observations made by our group earlier [10] which showed that miR-15b is only slightly expressed in colon during weaning compared to its expression in the ileum and jejunum. However, after weaning it increased again suggesting a correlation between weaning and expression of miR-15. Taken together, our approach based on paralleled in vivo expression analysis of miRNA and mRNA showed a clear anticorrelation supporting in vitro data. As a consequence, we assume that downregulation of hedgehog signaling peptides by the miR-15 family members leads to diminished maturation of enterocytes and higher proliferation. After an obvious downregulation of hedgehog signaling, we were able to observe a static phase between day 31–35 and day 35–56 which might facilitate the physiological homeostasis of cell proliferation and differentiation during weaning. The porcine intestine is an important model for the study of postnatal development of mammalian intestine. MiRNAs are promising candidates for diagnostic and therapeutic applications [19] as dysregulation of hedgehog signaling has been linked to intestinal cancer and celiac disease [2,3]. Results presented in this study promote improved understanding of the complex regulation of hedgehog signaling during intestinal development and disease.
Acknowledgement
Immune cells in the intestinal lamina propria are in a state of basal tolerance. In response to epithelial damage, a switch to an activated status is required to limit the consequences of exposure to potentially dangerous luminal content. This control is maintained in part by the immune system itself and in part by the epithelial tissue. The intact epithelium provides factors that mediate immune suppression under steady-state conditions. Upon tissue damage, the loss of epithelial cells results in loss of these factors and relief of the active immunosuppression. Several epithelium-derived factors have been identified that can suppress the mucosal immune response, including thymic stromal lymphopoietin, transforming growth factor-β, semaphorin 7a, and interleukin 25 (IL25)., , , These factors mainly exert their effects through their influence on the development of dendritic cells and macrophages with tolerogenic properties such as production of IL10 or inhibition of IL17 and tumor necrosis factor-α.