The circular RNA Cdr1as, via miR-7 and its targets, regulates insulin transcription and secretion in islet cells

Abstract

Among the identified thousands of circular RNAs (circRNA) in humans and animals, Cdr1as (also known as CiRS-7) was recently demonstrated to act as a powerful miR-7 sponge/inhibitor in developing midbrain of zebrafish, suggesting a novel mechanism for regulating microRNA functions. MiR-7 is abundantly expressed in islet cells, but overexpressing miR-7 in transgenic mouse β cells causes diabetes. Therefore, we infer that Cdr1as expression may inhibit miR-7 function in islet cells, which in turn improves insulin secretion. Here, we show the first characterization of Cdr1as expression in islet cells, which was upregulated by long-term forskolin and PMA stimulation, but not high glucose, indicating the involvement of cAMP and PKC pathways. Remarkably, both insulin content and secretion were significantly increased by overexpression of Cdr1as in islet cells. We further identified a new target Myrip in the Cdr1as/miR-7 pathway that regulates insulin granule secretion, and also another target Pax6 that enhances insulin transcription. Taken together, our findings revealed the effects of the strongly interacting pair of Cdr1as/miR-7 on insulin secretion, which may become a new target for improving β cell function in diabetes.

Figure 1. Characterization and functional analysis of Cdr1as.

(a) Cdr1as and miR-7 expression profile in neuroendocrine tissues and cell lines. Expression levels of Cdr1as and miR-7 are normalized to Gapdh mRNA levels. Experiments were independently performed five times (n = 5) in triplicates. (b) Schematic illustration of the Cdr1 locus with primers and validation of mouse Cdr1as. SD, splicing donor; SA, splicing acceptor. Sanger sequencing depicts the junction of Cdr1as.

Figure 2. Effects of stimulators on Cdr1as expression.

(a) Cdr1as expression in mouse islets treated by forskolin (10 μM). (b) PMA (1 μM) stimulated Cdr1as expression in mouse islets. (c) Effect of Glucose on Cdr1as expression in mouse islets. One-way ANOVA and Bonferroni test are used for statistical analysis. n = 3, *P < 0.05; **P < 0.01.

Figure 3. Impact of Cdr1as on insulin secretion.

Insulin secretion is measured by GSIS assay in MIN6 cell (a) and mouse islet cells (b). n = 5, *P < 0.05, **P < 0.01.

Figure 4. Insulin production is regulated by Cdr1as.

(a) Insulin content in MIN6 cells measured by ELISA. (b) Insulin content in islet cells determined by ELISA. n = 3, *P < 0.05, **P < 0.01. (c) Insulin levels in MIN6 cells are visualized by immunostaining with insulin antibody. (d) Insulin levels in mouse islet cells are estimated by immunostaining with insulin antibody.

Figure 5. Cdr1as regulates insulin1 and insulin2 transcriptional levels.

mRNA levels of insulin 1 and insulin 2 in MIN6 cells (a) or in mouse islets (b). n = 5, *P < 0.05, **P < 0.01.

Figure 6. Cdr1as regulates insulin pathways by elevating effect on Myrip and Pax6.

(a) The predicted miR-7a target sequence in the 3′-UTR sequence of wildtype (WT) or mutated (Mut) Myrip and Pax6 (upper panel). After co-transfected with miR-7 in 293T cells, luciferase activities of wildtype 3′-UTR in Myrip or Pax6 as well as mutated 3′-UTR in Myrip or Pax6 are measured (lower panel). (b) Effects of miR-7 and Cdr1as overexpression on endogenous mRNA levels of Myrip or Pax6 in MIN6 cells or islet cells. (c) Representative Western blots and quantitatively analysis show the effects of miR-7 and Cdr1as on the protein levels of Myrip and Pax6 in MIN6 cells. n = 3, *P < 0.05, **P < 0.01.

Figure 7. Working model of Cdr1as/miR-7-associated network in β cells.

Overexpression of exogenous Cdr1as or stimulation of forskolin or PMA can significantly increase the expression level of Cdr1as in islet cells, which in turn inhibits miR-7’s function in insulin biosynthesis and secretion. In particular, physiological and biological functions of two major target genes (e.g., Myrip and Pax6) of miR-7 have been well studied in islet cells. The product of the Myrip gene was found to link the Rab27A-associated and insulin-containing granules (i.e., dense-core vesicle, DCV) via Myosin 5A to the actin filaments for DCV transportation towards plasma membrane and insulin secretion. On the other hand, the transcription factor Pax6 was found to enhance insulin transcription by binding to the promoters of insulin gene 1 and 2.