miR-181a-5p mediates the effects of BMP4 on intestinal cell proliferation and differentiation

The intestinal mucosa undergoes a dynamic process of continual proliferation, differentiation and apoptosis. Delineating the mechanisms involved in intestinal epithelial cell (IEC) differentiation is crucial to our understanding of not only normal gut adaptation but also aberrant intestinal growth. BMP signaling is a crucial regulator of intestinal proliferation and differentiation. However, the molecular underpinnings of the BMP pathway in this context are not entirely known. Here we showed that treatment with BMP4 increased expression of enterocyte markers and decreased proliferation of IECs, and importantly, decreased the expression of miR-181a-5p in mouse and human intestinal organoids. Inhibition of miR-181a-5p, a member of miR-181 with highest expression in intestinal cells, significantly increased enterocyte differentiation as noted by increased expression of enterocyte markers in human and mouse intestinal organoids. In addition, miR-181a-5p miRCURY LNA inhibitor repressed the expression of endogenous miR-181a-5p and decreased intestinal stem cell self-renewal as noted by the decreased expression of Ki67, cyclin D1 and OLFM4 and organoid forming efficiency. In contrast, overexpression of miR-181a-5p mimics decreased the expression of enterocyte markers. Together, we provide evidence showing that miR-181a-5p inhibits intestinal enterocyte differentiation and promotes IEC proliferation. Overall design: To determine downstream molecules in mediating the effects of mir-181a-5p on intestinal cell proliferation and differentiation, mouse small intestinal organoids were infected with lentivirus expressing miR-181a-5p mimic (MLMIR0085, Sigma) or miRNA mimic control (NCLMIR002, Sigma). The infected organoids were cultured in the presence of puromycin (2 ug/ml) to select for organoids with stable overexpression of miR-181a-5p.