MiR-335 regulates exocytotic proteins and affects glucose-stimulated insulin secretion through decreased Ca2+-dependent exocytosis in beta cells

Background and aims: Ca2+-induced exocytosis is essential for insulin to be secreted from beta-cells, and in islets from type-2 diabetic (T2D) donors the expression of several genes coding for exocytotic proteins is reduced. Largely this phenomenon cannot be explained by polymorphism; rather it is likely due to epigenetic factors like microRNAs (miRNAs). Indeed, previous studies have identified a number of miRNAs with differential expression in the islets from T2D donors and the Goto- Kakizaki (GK) rat. One of the upregulatedmiRNAs in the GK rat is miR- 335, predicted to target several exocytotic genes amongst those Stxbp1 is a validated target. Here we aim to investigate whether miR-335 regulates the expression of exocytotic genes and affects insulin secretion and exocytosis in beta-cells. Materials and methods: Insulin secretion was measured by radio immuno assay. Exocytosis and docking of insulin granules was studied by capacitance measurements using the patch-clamp technique and by TIRF microscopy. Rat miR-335 was overexpressed using chemicallymodified mature microRNA mimic in INS-1 832/13 beta-cells by transfection. Gene knockdown was performed with RNAi. Protein and mRNA levels were analysed with Western Blot and RT-qPCR, respectively. Results: Overexpression of miR-335 (OE335) in INS-1 832/13 cells reduced insulin secretion at 16.7 mM glucose compared to control cells (SCR) (n=3;