Article

Targeted Inhibition of miRNA Maturation with Morpholinos Reveals a Role for miR-375 in Pancreatic Islet Development

Oregon State University, United States of America
PLoS Biology (Impact Factor: 9.34). 09/2007; 5(8):e203. DOI: 10.1371/journal.pbio.0050203
Source: PubMed

ABSTRACT

Several vertebrate microRNAs (miRNAs) have been implicated in cellular processes such as muscle differentiation, synapse function, and insulin secretion. In addition, analysis of Dicer null mutants has shown that miRNAs play a role in tissue morphogenesis. Nonetheless, only a few loss-of-function phenotypes for individual miRNAs have been described to date. Here, we introduce a quick and versatile method to interfere with miRNA function during zebrafish embryonic development. Morpholino oligonucleotides targeting the mature miRNA or the miRNA precursor specifically and temporally knock down miRNAs. Morpholinos can block processing of the primary miRNA (pri-miRNA) or the pre-miRNA, and they can inhibit the activity of the mature miRNA. We used this strategy to knock down 13 miRNAs conserved between zebrafish and mammals. For most miRNAs, this does not result in visible defects, but knockdown of miR-375 causes defects in the morphology of the pancreatic islet. Although the islet is still intact at 24 hours postfertilization, in later stages the islet cells become scattered. This phenotype can be recapitulated by independent control morpholinos targeting other sequences in the miR-375 precursor, excluding off-target effects as cause of the phenotype. The aberrant formation of the endocrine pancreas, caused by miR-375 knockdown, is one of the first loss-of-function phenotypes for an individual miRNA in vertebrate development. The miRNA knockdown strategy presented here will be widely used to unravel miRNA function in zebrafish.

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Available from: Anne Karine Lagendijk, Aug 13, 2014
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    • "To date, thousands of miRNA genes have been identified by a combination of cloning, direct sequencing and bioinformatic techniques. It has been proposed that these molecules influence insulin secretion [13], betacell differentiation [14], pancreatic islet development [15], and indirectly control glucose and lipid metabolism [16]. miR103/107 are highly expressed in diabetes. "

    Full-text · Dataset · Nov 2015
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    • "To date, thousands of miRNA genes have been identified by a combination of cloning, direct sequencing and bioinformatic techniques. It has been proposed that these molecules influence insulin secretion [13], betacell differentiation [14], pancreatic islet development [15], and indirectly control glucose and lipid metabolism [16]. miR103/107 are highly expressed in diabetes. "
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    ABSTRACT: Aims: A transposition of the long segment of distal ileum in obese Zucker rats improved glucose tolerance 6 months after IT. It was undertaken to compare the gene expression of miRNA-103, -107 and caveolin-1 in the liver of euglycemic groups of IT relative to SHAM operated rats. Main methods: Obese, male Zucker rats underwent either transposition of 50% distal ileum or sham surgery. For determining the gene expression, the Real-Time PCR for caveolin-1 and miRNA-103, -107 was performed. Plasma concentrations of LDL, HDL, TG and total cholesterol were measured with enzymatic colorimetric assays after optimization procedure. Key findings: The Cav-1 expression in liver tissue after ileal transposition was 1.22 times higher compared to the SHAM group (SHAM median 63.58, min 41.3, max 82.4; IT median 77.35, min 60.8, max 95.41, p < 0.001). miRNA-107 expression was significantly downregulated by 0.6-fold in the IT group compared to the SHAM group (SHAM median 507.51, min 236.42, max 721.29; IT median 355.2, min 278.15, max 478.15, p < 0.015. The level of TG was significantly higher after IT surgery (SHAM median 115, min 96, max 143; IT median 153, min 115, max 162, p = 0.001). The total cholesterol plasma levels decreased after IT (SHAM median 178, min 161, max 183; IT median 128, min 103, max 114, p < 0.000001). The LDL plasma level in IT was two-fold lower than in the SHAM (SHAM median 117, min 68, max 151; IT median 58, min 45, max 61, p < 0.000001). Significance: The transposition of 50% of the distal ileum lead to an increase in caveolin-1 and reduction in miR-107 expression compared to those of SHAM group. Endogenous miR-107 is more involved in regulation of the functions of insulin-target liver tissue than miRNA-103. Reduced LDL and cholesterol plasma levels suggest positive effects on lipid metabolism in long-term observations. The present study is the first to show a lack of IT effect regarding triglycerides six months after surgery.
    Full-text · Article · Oct 2015
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    • "This miRNA is one of the most abundant miR- NAs in primary b-cells and was first discovered as a negative regulator of insulin secretion (Poy et al. 2004). Later, miR-375 was shown to play a role in islet development (Kloosterman et al. 2007) and in maintenance of normal b-cell mass (Poy et al. 2009). Voltage-gated Na + channels are required for the initiation of action potentials in neurones and muscle cells, but the role of these channels in pancreatic bcells is more elusive. "
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    ABSTRACT: AimMiR-375 has been implicated in insulin secretion and exocytosis through incompletely understood mechanisms. Here we aimed to investigate the role of miR-375 in the regulation of voltage-gated Na+ channel properties and glucose-stimulated insulin secretion in insulin-secreting cells.Methods MiR-375 was overexpressed using double-stranded mature miR-375 in INS-1 832/13 cells (OE375) or downregulated using LNA (locked nucleic acid)-based anti-miR against miR-375 (LNA375). Insulin secretion was determined using RIA. Exocytosis and ion channel properties were measured using the patch clamp technique in INS-1 832/13 cells and beta-cells from miR-375KO mice. Gene expression was analyzed by RT-qPCR and protein levels were determined by western blot.ResultsVoltage-gated Na+ channels were found to be regulated by miR-375. In INS-1 832/13 cells, steady-state inactivation of the voltage-gated Na+ channels was shifted by ~6 mV to a more negative membrane potential upon downregulation of miR-375. In the miR-375 KO mouse voltage-gated Na+ channel inactivation was instead shifted by ~14 mV to a more positive membrane potential. Expression of suggested targets scn3a and scn3b in INS-1 832/13 cells was only slightly moderated by miR-375. Modulation of miR-375 levels in INS-1-832/13 cells did not significantly affect insulin release. However, Ca2+ dependent exocytosis was significantly reduced in OE375 cells.Conclusion We conclude that voltage-gated Na+ channels are regulated by miR-375 in insulin secreting cells, and validate that the exocytotic machinery is controlled by miR-375 also in INS–1 832/13 cells. Altogether we suggest miR-375 to be involved in a complex multifaceted network controlling insulin secretion and its different components.This article is protected by copyright. All rights reserved.
    Full-text · Article · Jan 2015 · Acta Physiologica
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