Abstract
Insulin release from pancreatic beta-cells plays an essential role in blood glucose homeostasis. Several proteins controlling insulin exocytosis have been identified, but the factors determining the expression of the components of the secretory machinery of beta-cells remain largely unknown. MicroRNAs are newly discovered small non-coding RNAs acting as repressors of gene expression. We found that overexpression of mir-9 in insulin-secreting cells causes a reduction in exocytosis elicited by glucose or potassium. We show that mir-9 acts by diminishing the expression of the transcription factor Onecut-2 and, in turn, by increasing the level of Granuphilin/Slp4, a Rab GTPase effector associated with beta-cell secretory granules that exerts a negative control on insulin release. Indeed, electrophoretic mobility shift assays, chromatin immunoprecipitation, and transfection experiments demonstrated that Onecut-2 is able to bind to the granuphilin promoter and to repress its transcriptional activity. Moreover, we show that silencing of Onecut-2 by RNA interference increases Granuphilin expression and mimics the effect of mir-9 on stimulus-induced exocytosis. Our data provide evidence that in insulin-producing cells adequate levels of mir-9 are mandatory for maintaining appropriate Granuphilin levels and optimal secretory capacity.
Highlights
MicroRNAs are newly discovered regulators of gene expression that act by targeting the 3Ј-untranslated region (3Ј-UTR) of mRNA sequences and by preventing the productive translation of the messengers [12,13,14,15]. miRNAs have been implicated in many processes in invertebrates, including cell proliferation, apoptosis (16 –18), fat metabolism [17], and neuronal patterning [19]
We found that raising the level of mir-9, an miRNA expressed in neurons, in -cells, and in the rat -cell line INS-1E results in drastic impairment of glucose-stimulated insulin release
Mir-9 Is Selectively Expressed in Brain and in Insulin-secreting Cells—We first analyzed by Northern blotting the expression of several miRNAs in two insulin-secreting cell lines (Fig. 1A), in freshly isolated rat pancreatic islets (Fig. 1B), in two cell lines unrelated to -cells (NIH3T3 and HeLa), and in two rat tissues (Fig. 1A)
Summary
Plasmid Construction and siRNA Duplexes—The expression vector directing the synthesis of mir-9 was prepared by introducing oligonucleotides corresponding to the precursor sequence of mir-9 in front of the H1-RNA promoter of the pSuper vector (Oligoengine, Seattle, WA). To generate the 3Ј-UTR-OC2-luc construct, the 3Ј-UTR segment of the rat oc gene was amplified by PCR from INS-1E genomic DNA and inserted in the multiple cloning site of the psiCHECK-1 plasmid (Promega, Madison, WI). Secretion Experiments—For the assessment of the secretory capacity, INS-1E cells (3 ϫ 105) plated in 24-wells dishes were transiently co-transfected with plasmids or RNA duplexes leading to the expression of miRNAs or siRNAs and with a construct encoding the human growth hormone (hGH). 5 l of DNA was used for PCR to detect the presence of specific DNA segments with the following primers: OC2granuphilin: sense, 5Ј-TGGGGGAGGGTATGGTAAAT-3Ј, and antisense, 5Ј-CCTTCTAGCACTCTGGAAGCA-3Ј; rat insulin promoter: sense, 5Ј-AGGAGGGGTAGGTAGGC-3Ј, and antisense, 5Ј-AAGTAGAGTTGTTGACG-3Ј Precleared cell lysates were incubated overnight at 4 °C with 5 g of GFP antibody (Molecular Probes) or 4 g of OC2 antibody. 5 l of DNA was used for PCR to detect the presence of specific DNA segments with the following primers: OC2granuphilin: sense, 5Ј-TGGGGGAGGGTATGGTAAAT-3Ј, and antisense, 5Ј-CCTTCTAGCACTCTGGAAGCA-3Ј; rat insulin promoter: sense, 5Ј-AGGAGGGGTAGGTAGGC-3Ј, and antisense, 5Ј-AAGTAGAGTTGTTGACG-3Ј
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