title: MicroRNA-29 impairs the early phase of reprogramming process by targeting active DNA demethylation enzymes and Wnt signaling.
abstract: Somatic cell reprogramming by transcription factors and other modifiers such as microRNAs has opened broad avenues for the study of developmental processes, cell fate determination, and interplay of molecular mechanisms in signaling pathways. However, many of the mechanisms that drive nuclear reprogramming itself remain yet to be elucidated. Here, we analyzed the role of miR-29 during reprogramming in more detail. Therefore, we evaluated miR-29 expression during reprogramming of fibroblasts transduced with lentiviral OKS and OKSM vectors and we show that addition of c-MYC to the reprogramming factor cocktail decreases miR-29 expression levels. Moreover, we found that transfection of pre-miR-29a strongly decreased OKS-induced formation of GFP+-colonies in MEF-cells from Oct4-eGFP reporter mouse, whereas anti-miR-29a showed the opposite effect. Furthermore, we studied components of two pathways which are important for reprogramming and which involve miR-29 targets: active DNA-demethylation and Wnt-signaling. We show that inhibition of Tet1, Tet2 and Tet3 as well as activation of Wnt-signaling leads to decreased reprogramming efficiency. Moreover, transfection of pre-miR-29 resulted in elevated expression of β-Catenin transcriptional target sFRP2 and increased TCF/LEF-promoter activity. Finally, we report that Gsk3-β is a direct target of miR-29 in MEF-cells. Together, our findings contribute to the understanding of the molecular mechanisms by which miR-29 influences reprogramming.
authors.affiliation: Department of Clinical Medicine, Faculty of Medicine, University of São Paulo (FMRP-USP), Brazil; National Institute of Science and Technology in Stem Cell and Cell Therapy (INCTC), Center for Cell Therapy (CTC), Regional Blood Center, Ribeirão Preto, Brazil; Translational Hepatology and Stem Cell Biology, REBIRTH Cluster of Excellence and Dept. of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Hannover, Germany. Electronic address: mariane.fraguas@ki.se.
authors.forename: Mariane Serra
authors.initials: MS
authors.lastname: Fráguas
authors.affiliation: Translational Hepatology and Stem Cell Biology, REBIRTH Cluster of Excellence and Dept. of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Hannover, Germany. Electronic address: eggenschwiler.reto@mh-hannover.de.
authors.forename: Reto
authors.initials: R
authors.lastname: Eggenschwiler
authors.affiliation: Translational Hepatology and Stem Cell Biology, REBIRTH Cluster of Excellence and Dept. of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Hannover, Germany. Electronic address: hoepfner.jeannine@mh-hannover.de.
authors.forename: Jeannine
authors.initials: J
authors.lastname: Hoepfner
authors.affiliation: National Institute of Science and Technology in Stem Cell and Cell Therapy (INCTC), Center for Cell Therapy (CTC), Regional Blood Center, Ribeirão Preto, Brazil. Electronic address: josililian@usp.br.
authors.forename: Josiane Lilian Dos Santos
authors.initials: JLDS
authors.lastname: Schiavinato
authors.affiliation: University of Brasília - DF (UNB), Brazil. Electronic address: haddad@unb.br.
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Haddad
authors.affiliation: National Institute of Science and Technology in Stem Cell and Cell Therapy (INCTC), Center for Cell Therapy (CTC), Regional Blood Center, Ribeirão Preto, Brazil. Electronic address: luhabib@yahoo.com.br.
authors.forename: Lucila Habib Bourguignon
authors.initials: LHB
authors.lastname: Oliveira
authors.affiliation: Department of Clinical Medicine, Faculty of Medicine, University of São Paulo (FMRP-USP), Brazil; National Institute of Science and Technology in Stem Cell and Cell Therapy (INCTC), Center for Cell Therapy (CTC), Regional Blood Center, Ribeirão Preto, Brazil. Electronic address: agoesaraujo@usp.br.
authors.forename: Amélia Góes
authors.initials: AG
authors.lastname: Araújo
authors.affiliation: Department of Clinical Medicine, Faculty of Medicine, University of São Paulo (FMRP-USP), Brazil; National Institute of Science and Technology in Stem Cell and Cell Therapy (INCTC), Center for Cell Therapy (CTC), Regional Blood Center, Ribeirão Preto, Brazil. Electronic address: marazago@usp.br.
authors.forename: Marco Antônio
authors.initials: MA
authors.lastname: Zago
authors.affiliation: Department of Clinical Medicine, Faculty of Medicine, University of São Paulo (FMRP-USP), Brazil; National Institute of Science and Technology in Stem Cell and Cell Therapy (INCTC), Center for Cell Therapy (CTC), Regional Blood Center, Ribeirão Preto, Brazil. Electronic address: rapane@gmail.com.
authors.forename: Rodrigo Alexandre
authors.initials: RA
authors.lastname: Panepucci
authors.affiliation: Translational Hepatology and Stem Cell Biology, REBIRTH Cluster of Excellence and Dept. of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Hannover, Germany. Electronic address: cantz.tobias@mh-hannover.de.
authors.forename: Tobias
authors.initials: T
authors.lastname: Cantz
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D000070416:Antagomirs / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D065150:Cellular Reprogramming; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D016415:Sequence Alignment; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D060449:Wnt Signaling Pathway / Q000502:physiology*
chemical_list: D020413:3' Untranslated Regions; D000070416:Antagomirs; D004268:DNA-Binding Proteins; D035683:MicroRNAs; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; C540839:TET1 protein, mouse; D014157:Transcription Factors; D000071679:Glycogen Synthase Kinase 3 beta
keywords: Cellular reprogramming; Ten-eleven translocated 1 (TET1) gene; WNT signaling; miR-29