βlinc1 encodes a long noncoding RNA that regulates islet β-cell formation and function

Abstract

Pancreatic β cells are responsible for maintaining glucose homeostasis; their absence or malfunction results in diabetes mellitus. Although there is evidence that long noncoding RNAs (lncRNAs) play important roles in development and disease, none have been investigated in vivo in the context of pancreas development. In this study, we demonstrate that βlinc1 (β-cell long intergenic noncoding RNA 1), a conserved lncRNA, is necessary for the specification and function of insulin-producing β cells through the coordinated regulation of a number of islet-specific transcription factors located in the genomic vicinity of βlinc1. Furthermore, deletion of βlinc1 results in defective islet development and disruption of glucose homeostasis in adult mice.

Keywords: long noncoding RNA; pancreas development; β cell.

Figure 1.

βlinc1 is a conserved endocrine-specific lncRNA. (A) βlinc1 is located in a large syntenic block on human chromosome 20 and mouse chromosome 2 (purple lines). The position and direction of βlinc1 and the nearest adjacent genes are indicated. (B) Snapshot depicting βlinc1 transcript structure generated by de novo assembly of RNA sequencing (RNA-seq) data from mouse embryonic day 14.5 (E14.5) pancreas and islet samples and 30-way Multiz Alignment and Conservation. The mouse βlinc1 locus spans 8 kb, located in a gene desert between Nkx2.2 and Pax1 on the long (q) arm of chromosome 2 (chr2: 147,030,314–147,038,352, mm9), with a 73.6% sequence conservation with the human βLINC1 locus as determined by LiftOver. (C) βlinc1 RNA expression was determined by quantitative RT–PCR (qRT–PCR) in a tissue panel isolated from E15.5 embryos and adult islets. (D) RNA in situ hybridization of βlinc1 in pancreatic sections of E18.5 embryos and adult pancreata showing enrichment of the βlinc1 transcript in the trunk endocrine compartment and adult islets. White dotted lines depict the endocrine area and islets. The image is representative of at least three experiments. (E) Cellular fractionation of MIN6 cells showing that βlinc1 is highly enriched in nuclear versus cytosolic fractions. Gapdh and Malat1 were included as negative and positive controls of nuclear transcript retention, respectively. Samples without the addition of reverse transcriptase (noRT) were included to control for genomic contamination. n = 4. (F) βlinc1 and Nkx2.2 expression in MIN6 cells treated with two different siRNAs against βlinc1. n = 4. Error bars represent ±SEM. (*) P < 0.05, Student's t-test.

Figure 2.

βlinc1 knockout mice are glucose-intolerant and have defects in endocrine specification. (A) βlinc1^−/− 16-wk-old mice are mildly glucose-intolerant. (AUC) Area under the curve. (B) Plasma insulin levels in 16-wk-old βlinc1^−/− mice (green) (n = 10) and littermate βlinc1^+/+ control mice (blue) (n = 7) at 0 and 30 min after glucose injection. (C) Immunofluorescence analysis of pancreas development at E15.5 showing a reduction of β cells and an increase of somatostatin-producing δ cells in βlinc1^−/− mice compared with βlinc1^+/+ and βlinc1^+/− mice. There are no apparent morphological defects in the exocrine compartment, which was visualized by immunostaining against CPA1. Images are representative of n > 3 mice. Bar, 50 µm. (D) Quantification of hormone-producing cells in E15.5 pancreata. n = 4. Error bars represent ±SEM. (*) P < 0.05 βlinc1^−/− versus βlinc1^+/+; (#) P < 0.05 βlinc1^−/− versus βlinc1^+/−, Student's t-test.

Figure 3.

βlinc1 regulates the expression of endocrine-specific genes. (A) qPCR analysis of hormones in E15.5 pancreata. General reduction of hormone expression with the up-regulation of somatostatin. n ≥ 5. (B) qPCR analysis of βlinc1 and several transcription factors involved in pancreas development in E15.5 pancreata. n ≥ 5. Error bars represent ±SEM. (*) P < 0.05 βlinc1^−/− versus βlinc1^+/+; (#) P < 0.05 βlinc1^+/− versus βlinc1^+/+, Student's t-test.

Figure 4.

βlinc1 predominantly regulates the expression of β-cell-enriched genes that map to the same chromosomal region as βlinc1. (A) Pie chart showing the total numbers and relative percentages of up-regulated and down-regulated genes (P < 0.05) in βlinc1^+/− versus βlinc1^+/+. (B) Fold change and chromosomal location of the 10 most significantly down-regulated genes in βlinc1^+/− compared with βlinc1^+/+. A large proportion of these genes is in chromosome 2 (Supplemental Fig. 10a). (C) Schematic representation of the mouse karyotype. The red box denotes the location of the dysregulated genes on chromosome 2 in βlinc1^+/− compared with βlinc1^+/+ mice. (D) qRT–PCR validation of RNA-seq results for the genes located in chromosome 2. (*) P < 0.05 versus βlinc1^+/+; (#) P < 0.05 versus βlinc1^+/−. (E) Down-regulation of βlinc1 with two different siRNAs in MIN6 cells recapitulates the increase in somatostatin expression and down-regulation of Scg5 and Pax6 seen in vivo. n = 4. Error bars represent ±SEM. (*) P < 0.05 versus scrambled. Student's t-test.