6 MicroRNA Mir-625 is a Negative Regulator of Human Apical Sodium Dependent Bile Acid Transporter (ASBT)

Abstract

Apical Sodium-Dependent Bile Acid Transporter (ASBT) is responsible for active absorption of bile acids mainly in the ileum and is a central modulator of body cholesterol homeostasis. Inhibition of intestinal bile acid absorption reduces high levels of plasma cholesterol associated with diseases such as diabetes mellitus. Recent clinical trials have demonstrated that blockers of bile acid absorption improve lipid and cholesterol profiles in diabetic patients. Thus, specific inhibitors of ASBT may prove to be more effective therapeutic targets to lower plasma cholesterol. It is therefore, critical to delineate molecular and cellular mechanisms involved in ASBT regulation to develop better therapeuticmodalities for treatment of hypercholesterolemia. Recent studies have reported that 3'-untranslated region (3'-UTR) of ASBT is critical for regulation of ASBT expression. However, the role of micro RNAs (miRNAs) in the regulation of ASBT expression is not known. Using in silico computational target prediction, we initially identified the putative binding site for miR-625 in the 3'UTR of human ASBT mRNA (255bp-251bp downstream of the stop codon) with a context score percentile of 99. Luciferase assays were performed in human intestinal HUTU-80 and Caco2 cells as well as in kidney HEK293 cells. Mimic of miR-625 was transiently transfected in the cells along with a dual luciferase reporter construct (pMIR-GLO vector) containing human ASBT 3'-UTR sequence. The 3'-UTR of ASBT constructs demonstrated a significant decrease in relative luciferase activity as compared to empty vector in HUTU-80 (42.47 ± 1.5%, P<0.01), Caco2 (48.64 ± 2.7%, P<0.01) and HEK293 cells (70.30 ± 4.5%, P<0.01). These data showed that 3'-UTR is important regulator of ASBT. The validity of miR-625 as a negative regulator of ASBT expression was evident by co-transfection with mimic of miR-625 that resulted in a significant decrease in luciferase activity in HUTU-80 (57.54 ± 5.2%, P<0.01), Caco2 (80.89 ± 3.1%, P<0.01) and HEK 293 (70.82 ± 3.6%, P<0.01) cells as compared to cells co-transfected with negative control mimic. To identify the putative binding site of miR-625, we generated three ASBT luciferase constructs: 3'-UTR2 (1476bp), UTR3 (753bp) and UTR4 (306bp). Overexpression of miR-625 equally inhibited the luciferase activity of the three deletion constructs (UTR2: 71.06 ± 6.6%, P< 0.01; UTR3: 70.04 ± 5.1%, P<0.01; UTR4: 60.67 ± 3.9%, P<0.01), reflecting the in in silico that miR-625 binding site is present in the smallest 3'-UTR region (UTR4). Taken together, our results show that miR-625 is a novel specific negative regulator of human ASBT. These findings highlight the use of miRNAs as inhibitors of ASBT expression that could be used as potential therapeutic tools for the treatment of hypercholesterolemia. (Supported by NIDDK & VA)