Matched miRNA and mRNA signatures from an hESC-based in vitro model of pancreatic differentiation reveal novel regulatory interactions

Summary The differentiation of human pluripotent stem cells (hPSCs) to insulin-expressing beta islet-like cells is a promising in vitro model system for studying the molecular signaling pathways underlying beta cell differentiation, as well as a potential source of cells for the treatment of type 1 diabetes. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate many biological processes, including cellular differentiation. We studied the miRNA and mRNA expression profiles of hPSCs at five stages of in vitro differentiation along the pancreatic beta cell lineage (definitive endoderm, primitive gut tube, posterior foregut, pancreatic progenitor and hormone-expressing endocrine cells) in the context of samples of primary human fetal pancreas and purified adult islet cells using microarray analysis. Bioinformatic analysis of the resulting data identified a unique miRNA signature in differentiated beta islet cells, and predicted the effects of key miRNAs on mRNA expression. Many of the predicted miRNA–mRNA interactions involved mRNAs known to play key roles in the epithelial–mesenchymal transition process and pancreatic differentiation. We validated a subset of the predictions using qRT-PCR, luciferase reporter assays and western blotting, including the known interaction between miR-200 and ZEB2 (involved in epithelial–mesenchymal transition) and the novel interaction between miR-200 and SOX17 (a key transcription factor in specification of definitive endoderm). In addition, we found that miR-30d and let-7e, two miRNAs induced during differentiation, regulated the expression of RFX6, a transcription factor that directs pancreatic islet formation. These findings suggest that precise control of target mRNA expression by miRNAs ensures proper lineage specification during pancreatic development.

[1]  K. Ohuchida,et al.  MicroRNA, hsa-miR-200c, is an independent prognostic factor in pancreatic cancer and its upregulation inhibits pancreatic cancer invasion but increases cell proliferation , 2010, Molecular Cancer.

[2]  N. Baroukh,et al.  MicroRNA-124a Regulates Foxa2 Expression and Intracellular Signaling in Pancreatic β-Cell Lines* , 2007, Journal of Biological Chemistry.

[3]  Donald C. Chang,et al.  Mir-302 reprograms human skin cancer cells into a pluripotent ES-cell-like state. , 2008, RNA.

[4]  E. Li,et al.  Synergistic Function of DNA Methyltransferases Dnmt3a and Dnmt3b in the Methylation of Oct4 and Nanog , 2007, Molecular and Cellular Biology.

[5]  P. Sethupathy,et al.  Abstract 24: MicroRNA-27b Is a Regulatory Hub in Lipid Metabolism and Is Altered in Dyslipidemia , 2012, Arteriosclerosis, Thrombosis, and Vascular Biology.

[6]  Dennis van Hoof,et al.  Derivation of insulin-producing cells from human embryonic stem cells. , 2009, Stem cell research.

[7]  D. Steiner,et al.  Expression profiling of pancreatic beta-cells: glucose regulation of secretory and metabolic pathway genes. , 2000, Diabetes.

[8]  D. Botstein,et al.  Cluster analysis and display of genome-wide expression patterns. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[9]  A. Bang,et al.  Cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells , 2011, Nature Biotechnology.

[10]  Ron Shamir,et al.  Comprehensive MicroRNA Profiling Reveals a Unique Human Embryonic Stem Cell Signature Dominated by a Single Seed Sequence , 2008, Stem cells.

[11]  Gene W. Yeo,et al.  A distinct microRNA signature for definitive endoderm derived from human embryonic stem cells. , 2010, Stem cells and development.

[12]  N. Baroukh,et al.  miR-375 Targets 3′-Phosphoinositide–Dependent Protein Kinase-1 and Regulates Glucose-Induced Biological Responses in Pancreatic β-Cells , 2008, Diabetes.

[13]  K. Dewar,et al.  Rfx6 Directs Islet Formation and Insulin Production in Mice and Humans , 2009, Nature.

[14]  R. Yeh,et al.  MicroRNA regulation of cell lineages in mouse and human embryonic stem cells. , 2008, Cell stem cell.

[15]  A. Chinnaiyan,et al.  Supplementary Figure S3 , 2012 .

[16]  Shadan Ali,et al.  Re-expression of miR-200 by novel approaches regulates the expression of PTEN and MT1-MMP in pancreatic cancer. , 2012, Carcinogenesis.

[17]  E. Kroon,et al.  Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo , 2008, Nature Biotechnology.

[18]  D. Srivastava,et al.  Critical factors for cardiac reprogramming. , 2012, Circulation research.

[19]  C. Orvain,et al.  Rfx6 is an Ngn3-dependent winged helix transcription factor required for pancreatic islet cell development , 2010, Development.

[20]  Simone Brabletz,et al.  The ZEB1/miR‐200 feedback loop controls Notch signalling in cancer cells , 2011, The EMBO journal.

[21]  T. Brabletz,et al.  A reciprocal repression between ZEB1 and members of the miR-200 family promotes EMT and invasion in cancer cells , 2008, EMBO reports.

[22]  Zhiwei Wang,et al.  Up-regulation of miR-200 and let-7 by natural agents leads to the reversal of epithelial-to-mesenchymal transition in gemcitabine-resistant pancreatic cancer cells. , 2009, Cancer research.

[23]  M. Behlke,et al.  Pancreas-enriched miRNA refines endocrine cell differentiation , 2012, Development.

[24]  Zhijie Jiang,et al.  MicroRNA signature of the human developing pancreas , 2010, BMC Genomics.

[25]  D. Steiner,et al.  Expression profiling of pancreatic beta cells: glucose regulation of secretory and metabolic pathway genes. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[26]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[27]  N. Rajewsky,et al.  A pancreatic islet-specific microRNA regulates insulin secretion , 2004, Nature.

[28]  Camillo Ricordi,et al.  The unique cytoarchitecture of human pancreatic islets has implications for islet cell function , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[29]  G. Rutter,et al.  miR-29a and miR-29b Contribute to Pancreatic β-Cell-Specific Silencing of Monocarboxylate Transporter 1 (Mct1) , 2011, Molecular and Cellular Biology.

[30]  Douglas A. Melton,et al.  In vivo reprogramming of adult pancreatic exocrine cells to β-cells , 2008, Nature.

[31]  Raquel Norel,et al.  MicroRNA‐23b cluster microRNAs regulate transforming growth factor‐beta/bone morphogenetic protein signaling and liver stem cell differentiation by targeting Smads , 2009, Hepatology.

[32]  Michael T. McManus,et al.  MicroRNA Expression Is Required for Pancreatic Islet Cell Genesis in the Mouse , 2007, Diabetes.

[33]  S. Kulp,et al.  Targeting endoplasmic reticulum stress and Akt with OSU-03012 and gefitinib or erlotinib to overcome resistance to epidermal growth factor receptor inhibitors. , 2008, Cancer research.

[34]  Todd R. Golub,et al.  MicroRNA miR-125a controls hematopoietic stem cell number , 2010, Proceedings of the National Academy of Sciences.

[35]  Ping Jin,et al.  MicroRNA and gene expression patterns in the differentiation of human embryonic stem cells , 2009, Journal of Translational Medicine.

[36]  John G Doench,et al.  Specificity of microRNA target selection in translational repression. , 2004, Genes & development.

[37]  N. Tørring,et al.  Supplementary data Table 2 , 2007 .

[38]  Maikun Teng,et al.  MicroRNA Cluster 302–367 Enhances Somatic Cell Reprogramming by Accelerating a Mesenchymal-to-Epithelial Transition* , 2011, The Journal of Biological Chemistry.

[39]  D. Bartel.  MicroRNAs: Target Recognition and Regulatory Functions , 2009, Cell.

[40]  M. Hebrok,et al.  Stem cells to pancreatic beta-cells: new sources for diabetes cell therapy. , 2009, Endocrine reviews.

[41]  C. Burge,et al.  Prediction of Mammalian MicroRNA Targets , 2003, Cell.

[42]  Robert L. Judson,et al.  Embryonic stem cell–specific microRNAs promote induced pluripotency , 2009, Nature Biotechnology.

[43]  Y. Dor,et al.  miRNAs control insulin content in pancreatic β‐cells via downregulation of transcriptional repressors , 2011, The EMBO journal.

[44]  S. Ozcan.  MiR-30 family and EMT in human fetal pancreatic islets. , 2009, Islets.

[45]  Alvin C. Powers,et al.  Assessment of Human Pancreatic Islet Architecture and Composition by Laser Scanning Confocal Microscopy , 2005, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[46]  E. Kroon,et al.  Production of pancreatic hormone–expressing endocrine cells from human embryonic stem cells , 2006, Nature Biotechnology.

[47]  K. Yoshiura,et al.  Identification of pregnancy-associated microRNAs in maternal plasma. , 2010, Clinical chemistry.

[48]  S. Lipton,et al.  Direct reprogramming of adult human fibroblasts to functional neurons under defined conditions. , 2011, Cell stem cell.

[49]  M. Joglekar,et al.  The miR-30 family microRNAs confer epithelial phenotype to human pancreatic cells , 2009, Islets.

[50]  Julia Schüler,et al.  The EMT-activator ZEB1 promotes tumorigenicity by repressing stemness-inhibiting microRNAs , 2009, Nature Cell Biology.

[51]  Xiaoqing Tang,et al.  Identification of glucose-regulated miRNAs from pancreatic {beta} cells reveals a role for miR-30d in insulin transcription. , 2009, RNA.

[52]  U. Hofmann,et al.  Pivotal roles for eomesodermin during axis formation, epithelium-to-mesenchyme transition and endoderm specification in the mouse , 2008, Development.

[53]  T. Han,et al.  Differential expression profile of microRNAs in human placentas from preeclamptic pregnancies vs normal pregnancies. , 2009, American journal of obstetrics and gynecology.

[54]  Robert Blelloch,et al.  Embryonic Stem Cell Specific MicroRNAs Regulate the G1/S Transition and Promote Rapid Proliferation , 2008, Nature Genetics.

[55]  Jonathan Schug,et al.  Dynamic chromatin remodeling mediated by polycomb proteins orchestrates pancreatic differentiation of human embryonic stem cells. , 2013, Cell stem cell.

[56]  G. Goodall,et al.  The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1 , 2008, Nature Cell Biology.

[57]  N. Chao.  Faculty Opinions recommendation of In vivo reprogramming of adult pancreatic exocrine cells to beta-cells. , 2008 .

[58]  S. Olthof,et al.  Genetic screen identifies microRNA cluster 99b/let-7e/125a as a regulator of primitive hematopoietic cells. , 2012, Blood.