title: Effects of in vivo transfection with anti-miR-214 on gene expression in murine molar tooth germ.
abstract: MicroRNAs (miRNAs) are an abundant class of noncoding RNAs that are believed to be important in many biological processes through regulation of gene expression. Little is known of their function in tooth morphogenesis and differentiation. MicroRNA-214 (miR-214), encoded by the polycistronic Dnm30os gene, is highly expressed during development of molar tooth germ and was selected as a target for silencing with anti-miR-214. Mandibular injection of 1-100 pmol of anti-miR-214 close to the developing first molar in newborn mice resulted in significant decrease in expression of miR-214, miR-466h, and miR-574-5p in the tooth germ. Furthermore, levels of miR-199a-3p, miR-199a-5p, miR-690, miR-720, and miR-1224 were significantly increased. Additionally, the expression of 863 genes was significantly increased and the expression of 305 genes was significantly decreased. Among the genes with increased expression was Twist-1 and Ezh2, suggested to regulate expression of miR-214. Microarray results were validated using real-time RT-PCR and Western blotting. Among genes with decreased expression were Amelx, Calb1, Enam, and Prnp; these changes also being reflected in levels of corresponding encoded proteins in the tooth germ. In the anti-miR-214-treated molars the enamel exhibited evidence of hypomineralization with remnants of organic material and reduced surface roughness after acid etching, possibly due to the transiently decreased expression of Amelx and Enam. In contrast, several genes encoding contractile proteins exhibited significantly increased expression. mRNAs involved in amelogenesis (Ambn, Amelx, Enam) were not found among targets of miRNAs that were differentially expressed following treatment with anti-miR-214. It is therefore suggested that effects of miR-214 on amelogenesis are indirect, perhaps mediated by the observed miR-214-dependent changes in levels of expression of numerous transcription factors.
authors.affiliation: Department of Oral Biology, University of Oslo, Oslo, Norway. amer.sehic@odont.uio.no
authors.forename: Amer
authors.initials: A
authors.lastname: Sehic
authors.forename: Steinar
authors.initials: S
authors.lastname: Risnes
authors.forename: Cuong
authors.initials: C
authors.lastname: Khuu
authors.forename: Qalb-E-Saleem
authors.initials: QE
authors.lastname: Khan
authors.forename: Harald
authors.initials: H
authors.lastname: Osmundsen
mesh_terms: D000566:Amelogenesis / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D002454:Cell Differentiation; D000071221:Enhancer of Zeste Homolog 2 Protein; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics; D051379:Mice; D008815:Mice, Inbred Strains; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D008963:Molar / Q000196:embryology / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D063151:Polycomb Repressive Complex 2; D014083:Tooth Germ / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection; D051879:Twist-Related Protein 1 / Q000235:genetics
chemical_list: D035683:MicroRNAs; C534781:Mirn214 microRNA, mouse; D009687:Nuclear Proteins; D014157:Transcription Factors; D051879:Twist-Related Protein 1; C067250:Twist1 protein, mouse; D000071221:Enhancer of Zeste Homolog 2 Protein; C498410:Ezh2 protein, mouse; D011495:Histone-Lysine N-Methyltransferase; D063151:Polycomb Repressive Complex 2