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.

HMGA1pseudogenes as candidate proto-oncogenic competitive endogenous RNAs

“…A missense mutation of the initiator methionine codon prevents translation of HMGA1P7 whereas HMGA1P6 is non-protein coding since it carries a mutation in the stop codon and so generates a non-translatable mRNA. Within the high homology regions, we found perfectly conserved seed matches for miRNAs that have been predicted (miR-103, miR-142-3p, miR-370, and miR-432) or already demonstrated (miR-15 [31], miR-16 [31], miR-26a [32], miR-214 [33], miR-548c-3p [34] and miR-761 [33]) to target the HMGA1 gene (Figure 1B and 1C). …”

MicroRNAome and Expression Profile of Developing Tooth Germ in Miniature Pigs

“…MiR-200b and miR-200c are both important regulatory miRNAs, but only miR-200b regulates the porcine TGF-beta signaling pathway. MiR-214 is the most highly expressed miRNA, and the most likely “tooth-specific” miRNA, but recent studies of the role of miR-214 in tooth germs suggest that miR-214 indirectly affects amelogenesis [26].…”

MicroRNA 224 Regulates Ion Transporter Expression in Ameloblasts To Coordinate Enamel Mineralization

“…Changes in the crystal microstructure might not be detectable by eye or with a stereomicroscope. A recent study using in vivo transfection of anti-miR214 also showed no obvious enamel morphological changes observed with a stereomicroscope, but further SEM analysis indicated alterations in the prism decussation structure (59).…”