title: Modulation of microRNA-375 expression alters voltage-gated Na(+) channel properties and exocytosis in insulin-secreting cells.
abstract: OBJECTIVE
  MiR-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 locked nucleic acid (LNA)-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 analysed by RT-qPCR, and protein levels were determined by Western blot.
  
  
  RESULTS
  Voltage-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 approx. 6 mV to a more negative membrane potential upon down-regulation of miR-375. In the miR-375 KO mouse, voltage-gated Na(+) channel inactivation was instead shifted by approx. 14 mV to a more positive membrane potential. Potential targets differed among species and 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, Ca(2+) dependent exocytosis was significantly reduced in OE375 cells.
  
  
  CONCLUSIONS
  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.
authors: Unit of Islet Cell Exocytosis, Department of Clinical Sciences in Malmö, Lund University Diabetes Centre, Clinical Research Centre, Lund University, SUS Malmö, Malmö, Sweden.
authors: V A
authors: VA
authors: Salunkhe
authors: J L S
authors: JL
authors: Esguerra
authors: J K
authors: JK
authors: Ofori
authors: I G
authors: IG
authors: Mollet
authors: M
authors: M
authors: Braun
authors: M
authors: M
authors: Stoffel
authors: A
authors: A
authors: Wendt
authors: L
authors: L
authors: Eliasson
mesh_terms: D000818:Animals; D002118:Calcium; D055724:Electrophysiological Phenomena; D005786:Gene Expression Regulation / Q000502:physiology*; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism; D007515:Islets of Langerhans / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D061566:Voltage-Gated Sodium Channels / Q000235:genetics / Q000378:metabolism*
chemical_list: D007328:Insulin; D035683:MicroRNAs; C540349:Mirn375 microRNA, mouse; D061566:Voltage-Gated Sodium Channels; D005947:Glucose; D002118:Calcium
keywords: Scn3a; exocytosis; insulin secretion; microRNA; patch-clamp; voltage-dependent channels
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