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 a key role for BMP4/miR-181/glycolysis signaling pathway in the maintenance of intestinal epithelial cell proliferation and differentiation. 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. Moreover, BMP4 treatment or inhibition of miR-181a-5p repressed HK1 expression and inhibited glycolysis. In line of this, knockdown of HK1 or inhibition of glycolysis using 2-DG promoted enterocyte maturation and inhibited proliferation of IECs. Together, we provide evidence showing that miR-181a-5p inhibits intestinal enterocyte differentiation and promotes IEC proliferation thorough HK1-dependent glycolysis. Importantly, our findings identified miR-181a-5p as downstream in mediating BMP4 induction of enterocyte differentiation and inhibition of proliferation in IECs. To determine downstream molecules in mediating the effects of BMP4 on intestinal cell proliferation and differentiation, we treated mouse intestinal organoids with BMP4 for 4 days. We performed miRNA expression profiling analysis using data obtained from miRNA-seq of mouse intestinal organoids treated with PBS control or BMP4.