Identification of glucose-regulated miRNAs from pancreatic {beta} cells reveals a role for miR-30d in insulin transcription

Abstract

MicroRNAs (miRNAs) are small noncoding ribonucleotides that bind mRNAs and function mainly as translational repressors in mammals. MicroRNAs have been implicated to play a role in many diseases, including diabetes. Several reports indicate an important function for miRNAs in insulin production as well as insulin secretion. We have recently carried out a screen in the pancreatic beta-cell line MIN6 to identify miRNAs with altered abundance in response to changes in glucose concentrations. This screen resulted in identification of 61 glucose-regulated miRNAs from a total of 108 miRNAs detectable in MIN6 cells. Many of the identified miRNAs, including miR-124a, miR-107, and miR-30d were up-regulated in the presence of high glucose. Only a few of the miRNAs, including miR-296, miR-484, and miR-690 were significantly down-regulated by high glucose treatment. Interestingly, we found that overexpression of miR-30d, one of the miRNAs up-regulated by glucose, increased insulin gene expression, while inhibition of miR-30d abolished glucose-stimulated insulin gene transcription. Overexpression or inhibition of miR-30d did not have any effect on insulin secretion. These data suggest that the putative target genes of miR-30d may be negative regulators of insulin gene expression.

FIGURE 1.

Identification of glucose-regulated miRNAs from the pancreatic β-cell line MIN6 by miRNA array analysis. (A) Total RNA isolated from MIN6 cells treated with 20 mM pyruvate, 1 or 25 mM glucose for 16 h was separated on a 15% PAGE gel. After isolation of small RNAs from the gel, they were radiolabeled and hybridized to the array membrane. (B) Hierarchical clustering of the identified miRNAs based on their expression profiles using Gene Cluster 3.0 (average linkage and Euclidean distance as similarity measure). The data obtained for each miRNA were normalized to external and internal controls and median centered prior to clustering. The expression levels ranged from +1.5 log¹⁰ to −1.5 log¹⁰.

FIGURE 2.

Northern blot validation of the glucose-regulated miRNAs from the array data. Total RNA (100 μg per sample) from MIN6 cells treated with 20 mM pyruvate (no glucose control), 1 or 25 mM glucose was separated on a 15% PAGE gel under denaturing conditions, blotted, and hybridized with ³²P-labeled miRNA probes. 5S rRNA stained with ethidium bromide was used as a loading control and miRNA levels were compared with 1 mM glucose, which was set as onefold. The expression patterns of miR-124a (A), miR-107 (B), miR-30d (C), miR-690 (D), miR-375 (E), and let7 (F) were consistent with the array data. The array bar for each miRNA was exported from the clustering tree. Red bar indicates increased levels, while a black bar means no signal was detected in the array.

FIGURE 3.

Glucose-induced miR-30d expression in isolated mouse islets. Mouse pancreatic islets were isolated, purified, and incubated with 1 or 16.7 mM glucose in RPMI for 16 h. After this incubation period, RNA was extracted and used for real-time PCR to measure miR-30d levels. The level of miR-30d expression was normalized to the β-cell-specific transcription factor NeuroD1 mRNA levels. The presented data are the average of three independent experiments ± SD; (*) P < 0.05.

FIGURE 4.

MiR-30d affects insulin gene transcription, but not insulin secretion in MIN6 cells. MIN6 cells were transfected with miR-30d precursor or miR-30d inhibitor and with corresponding negative controls. After 48 h, the cells were incubated with 1 or 25 mM glucose and insulin secretion (A) and insulin I mRNA levels (B) were quantified using mouse insulin ELISA and real-time PCR, respectively. The presented data are the average of three independent experiments ± SD.

FIGURE 5.

Overexpression of miR-30d has no effect on Pdx1 and NeuroD1 mRNA and protein levels. MIN6 cells were transfected with miR-30d precursor or control precursor miRNA. After 48 h, cells were incubated with 1 or 25 mM glucose in DMEM for 16 h. (A) Total RNA was extracted and used for real-time PCR to measure Pdx1 and NeuroD1 mRNA levels. The data shown are the average of three independent experiments ± SD. (B) Western blot analysis of Pdx-1 and NeuroD1 protein levels in 1 or 25 mM glucose incubated MIN6 cells after transfection with the miR-30d precursor or control precursor.