Role of MicroRNA-423-5p in posttranscriptional regulation of the intestinal riboflavin transporter-3.

Riboflavin (RF) is essential for normal cellular functions and health. Humans obtain RF from exogenous sources via intestinal absorption that involves a highly specific carrier-mediated process. We have recently established that the riboflavin transporter-3 (RFVT3) is vital for the normal intestinal RF uptake process and have characterized certain aspects of its transcriptional regulation. Little is known, however, about how this transporter is regulated at the posttranscriptional level. We address this issue by focusing on the role of microRNAs. Using bioinformatics, we identified two potential interacting miRNAs with the human (h) RFVT3-3'-UTR, and showed (using pmirGLO-hRFVT3-3'-UTR) that the hRFVT3-3'-UTR is, indeed, a target for miRNA effect. Of the two putative miRNAs identified, miR-423-5p was found to be highly expressed in intestinal epithelial cells and that its mimic affected luciferase reporter activity of the pmirGLO-hRFVT3-3'-UTR construct, and also led to inhibition in RF uptake by intestinal epithelial Caco-2 and HuTu-80 cells. Furthermore, cells transfected with mutated seed sequences for miR-423-5p showed an abrogation in inhibitory effect of the miR-423-5p mimic on luciferase activity. While miR-423-5p did not affect the level of expression of the hRFVT3 mRNA, it did lead to a significant inhibition in the level of expression of its protein. Similarly, miR-423-5p was found to affect the level of expression of the mouse RFVT3 in cultured intestinal enteroids. These findings demonstrate, for the first time, that the RFVT3 is a target for posttranscriptional regulation by miRNAs in intestinal epithelial cells and that this regulation has functional consequences on intestinal RF uptake.NEW & NOTEWORTHY Our findings show for the first time that RFVT3 is a target for posttranscriptional regulation by miR-423-5p in intestinal epithelial cells, and this regulation has functional consequences on intestinal riboflavin (RF) uptake process.