Hypoxia inducible microRNA-155 negatively regulates epithelial barrier in Eosinophilic Esophagitis by suppressing Tight Junction Claudin-7
MicroRNA (miRNA)-mediated mRNA regulation directs many homeostatic and pathological processes, but how miRNAs coordinate aberrant esophageal inflammation during eosinophilic esophagitis (EoE) is poorly understood. Here, we report a deregulatory axis where microRNA-155 (miR-155) regulates epithelial barrier dysfunction by selectively constraining tight junction CLDN7 (claudin-7). MiR-155 is elevated in the esophageal epithelium of biopsies from patients with active EoE and in cell culture models. miR-155 localisation using in situ hybridisation (ISH) in patient biopsies, and intra-epithelial compartmentalisation of miR-155 shows expression predominantly within the basal epithelia. Epithelial miR-155 activity was evident through diminished target gene expression in 3D organotypic cultures, particularly in relatively undifferentiated basal cell states. Mechanistically, generation of a novel cell line with enhanced epithelial miR-155 stable overexpression induced a functionally deficient epithelial barrier in 3D air-liquid interface epithelial cultures measured by transepithelial electrical resistance (TEER). Histological assessment of 3D esophageal organoid cultures overexpressing miR-155 showed notable dilated intra-epithelial spaces. Unbiased RNA-sequencing analysis and immunofluorescence determined a defect in epithelial barrier tight junctions and revealed a selective reduction in the expression of critical esophageal tight junction molecule, claudin-7. Together, our data reveal a previously unappreciated role for miR-155 in mediating epithelial barrier dysfunction in esophageal inflammation. Overall design: Esophageal epithelial cells (EPC2-hTERTs) were transfected via lentiviral-mediated overexpression of miR-155-5p to genrated a stable overexpressing miR-155-5p cell line. miR-155-5p overexpressing (miR-155OE) and control (CTRL) cells were cultured in physiologically relevant 3D-ALI cultures to investigate the function of miR-155-5p overexpression on epithelial barrier and overall epithelial biology. RNA-seq was carried out using these miR-155OE and CTRL cells. Comparative gene expression profiling of RNA-seq data for miR-155OE and CTRL cells was conducted.