miR-122-5p is involved in posttranscriptional regulation of the mitochondrial thiamin pyrophosphate transporter (SLC25A19) in pancreatic acinar cells

Abstract

Thiamin (vitamin B1) plays a vital role in cellular energy metabolism/ATP production. Pancreatic acinar cells (PACs) obtain thiamin from circulation and convert it to thiamin pyrophosphate (TPP) in the cytoplasm. TPP is then taken up by the mitochondria via a carrier-mediated process that involves the mitochondrial TPP transporter (MTPPT; encoded by the gene SLC25A19). We have previously characterized different aspects of the mitochondrial carrier-mediated TPP uptake process, but nothing is known about its possible regulation at the posttranscriptional level. We address this issue in the current investigations focusing on the role of miRNAs in this regulation. First, we subjected the human (and rat) 3'-untranslated region (3'-UTR) of the SLC25A19 to three in-silico programs, and all have identified putative binding sites for miR-122-5p. Transfecting pmirGLO-hSLC25A19 3'-UTR into rat PAC AR42J resulted in a significant reduction in luciferase activity compared with cells transfected with pmirGLO-empty vector. Mutating as well as truncating the putative miR-122-5p binding sites in the hSLC25A19 3'-UTR led to abrogation of inhibition in luciferase activity in PAC AR42J. Furthermore, transfecting/transducing PAC AR42J and human primary PACs with mimic of miR-122-5p led to a significant inhibition in the level of expression of the MTPPT mRNA and protein as well as in mitochondrial carrier-mediated TPP uptake. Conversely, transfecting PAC AR42J with an inhibitor of miR-122-5p increased MTPPT expression and function. These findings show, for the first time, that expression and function of the MTPPT in PACs are subject to posttranscriptional regulation by miR-122-5p.NEW & NOTEWORTHY This study shows that the expression and function of mitochondrial TPP transporter (MTPPT) are subject to posttranscriptional regulation by miRNA-122-5p in pancreatic acinar cells.

Keywords: SLC25A19; miRNA; mitochondrial TPP transporter; posttranscriptional regulation.

Graphical abstract

Figure 1.

Prediction and localization of putative miRNA binding sites in the 3′-UTRs of SLC25A19. miRNA binding sites in human (A) and rat SLC25A19 3′-UTRs (B) as predicted by TargetScan (the depicted figure is based on http://www.targetscan.org).

Figure 2.

Determination of luciferase activity of pmirGLO-hSLC25A19 3′-UTR in PAC AR42J. Schematic representation of human SLC25A19 gene showing the 3′-UTR (458 bp) cloned in pmirGLO vector (A). PAC AR42J were transiently transfected with pmirGLO vector or with pmirGLO-hSLC25A19 3′-UTR constructs for 48 h (B). Activity of luciferase was then determined then normalized relative to Renilla luciferase activity. Data were expressed as percentage relative to control (pmirGLO vector). Data are means ± SE of 3–5 independent determinations (*P < 0.01).

Figure 3.

Effect of mutating the putative binding sites for miR-122-5p in the hSLC25A19 3′-UTR on luciferase activity in PAC AR42J. Cells were transfected with either pmirGLO empty vector or with pmirGLO-hSLC25A19 3′-UTR wild-type (WT) and mutated constructs (S1, S2, S1 and S2; see Methods) for 48 h. Activity of luciferase was then determined and normalized relative to Renilla luciferase activity. Data are means ± SE of four independent determinations (*P < 0.01).

Figure 4.

Effect of truncating the putative binding sites for miR-122-5p in the hSLC25A19 3′-UTR on luciferase activity in PAC AR42J. Schematic representation of different hSLC25A19 3′-UTR constructs [PCR amplified and cloned (see methods)] in pmirGLO vector (A). PAC AR42J were transfected with pmirGLO vector or with pmirGLO [full-length and truncated constructs (298 bp and 145 bp)] for 48 h (B). Activity of luciferase was then determined and normalized relative to Renilla luciferase activity. Data are means ± SE of 3–4 independent determinations (*P < 0.01). NS, not significant.

Figure 5.

Effect of transfection of PAC AR42J with mimic miR-122-5p on MTPPT expressions and on mitochondrial carrier-mediated TPP uptake. Cells were transfected with mimic miRNAs [mimic negative control or miR-122-5p (200 nM; 48 h)] followed by determination of level of expression of MTPPT mRNA (by RT-qPCR) (A) and protein (by Western blotting) (B) as well as mitochondrial carrier-mediated uptake of [³H]TPP (C). Data of RT-qPCR and Western blotting were normalized relative to internal controls (18S rRNA and PDH, respectively). Data are means ± SE of 3–4 independent determinations (**P < 0.01; *P < 0.05).

Figure 6.

Effect on MTPPT expressions and mitochondrial carrier-mediated TPP uptake of transducing hPACs with lentiviral miR-122-5p mimic. Mimic miRNAs (lenti-control or lenti-122-5p, 48 h) were transduced into hPACs, followed by determination of MTPPT mRNA (by RT-qPCR) (A) and protein (Western blotting) expression (B), as well as mitochondrial carrier-mediated uptake of [³H]TPP (C). Data of RT-qPCR and Western blotting were normalized relative to internal controls (β-actin and PDH, respectively). Data are means ± SE of 3–5 independent determinations (**P < 0.01, *P < 0.05).

Figure. 7.

Effect on MTPPT expressions and mitochondrial carrier-mediated TPP uptake of transfecting PAC AR42J with miR-122-5p inhibitor. Negative (control) inhibitor or miR-122-5p inhibitor (200 nM; 48 h) were transfected into PAC AR42J followed by determination of MTPPT mRNA (by RT-qPCR) (A) and protein (Western blotting) expression (B), as well as mitochondrial carrier-mediated uptake of [³H]TPP (C). Data of RT-qPCR and Western blotting were normalized with internal controls of 18S rRNA and PDH, respectively. Data are means ± SE of 3–4 independent determinations (**P < 0.01; *P < 0.05).