Regulation of Pancreatic microRNA-7 Expression

Genome-encoded microRNAs (miRNAs) provide a posttranscriptional regulatory layer, which is important for pancreas development. Differentiation of endocrine cells is controlled by a network of pancreatic transcription factors including Ngn3 and NeuroD/Beta2. However, how specific miRNAs are intertwined into this transcriptional network is not well understood. Here, we characterize the regulation of microRNA-7 (miR-7) by endocrine-specific transcription factors. Our data reveal that three independent miR-7 genes are coexpressed in the pancreas. We have identified conserved blocks upstream of pre-miR-7a-2 and pre-miR-7b and demonstrated by functional assays that they possess promoter activity, which is increased by the expression of NeuroD/Beta2. These data suggest that the endocrine specificity of miR-7 expression is governed by transcriptional mechanisms and involves members of the pancreatic endocrine network of transcription factors

Dysregulation of Dicer1 in Beta Cells Impairs Islet Architecture and Glucose Metabolism

microRNAs (miRNAs) play important roles in pancreas development and in regulation of insulin expression in the adult. Here we show that loss of miRNAs activity in beta-cells during embryonic development results in lower beta-cell mass and in impaired glucose tolerance. Dicer1-null cells initially constitute a significant portion of the total beta-cell population. However, during postnatal development, Dicer1-null cells are depleted. Furthermore, wild-type beta cells are repopulating the islets in complex compensatory dynamics. Because loss of Dicer1 is also associated with changes in the distribution of membranous E-cadherin, we hypothesized that E-cadherin activity may play a role in beta cell survival or islet architecture. However, genetic loss of E-cadherin function does not impair islet architecture, suggesting that miRNAs likely function through other or redundant effectors in the endocrine pancreas

Machine learning workflows identify a microRNA signature of insulin transcription in human tissues

Dicer knockout mouse models demonstrated a key role for microRNAs in pancreatic β-cell function. Studies to identify specific microRNA(s) associated with human (pro-)endocrine gene expression are needed. We profiled microRNAs and key pancreatic genes in 353 human tissue samples. Machine learning workflows identified microRNAs associated with (pro-)insulin transcripts in a discovery set of islets (n = 30) and insulin-negative tissues (n = 62). This microRNA signature was validated in remaining 261 tissues that include nine islet samples from individuals with type 2 diabetes. Top eight microRNAs (miR-183-5p, -375-3p, 216b-5p, 183-3p, -7-5p, -217-5p, -7-2-3p, and -429-3p) were confirmed to be associated with and predictive of (pro-)insulin transcript levels. Use of doxycycline-inducible microRNA-overexpressing human pancreatic duct cell lines confirmed the regulatory roles of these microRNAs in (pro-)endocrine gene expression. Knockdown of these microRNAs in human islet cells reduced (pro-)insulin transcript abundance. Our data provide specific microRNAs to further study microRNA-mRNA interactions in regulating insulin transcription

The Promoter of the pri-miR-375 Gene Directs Expression Selectively to the Endocrine Pancreas

microRNAs (miRNAs) are known to play an essential role in controlling a broad range of biological processes including animal development. Accordingly, many miRNAs are expressed preferentially in one or a small number of cell types. Yet the mechanisms responsible for this selectivity are not well understood. The aim of this study was to elucidate the molecular basis of cell-specific expression of the pri-miR-375 gene, which is selectively expressed in pancreatic islets, and has been implicated both in the development of islets, and the function of mature pancreatic beta cells. An evolutionarily conserved 768 bp region of DNA upstream of the pri-miR-375 gene was linked to GFP and luciferase reporter genes, and expression monitored in transgenic mice and transfected cultured cells. Deletion and targeted mutagenesis analysis was used to evaluate the functional significance of sequence blocks within the upstream fragment. 5′-RACE analysis was used for mapping the pri-miR-375 gene transcription start site. The conserved 768 bp region was able to direct preferential expression of a GFP reporter gene to pancreatic islets in transgenic mice. Deletion analysis using a luciferase reporter gene in transfected cultured cell lines confirmed the cell specificity of the putative promoter region, and identified several key cis-elements essential for optimal activity, including E-boxes and a TATA sequence. Consistent with this, 5′-RACE analysis identified a transcription start site within this DNA region, 24 bp downstream of the TATA sequence. These studies define the promoter of the pri-miR-375 gene, and show that islet-specific expression of the pri-miR-375 gene is controlled at the transcriptional level. Detailed analysis of the transcriptional mechanisms controlling expression of miRNA genes will be essential to permit a comprehensive understanding of the complex role of miRNAs such as miR-375 in developmental processes

Changes in miRNA expression during BCD cell expansion in vitro.

<p>A. miRNA microarray analysis. Values represent the ratio between levels in expanded islet cells at passage 2 and isolated human islets. Data are mean of results from 2 donors. B. qRT-PCR analysis of RNA extracted from sorted GFP⁺ BCD cells at passage 2. Values are mean±SE, relative to islets (n = 3 donors). *p ≤0.05; **p≤0.01; ***p≤0.001.</p

Primers for qRT-PCR analyses.

<p>Primers for qRT-PCR analyses.</p

Effect of miR-375 overexpression on BCD cell redifferentiation.

<p>A. miR-375 <i>in-situ</i> hybridization in human islets. DNA was stained with DAPI. Bar = 75 μm. B. Overexpression of miR-375. Expanded islet cells, or sorted GFP⁺ BCD cells, at passages 5–12 were infected with miR-375 or empty viral vectors and analyzed 5 days later by qPCR. Data are mean±SE (for expanded islet cells, n = 8 donors; for BCD cells, n = 3 donors), relative to empty viral vector, and normalized to miR-24 and U6-snRNA. C, D. Changes in expression of mesenchymal genes (C) and islet cell genes (D) in expanded islet cells infected at passages 4–12 with miR-375 or empty viral vectors, and analyzed by qRT-PCR. Data are mean±SE (n = 4–8 donors). E. Changes in expression of β-cell genes in sorted GFP⁺ BCD cells infected at passages 4–7 with miR-375 or empty viral vectors, and analyzed 5 days later by qPCR. Data are mean±SE (n = 3 donors), relative to empty viral vector. F-H. Immunofluorescence analysis of C-peptide in expanded islet cells infected at passages 5–6 with miR-375 or empty viral vectors. F,G. Quantitation of C-peptide⁺ cells among total expanded islet cells (F), or GFP⁺ BCD cells (G). Values are mean±SD (n = 3 donors), based on counting >500 cells in each condition. H. Bar = 30 μm. I. Changes in proliferation of expanded islet cells infected at passages 3–5 with miR-375 or empty viral vectors, and analyzed 5 days later by immunofluorescence for Ki67. Values are mean±SD (n = 4 donors), based on counting >500 cells in each condition.</p

miR-375 overexpression downregulates GSK3.

<p>A. Proteomic profiling of sorted GFP⁺ BCD cells infected at passages 4–6 with miR-375 or empty viral vectors. Proteins changed >2-fold are shown. p≤0.05 (n = 3 donors, each analyzed in duplicates). B-D. Immunoblotting of phospohorylated GSK-3α and GSK-3β inactive or active forms in expanded islet cells at the indicated passages. Values are mean±SE (n = 3 donors). *p≤0.05; **p≤0.01. E. Immunoblotting of total GSK-3α and GSK-3β proteins in expanded islet cells infected at passages 3–5 with miR-375 or empty viral vectors. Values are mean±SE (for GSK-3α, n = 3 donors; for GSK-3β, n = 5 donors). F. Immunoblotting of active forms of GSK-3α and GSK-3β. Values are mean±SE (n = 6 donors). G. Immunoblotting of the inactive form of GSK-3β. Values are mean±SE (n = 4 donors). H. Immunoblotting of inactive form of GSK-3α in expanded islet cells infected at passages 3–4 with miR-375 or empty viral vectors. Values are mean±SE (n = 6 donors).</p

Islet donors used in the study.

<p>Islet donors used in the study.</p

Synergistic effects of miR-375 overexpression and RC on BCD cell redifferentiation.

<p>A. qPCR analysis of changes in miR-375 levels in expanded islet cells treated at passages 5–7 with RC for the indicated times. Values are mean±SE (n = 3 donors), relative to d0, and normalized to miR-24 and U6-snRNA. B. <i>In-situ</i> hybridization with miR-375 or scrambled probe following a 4-day treatment with RC of expanded islet cells at passage 5 labeled with the β-cell lineage tracing vectors. DNA was stained with DAPI. Bar = 75 μm for miR-375, 50 μm for scrambled probe. C,D. qPCR analysis of changes in expression of β-cell genes in expanded islet cells (C) infected at passages 4–12, and sorted GFP⁺ BCD cells (D) infected at passages 4–7, with miR-375 or empty viral vectors and treated with RC for 4–6 days. Data are mean±SE (n = 3–9 donors in C, n = 3 donors in D), relative to cells infected with empty vector. UI, uninfected. E,F. Quantitation of immunofluorescence analysis of C-peptide in expanded islet cells infected at passages 5–6 with miR-375 or empty viral vectors and treated with RC for 4 days. Values are mean±SD (n = 3 donors), based on counting >500 cells in each condition.</p