Role of microRNA miR-122-5p in post-transcriptional regulation of the mitochondrial thiamin pyrophosphate transporter (MTPPT; SLC25A19) in pancreatic acinar cells (PACs)

Abstract

Thiamin (vitamin B1; also known as the “energy vitamin”) plays a critical role in normal energy metabolism and ATP production. Like all other human/mammalian cells, pancreatic acinar cells (PACs) cannot synthesize thiamin, and thus, must obtain the vitamin from circulation. Internalized free thiamin is then converted to TPP in the cytoplasm and transported into mitochondria for utilization via a carrier-mediated process that is mediated by the MTPPT (product of the SLC25A19 gene). Previous studies from our laboratory have characterized different aspects of the mitochondrial TPP uptake process in PACs; we have also delineated aspects of the MTPPT transcriptional regulation. To date, however, there has been nothing known about post-transcriptional regulation of MTPPT expression and function in PACs. We began to address these issues and, in this study, we examined the potential role of miRNAs in this regulation. First, we subjected the 458 bp long hMTPPT-3' untranslated region (UTR) to different in silico analysis (namely: miRDB, Targetscan, and DIANA-microTCDS), which identified multiple putative miRNA binding sites (including sites for miR-122-5p) in this region. These putative miRNA binding sites were found to be conserved in the human and rat MTPPTs. We then examined the effect of transfecting pmirGLO-hMTPPT-3'UTR into PAC (rat-derived AR42J cells) and observed a significant reduction in luciferase activity compared with cells transfected with pmirGLO empty vector. Focusing on the putative miR-122-5p (since it is expressed in normal human pancreatic cells and that its level is induced in pancreatitis), we found that transfecting AR42J cells as well as human native primary PACs (obtained from normal human organ donors) with a mimic of miR-122-5p to lead to a significant inhibition in level of MTPPT mRNA and protein expression as well as in TPP uptake by isolated mitochondria. On the other hand, inhibiting the miR-122-5p with the use of miR-122-5p inhibitor was found to lead to an increase in MTPPT mRNA and protein expression as well as in TPP uptake by isolated mitochondria. Additionally, truncating the region that carries the putative miR-122-5p binding site(s) in the 3'UTR of MTPPT as well as mutating those sites were found to lead to an abrogation in the inhibitory effect of this miRNA on luciferase activity in PACs. In conclusion, our results demonstrate, for the first time, that expression and function of the MTPPT are subject to post-transcriptional regulation by miR-122-5p. Supported by NIH grants AA-018071 & DK-56061, and VA grants 101BX001142 & IK6BX006189 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.