The Ia-2β intronic miRNA, miR-153, is a negative regulator of insulin and dopamine secretion through its effect on the Cacna1c gene in mice

Abstract

Aims/hypothesis: miR-153 is an intronic miRNA embedded in the genes that encode IA-2 (also known as PTPRN) and IA-2β (also known as PTPRN2). Islet antigen (IA)-2 and IA-2β are major autoantigens in type 1 diabetes and are important transmembrane proteins in dense core and synaptic vesicles. miR-153 and its host genes are co-regulated in pancreas and brain. The present experiments were initiated to decipher the regulatory network between miR-153 and its host gene Ia-2β (also known as Ptprn2).

Methods: Insulin secretion was determined by ELISA. Identification of miRNA targets was assessed using luciferase assays and by quantitative real-time PCR and western blots in vitro and in vivo. Target protector was also employed to evaluate miRNA target function.

Results: Functional studies revealed that miR-153 mimic suppresses both glucose- and potassium-induced insulin secretion (GSIS and PSIS, respectively), whereas miR-153 inhibitor enhances both GSIS and PSIS. A similar effect on dopamine secretion also was observed. Using miRNA target prediction software, we found that miR-153 is predicted to target the 3'UTR region of the calcium channel gene, Cacna1c. Further studies confirmed that Cacna1c mRNA and protein are downregulated by miR-153 mimics and upregulated by miR-153 inhibitors in insulin-secreting freshly isolated mouse islets, in the insulin-secreting mouse cell line MIN6 and in the dopamine-secreting cell line PC12.

Conclusions/interpretation: miR-153 is a negative regulator of both insulin and dopamine secretion through its effect on Cacna1c expression, which suggests that IA-2β and miR-153 have opposite functional effects on the secretory pathway.

Keywords: Cav1.2; IA-2; Insulin secretion; Intronic miRNA; MicroRNAs.

Fig. 1

Impact of miR-153 on insulin and dopamine secretion. After stimulation with 25 mmol/l glucose (a, b) or 50 mmol/l KCl (c, d) in MIN6 cells, or primary mouse islets transfected with scrambled control, miR-153 mimic or miR-153 inhibitor for 72 h, insulin secretion was measured. Data are presented as fold change compared with the level before stimulation. Experiments were performed six times (n=6) in triplicate. (e, f) Dopamine secretion (fold change) in PC12 cells stimulated with 25 mmol/l KCl with (e) or without (f) PMA compared with basal levels, 72 h after transfection with miR-153 mimic or miR-153 inhibitor (n=3). (g) Change in mouse islet [Ca²⁺] responses after islet transfection with scrambled controls, miR-153 mimic or miR-153 inhibitor for 72 h, followed by stimulation with 30 mmol/l KCl. *p<0.05 and **p<0.01 vs controls

Fig. 2

Effect of miR-153 mimic or inhibitor on calcium genes: putative miR-153 target site in the Cacna1c 3′-UTR. (a) Expression analysis of miR-153 predicted targets by quantitative real-time PCR 72 h after transfection with scrambled control (black bars), miR-153 mimic (grey bars) or miR-153 inhibitor (white bars). All data were normalised to Gapdh (n=3). (b) MiR-153 predicted target site in mouse Cacna1c 3′-UTR and mutated 3′-UTR sequence with four nucleotide replacements (lower case letters). There is strong sequence conservation in mice, rats and humans. (c) Luciferase reporter assay demonstrating functional activity of miR-153 mimic on WT Cacna1c 3′-UTR in 293T cells, but not on mutated Cacna1c 3′-UTR. Normalised to Renilla luciferase activity (n=3). *p<0.05 and **p<0.01 vs controls

Fig. 3

Regulation of Cacna1c expression by miR-153. Effects of miR-153 mimic and miR-153 inhibitor on Cacna1c expression by quantitative real-time PCR: (a) MIN6 cells; (b) mouse islets; (c) PC12 cells. (n=3). Western blots for CACNA1C and α-tubulin protein levels in MIN6 cells (d) and PC12 cells (e). The relative expression values were determined by Image J (n=3). Ia-2β mRNA levels were unchanged in MIN6 cells (f), mouse islets (g) and PC12 cells (h) following transfection with miR-153 mimic or inhibitor. (n=3), *p<0.05 and **p<0.01 vs controls

Fig. 4

Effect of miR-153-Cacna1c target protector. Glucose stimulates insulin secretion (fold change) in MIN6 cells (a) and mouse islets (b), after transfection with miR-153 mimic with and without miR-153-Cacna1c target protector (TP), showing that the target protector partly abrogated the inhibitory effect of miR-153 on GSIS (n=3). Cacna1c expression analysis by quantitative real-time PCR in MIN6 cells (c) and mouse islets (d) after transfection with miR-153 mimic with and without its target protector. All data were normalised to Gapdh. n=3, *p<0.05 vs controls, †p<0.05, miR-153 mimic vs TP+miR-153 mimic

Fig. 5

miR-153 and Cacna1c expression in Ia-2β KO mice. Quantitative real-time PCR analysis of miR-153 levels (a, b) and Cacna1c mRNA (c, d) in brain and islets from WT, Ia-2 KO, Ia-2β KO and DKO mice, normalised to Gapdh. (e, f) CACNA1C expression was determined by western blot in pancreas and brain from WT, Ia-2 KO, Ia-2β KO and DKO mice. The relative expression values were determined by Image J. n=5, *p<0.05 and **p<0.01 vs controls

Fig. 6

Model illustrating the dual, but opposite, effects on secretion resulting from the stimulation of both the Ia-2β gene and the expression of its intronic microRNA, miR-153. The increase in IA-2β protein facilitates secretion by increasing the number of DCV, whereas an increase in the expression of miR-153, a negative regulator, decreases secretion by inhibiting the expression of calcium channel gene, Cacna1c