MicroRNA-223 is essential for maintaining functional β-cell mass during diabetes through inhibiting both FOXO1 and SOX6 pathways

Abstract

The initiation and development of diabetes are mainly ascribed to the loss of functional β-cells. Therapies designed to regenerate β-cells provide great potential for controlling glucose levels and thereby preventing the devastating complications associated with diabetes. This requires detailed knowledge of the molecular events and underlying mechanisms in this disorder. Here, we report that expression of microRNA-223 (miR-223) is up-regulated in islets from diabetic mice and humans, as well as in murine Min6 β-cells exposed to tumor necrosis factor α (TNFα) or high glucose. Interestingly, miR-223 knockout (KO) mice exhibit impaired glucose tolerance and insulin resistance. Further analysis reveals that miR-223 deficiency dramatically suppresses β-cell proliferation and insulin secretion. Mechanistically, using luciferase reporter gene assays, histological analysis, and immunoblotting, we demonstrate that miR-223 inhibits both forkhead box O1 (FOXO1) and SRY-box 6 (SOX6) signaling, a unique bipartite mechanism that modulates expression of several β-cell markers (pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1), and urocortin 3 (UCN3)) and cell cycle-related genes (cyclin D1, cyclin E1, and cyclin-dependent kinase inhibitor P27 (P27)). Importantly, miR-223 overexpression in β-cells could promote β-cell proliferation and improve β-cell function. Taken together, our results suggest that miR-223 is a critical factor for maintaining functional β-cell mass and adaptation during metabolic stress.

Keywords: SRY-box 6 (SOX6); beta cell (B-cell); cell proliferation; diabetes; forkhead box O1 (FOXO1); gene regulation; insulin; metabolic disorder; microRNA (miRNA); microRNA-223; β-cell proliferation.

Figure 1.

miR-223 is up-regulated in β-cells treated with TNFα or high glucose and islets from diabetic mouse model and human patients. A and B, expression of miR-223 in islets of mice with 18 weeks of HFD feeding (A) and db/db mice (B). C and D, mRNA levels of miR-223 in Min6 insulinoma β-cells treated with 25 mm glucose (C) or 5 ng/ml TNFα (D). E, levels of miR-223 were detected in islets from healthy, obese, and type 2 diabetic human donors. Data are shown as mean ± S.E. (error bar).*, p < 0.05; ***, p < 0.001 versus controls by t test.

Figure 2.

Ablation of miR-223 exacerbates β-cell dysfunction in diabetic condition. A, deletion of miR-223 in KO mice was confirmed by 3% agarose gel electrophoresis. B and C, fasting plasma glucose (B) and insulin levels (C) were measured. D and E, HOMA index for insulin resistance (D) and β-cell function (E) were calculated based on fasting blood glucose and insulin levels. F and G, IPITT was performed after 6-h fasting (F) and quantification (G). H and I, IPGTT was performed after overnight fasting (H) and quantification (I). Data are shown as mean ± S.E. (error bar).*, p < 0.05; ***, p < 0.001 versus controls by t test.

Figure 3.

miR-223 deficiency leads to maladaptive β-cell proliferation. A–C, pancreatic tissues from WT and miR-223 KO mice after HFD or STZ injection were stained with insulin and K_i-67 antibodies. Quantification of insulin-positive area (B), and percent of K_i-67 positive cells within insulin-positive area (C) were shown. D, flow cytometry analysis of pancreatic tissue from WT and KO mice showed decreased K_i-67 signal within insulin-positive population. E, H&E staining and quantification of pancreatic tissue were shown. Scale bar, 100 μm. Data are shown as mean ± S.E. (error bar).*, p < 0.05; **, p < 0.01; ***, p < 0.001 versus controls by t test.

Figure 4.

miR-223 directly targets Foxo1 and Sox6 pathways. A and B, the putative miR-223–binding sites in the 3′-UTR regions of Foxo1 (A) and Sox6 (B) are conserved among mammalian species (human, mouse, and rat). C and D, luciferase reporter assays showed that Foxo1 (C) and Sox6 (D) were authentic targets of miR-223 in HEK293 cells. E and F, similar Luciferase reporter assays performed using Min6 β-cells. β-Gal was used as a transfection control. Similar results were observed in three additional independent experiments. Ctl, control. Data are shown as mean ± S.E. (error bar).*, p < 0.05; **, p < 0.01 versus controls by t test.

Figure 5.

Foxo1 and Sox6 signaling cascades are activated upon miR-223 deletion. A, immunofluorescent analysis of pancreatic tissue exhibited higher Foxo1 signal and localization to nucleus. B, Western blotting results showed that Foxo1 and Sox6 were increased by at least 2-fold in pancreas of KO mice, and protein levels of phosphorylated Akt at Thr(p)308 and Ser(p)473 sites as well as phosphorylated Foxo1 protein levels were significantly decreased. C, KO mice showed decreased Pdx1 and Glut2 protein levels, whereas total p27 levels were increased. D, mRNA levels of β-cell marker (Pdx1, Ucn3, Nkx6.1, Mafa) and progenitor marker (Ngn3) were measured. Scale bar, 100 μm. Data are shown as mean ± S.E. (error bar).*, p < 0.05; **, p < 0.01; ***, p < 0.001 versus controls by t test.

Figure 6.

miR-223 regulates β-cell proliferation and identity in cell-autonomous manner. A, representative images of Ad.GFP- or Ad.223off-infected Min6 β-cells. B, expression of miR-223 was decreased after knockdown as measured by qPCR. C and D, representative FACS scatter plots (C) and quantification (D) of K_i-67 positive cells. E, MTS assay of Min6 β-cells after miR-223 knockdown, followed by 1 h H₂O₂ (200 mm) and 24 h palmitate treatment. F and G, protein levels of Foxo1, Sox6, and p27 were increased, whereas p-Foxo1, Pdx1, and Glut2 levels were reduced 48 h after Ad.223off infection. H, expression of key genes related to β-cell identity and proliferation were measured. Data are shown as mean ± S.E. (error bar).*, p < 0.05; **, p < 0.01; ***, p < 0.001 versus controls by t test

Figure 7.

Overexpression of miR-223 rescues Min6 β-cell by improving proliferation response and viability. A, representative images showed Ad.GFP- or Ad.miR-223–infected Min6 β-cells. B, expression of miR-223 mRNA was measured by qPCR. C and D, proliferation of Min6 cells was significantly increased upon up-regulation of miR-223 as shown by representative FACS scatter plots (C) and quantification (D) of K_i-67–positive cells. E, MTS assay showed that Min6 cell viability was increased after miR-223 overexpression followed by 24 h palmitate (0.5 mm) or 1 h H₂O₂ (200 mm) treatments. F, protein levels of Foxo1, Sox6, and p27 were decreased, whereas p-Foxo1, Pdx1 and Glut2 levels were increased 48 h after Ad.miR223 infection. H, key genes related to β-cell identity and proliferation were measured after overexpressing miR-223. I, glucose-stimulated insulin secretion assay showed that miR-223 down-regulation impaired insulin secretion, which could be rescued by overexpression of miR-223. Data are shown as mean ± S.E. (error bar).*, p < 0.05; **, p < 0.01; ***, p < 0.001 versus controls by t test.

Figure 8.

Graphical summary. Proposed scheme for the mechanism illustrating the regulation of β-cell function plasticity by miR-223.