Putative anion transporter 1 (PAT1， SLC26A6)， an intestinal epithelial Cl/ exchanger， also plays a key role in oxalate homeostasis via mediating intestinal oxalate secretion. Indeed， Slc26a6-null mice showed defect in intestinal oxalate secretion and high incidence of kidney stones. Recent emergence of PAT-1 as a novel therapeutic target for nephrolithiasis warrants detailed understanding of the mechanisms of PAT-1 regulation in health and disease. Therefore， we investigated the regulation of PAT-1 expression by microRNAs (miRNA)， as they have been shown to play key role in modulating expression of other ion transporters. In silico analysis of PAT-1 3'-untranslated region (UTR) revealed potential b... More
Putative anion transporter 1 (PAT1， SLC26A6)， an intestinal epithelial Cl/ exchanger， also plays a key role in oxalate homeostasis via mediating intestinal oxalate secretion. Indeed， Slc26a6-null mice showed defect in intestinal oxalate secretion and high incidence of kidney stones. Recent emergence of PAT-1 as a novel therapeutic target for nephrolithiasis warrants detailed understanding of the mechanisms of PAT-1 regulation in health and disease. Therefore， we investigated the regulation of PAT-1 expression by microRNAs (miRNA)， as they have been shown to play key role in modulating expression of other ion transporters. In silico analysis of PAT-1 3'-untranslated region (UTR) revealed potential binding sites for several miRNAs， suggesting the role of miRNAs in modulating PAT1 expression. miRNAs showing highest context scores (125a-5p， 339-5p， 423-5p， 485-5p， and 501-3p) were selected as candidates for their effects on the activity of a 263-bp PAT-1 3'-untranslated region (UTR) fragment cloned into pmirGLO vector upstream of luciferase. The 3'-UTR activity was measured by dual luciferase reporter assay in Caco-2， T-84， HT-29， and SK-CO15 cells. Transient transfection of PAT-1 3'-UTR significantly decreased the relative luciferase activity compared with the empty vector suggesting binding of potential miRNA(s) to the PAT-1 3'-UTR. Among all the selected candidates， cotransfection with miRNA mimics 125a-5p and 423-5p further decreased PAT-1 3'-UTR activity. Furthermore， increasing miR-125a-5p abundance via mimic transfection in Caco-2 cells decreased both mRNA and protein levels of PAT-1. Our results demonstrate a novel regulatory mechanism of intestinal PAT-1 expression via miR-125a-5p that could be of therapeutic importance in disorders associated with decreased PAT-1 expression and function.
