MicroRNA-124a Regulates Foxa2 Expression and Intracellular Signaling in Pancreatic β-Cell Lines

Abstract

MicroRNAs (miRNAs) are short non-coding RNAs that have been implicated in fine-tuning gene regulation, although the precise roles of many are still unknown. Pancreatic development is characterized by the complex sequential expression of a gamut of transcription factors. We have performed miRNA expression profiling at two key stages of mouse embryonic pancreas development, e14.5 and e18.5. miR-124a2 expression was strikingly increased at e18.5 compared with e14.5, suggesting a possible role in differentiated ␤-cells. Among the potential miR124a gene targets identified by biocomputation, Foxa2 is known to play a role in ␤-cell differentiation. To evaluate the impact of miR-124a2 on gene expression, we overexpressed or down-regulated miR-124a2 in MIN6 ␤-cells. As predicted, miR-124a2 regulated Foxa2 gene expression, and that of its downstream target, pancreatic duodenum homeobox-1 (Pdx-1). Foxa2 has been described as a master regulator of pancreatic development and also of genes involved in glucose metabolism and insulin secretion, including the ATP-sensitive K ؉ (K ATP ) channel subunits, Kir6.2 and Sur-1. Correspondingly, miR-124a2 overexpression decreased, and anti-miR-124a2 increased Kir6.2 and Sur-1 mRNA levels. Moreover, miR-124a2 modified basal and glucose-or KCl-stimulated intracellular free Ca 2؉ concentrations in single MIN6 and INS-1 (832/13) ␤-cells, without affecting the secretion of insulin or co-transfected human growth hormone, consistent with an altered sensitivity of the ␤-cell exocytotic machinery to Ca 2؉ . In conclusion, whereas the precise role of microRNA-124a2 in pancreatic development remains to be deciphered, we identify it as a regulator of a key transcriptional protein network in ␤-cells responsible for modulating intracellular signaling.Studies implicating small regulatory RNAs in the control of gene expression have demonstrated that transcriptional regulation is controlled not only by protein factors, but also by small endogenous RNA molecules of ϳ19 -23 nucleotides in length called microRNAs (miRNAs or miRs) 3 (1, 2). The first miRNAs discovered were lin-4 and let-7, which are crucial for regulating the developmental timing in the nematode, Caenorhabditis elegans (1, 3). Since this initial report, several hundred miRNAs have been identified in plants and animals that regulate diverse biological processes ranging from cell metabolism to cell differentiation and growth, apoptosis, and immune responses (4 -8). Moreover, it has been shown that miRNAs are characterized by differential spatial and temporal expression patterns supporting their role in such processes (3, 9). miRNAs serve as regulators of gene expression by binding to complementary sites on their target transcripts and, by an ill-defined mechanism, significantly induce the cleavage of mRNA or the repression of translation, depending on the partial or complete sequence homology, respectively (2, 10 -12). It has been estimated that miRNA genes represent ϳ1% of the genome of complex organisms. It appears that they share a certain...

Identification of microRNAs with a role in glucose stimulated insulin secretion by expression profiling of MIN6 cells

“…Previous studies have reported mir-375, mir-9, mir-124a and mir-96 to play a role in regulation of insulin secretion in rodent pancreatic beta cell lines MIN6, INS-IE and MIN6 B1 cells [1][2][3][4]; however, in this study, mir-375, mir-9, mir-124a and mir-96…”

“…Much is known of the mechanisms of GSIS and the proteins involved therein; however, the specific regulation of exocytosis-related proteins remains less well understood. A small number of miRNAs have been shown to play a role in regulation of components of the insulin secretory pathway [1][2][3]. This study aimed to investigate the feasibility of identifying other miRNAs involved in the process of GSIS.…”

“…In recent years, miRNAs have been implicated to play a role in regulation of insulin secretion [1][2][3][4], pancreatic islet development and cell differentiation [5][6][7][8][9][10][11], insulin resistance [12][13][14][15], and have been associated with secondary complications of diabetes, such as diabetic nephropathy [16,17] and cardiovascular disease [18,19].…”

“…Overexpression of mir-124a leads to increased Ca 2+ levels within MIN6 cells, possibly due to dysregulation of Kir6.2 and sur1 subunits [2]. Overexpression of mir-124a in MIN6 B1 cells led to increased insulin secretion in response to basal ACCEPTED MANUSCRIPT 4 glucose concentrations and reduced secretion in response to stimulatory glucose concentrations [4].…”

Expression profiling of microRNA using oligo DNA arrays

“…They have been shown to regulate the Caenorhabditis elegans developmental timing (6,36), as well as the embryonic and post embryonic differentiation in rodents (37,38) and in human beings (39)(40)(41). MiRNAs have been involved in physiological processes, such as insulin regulation (42,43), lipid metabolism (44) and synaptic activity, (45).…”