Intestinal epithelial cells (IECs) are exposed to the low-oxygen environment present in the lumen of the gut. These hypoxic conditions on one hand are fundamental for the survival of the commensal microbiota and, on the other hand, favor the formation of a selective semipermeable barrier, allowing IECs to transport essential nutrients/water while keeping the sterile internal compartments separated from the lumen containing commensals. The hypoxia-inducible factor (HIF) complex, which allows cells to respond and adapt to fluctuations in oxygen levels, has been described as a key regulator in maintaining IEC barrier function by regulating their tight junction integrity. In this study, we sought to better evaluate the mechanisms by which low oxygen conditions impact the barrier function of human IECs. By profiling miRNA expression in IECs under hypoxia, we identified microRNA 320a (miRNA-320a) as a novel barrier formation regulator. Using pharmacological inhibitors and short hairpin RNA-mediated silencing, we could demonstrate that expression of this microRNA (miRNA) was HIF dependent. Importantly, using overexpression and knockdown approaches of miRNA-320a, we could confirm its direct role in the regulation of barrier function in human IECs. These results reveal an important link between miRNA expression and barrier integrity, providing a novel insight into mechanisms of hypoxia-driven epithelial homeostasis.

38Intestinal epithelial cells (IECs) are exposed to the low-oxygen environment present in the 39 lumen of the gut. These hypoxic conditions are on one hand fundamental for the survival of 40 the commensal microbiota, and on the other hand, favor the formation of a selective 41 semipermeable barrier allowing IECs to transport essential nutrients/water while keeping the 42 sterile internal compartments separated from the lumen containing commensals. The 43 hypoxia-inducible factor (HIF) complex, which allows cells to respond and adapt to fluctuations 44 in oxygen levels, has been described as a key regulator in maintaining IEC barrier function by 45 regulating their tight junction integrity. In this study, we sought to better evaluate the 46 mechanisms by which low oxygen conditions impact the barrier function of human IECs. By 47 profiling miRNA expression in IECs under hypoxia, we identified miRNA-320a as a novel barrier 48 formation regulator. Using pharmacological inhibitors and short hairpin RNA-mediated 49 silencing we could demonstrate that expression of this miRNA was HIF-dependent. 50 Importantly, using over-expression and knock-down approaches of miRNA-320a we could 51 confirm its direct role in the regulation of barrier functions in human IECs. These results reveal 52 an important link between miRNA expression and barrier integrity, providing a novel insight 53 into mechanisms of hypoxia-driven epithelial homeostasis. 54