miR-216a-targeting theranostic nanoparticles promote proliferation of insulin-secreting cells in type 1 diabetes animal model

Abstract

Aberrant expression of miRNAs in pancreatic islets is closely related to the development of type 1 diabetes (T1D). The aim of this study was to identify key miRNAs dysregulated in pancreatic islets during T1D progression and to develop a theranostic approach to modify their expression using an MRI-based nanodrug consisting of iron oxide nanoparticles conjugated to miRNA-targeting oligonucleotides in a mouse model of T1D. Isolated pancreatic islets were derived from NOD mice of three distinct age groups (3, 8 and 18-week-old). Total RNA collected from cultured islets was purified and global miRNA profiling was performed with 3D-Gene global miRNA microarray mouse chips encompassing all mouse miRNAs available on the Sanger miRBase V16. Of the miRNAs that were found to be differentially expressed across three age groups, we identified one candidate (miR-216a) implicated in beta cell proliferation for subsequent validation by RT-PCR. Alterations in miR-216a expression within pancreatic beta cells were also examined using in situ hybridization on the frozen pancreatic sections. For in vitro studies, miR-216a mimics/inhibitors were conjugated to iron oxide nanoparticles and incubated with beta cell line, βTC-6. Cell proliferation marker Ki67 was evaluated. Expression of the phosphatase and tensin homolog (PTEN), which is one of the direct targets of miR-216a, was analyzed using western blot. For in vivo study, the miR-216a mimics/inhibitors conjugated to the nanoparticles were injected into 12-week-old female diabetic Balb/c mice via pancreatic duct. The delivery of the nanodrug was monitored by in vivo MRI. Blood glucose of the treated mice was monitored post injection. Ex vivo histological analysis of the pancreatic sections included staining for insulin, PTEN and Ki67. miRNA microarray demonstrated that the expression of miR-216a in the islets from NOD mice significantly changed during T1D progression. In vitro studies showed that treatment with a miR-216a inhibitor nanodrug suppressed proliferation of beta cells and increased the expression of PTEN, a miR-216a target. In contrast, introduction of a mimic nanodrug decreased PTEN expression and increased beta cell proliferation. Animals treated in vivo with a mimic nanodrug had higher insulin-producing functionality compared to controls. These observations were in line with downregulation of PTEN and increase in beta cell proliferation in that group. Our studies demonstrated that miR-216a could serve as a potential therapeutic target for the treatment of diabetes. miR-216a-targeting theranostic nanodrugs served as exploratory tools to define functionality of this miRNA in conjunction with in vivo MR imaging.

Figure 1

Schematic representation of dextran‐coated magnetic nanoparticles conjugated with the near infrared fluorescent dye Cy5.5 and miR-216a mimic or inhibitor.

Figure 2

miRNA profile of pancreatic islets from NOD mice. (A) A heatmap of miRNA expression in pancreatic islets from 3-, 8- and 18-week old NOD mice (n = 9). Red and green represent high and low microRNA expression; (B) miRNA216a, 29b, 375, and 7 expression by real-time quantitative RT-PCR in pancreatic islets from 3-, 8- and 18-week old NOD mice. All experiments were performed in triplicate.

Figure 3

Verification of miR-216 and PTEN expressions in pancreatic islets performed on consecutive sections from NOD mice. All experiments were performed in triplicate. (A) In situ hybridization analysis of miR-216a expression in pancreatic islets from 3-, 8- and 18-week old NOD mice (bottom, miR-216a: green, magnification bar = 50 μm); Insulin (red) and CD3 (green) immunostaining of consecutive sections (top, cell nucleus – blue). (B) Immunostaining of PTEN (green) and CD3 (red) expression in pancreatic islets from 3-, 8- and 18-week old NOD mice (bottom, cell nucleus - blue, magnification bar = 50 μm); Immunostaining of Ki67 (green) and CD3 (red) expression in pancreatic islets from 3-, 8- and 18-week old NOD mice (top, cell nucleus – blue). Note Ki67 positive cells located in the periphery of the damaged islets of 3 and 8 wks old mice (yellow arrows). In contrast, in 18 wks old mice Ki67 positive cells (red arrow) were located in the central area of the damaged islet, where the residual insulin producing cells are located on the consecutive section in (A).

Figure 4

Changes in PTEN expression and cell proliferation caused by miRNA-216 targeting nanodrugs. (A) Western blotting analysis of PTEN expression in beta-TC6 cells treated with MN-ASO inhibiting nanodrug or MN-ASOscr nanodrug (top); Note the increase of PTEN expression after treatment with the MN-ASO. Western blot analysis of PTEN expression in beta-TC6 cells treated with MN-miRNA mimic nanodrug or MN-miRNAscr nanodrug (bottom), PTEN expression was compared in beta-TC6 cells incubated with or without FBS (serum starvation condition). Elevated PTEN expression in beta-TC6 cells under serum starvation (SS) was significantly suppressed after treatment with the miR216a mimic nanodrug. All experiments were performed in duplicate. (B) Western blot analysis of PETN expression in islets isolated from BALB/c and treated with MN-ASO inhibiting and MN-miRNA mimic nanodrug. All experiments were performed in duplicate. For western blot results, cropped blots are displayed, uncropped blots are included in the Supplemental Information File. beta-TC6 cells (not serum starved) were treated with either MN-ASO inhibiting nanodrug (left) or MN-miRNA mimic nanodrug (right, magnification bar = 25 μm). Note downregulation of PTEN after treatment with the mimic nanodrug. All experiments were performed in triplicate; (D) beta-TC6 cells were treated with either MN-ASO inhibiting nanodrug (left) or with MN-miRNA mimic nanodrug (right, magnification bar = 50 μm). Note increased proliferation after treatment with the mimic nanodrug due to downregulation of PTEN, a miR-216a target. All experiments were performed in triplicate.

Figure 5

In vivo MRI of nanodrugs delivery. (A) Experimental flowchart of diabetes induction using streptozotocin (STZ), intra-pancreatic ductal injection of the nanodrugs, magnetic resonance imaging (MRI) and post-injection testing. (B) Gross anatomic images of the pancreatic tissue (green arrow) pre‐ and post- intra-pancreatic ductal injection. Note the pancreatic area turning dark post injection, consistent with diffusion of the nanoparticle solution. (C) Representative MRI of the nanodrug delivery to the pancreas (MN-miRNA is shown). Axial T2‐weighted images showing loss of signal intensity in the pancreatic tissue (red outline) of diabetic animals 72 hours post injection.

Figure 6

Fluorescence microscopy of consecutive frozen pancreatic sections from STZ-induced diabetic mice injected with MN-miRNA, MN-ASO, MN-miRNAscr and MN-ASOscr. Animals injected with MN-miRNA showed higher insulin expression in pancreatic islets (top: green, insulin; red, Cy5.5; blue, cell nucleus) compared to the animals injected with MN-ASO or control nanodrugs. These animals also showed downregulated PTEN expression in their islets (middle: green, PTEN; red, Cy5.5; blue, cell nucleus) compared to the animals injected with MN-ASO or control nanodrugs. Finally, there was a notably higher cell proliferation in the islets of these animals compared to controls (bottom green, Ki67; red, Cy5.5; blue, cell nucleus); Magnification bar = 40 μm. All experiments were performed in triplicate.

Figure 7

Therapeutic effect of the nanodrugs and toxicity testing in STZ-induced diabetic mice. (A) Results of IPGTT in mice injected with MN-ASO, MN-miRNA and controls. There was a significant difference in the glucose disposal curves between the group injected with MN-miRNA and the other three groups. These mice showed significantly lower blood glucose levels at 60 mins after the challenge compared to the other groups (n = 5, *P < 0.05). (B) Blood chemistry panel of diabetic animals (n = 5) injected with miR-216-specific nanodrug showed no indication of hepatic, renal or pancreatic toxicity. Albumin (ALB), alanine aminotransferase (ALT), total bilirubin (TBIL), blood urea nitrogen (BUN), creatinine (CREA), amylase (AMYL), lipase (LIPA).