Unknown,Transcriptomics,Genomics,Proteomics

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Effects of in vivo transfection with anti-miR-214 on gene expression in murine molar tooth germ

ABSTRACT: The tooth is derived from both the ectoderm and the neural crest (ectomesenchyme). It is often used as a model for studies of the basic mechanisms of organ development, including differentiation, cellular interaction, morphogenesis, and mineralization of extracellular matrices. In mouse, tooth development begins at embryonic day 11.5 (E11.5) by thickening of the dental epithelium, whereas mineralization of enamel and dentin in first molars starts at postnatal day 0 (P0). Tooth development entails a multistep and complex process of gene expression. Studies with genetically modified mice have contributed substantially to our understanding of genes that regulate tooth development and morphology. MicroRNAs (miRNA) are a class of non-coding RNAs that regulate gene expression at a post-transcriptional level, and are considered as important regulatory molecules during foetal development. By binding to target mRNAs, miRNA induce mRNA decay or translation repression. Recent bioinformatic predictions of miRNA targets in vertebrates indicate that hundreds of miRNAs are responsible for regulation of expression of up to 30% of the human protein-coding genes. Recent experiments has verified that miRNAs can regulate expression of hundreds of mRNAs in cultured cells. Previous study has also shown that microarrays can be used to detect physiologically relevant effects of miRNAs on expression of mRNAs in vertebrates; miRNAs have been shown to alter expression of a greater number of transcripts than previously appreciated, primarily through interaction with 3M-4UTRs. Understanding the biological function of miRNAs require knowledge of their mRNA targets. Bioinformatic approaches have been used to predict mRNA targets, among which both transcription factors and pro-apoptopic genes were prominent candidates. The precise molecular function of miRNAs remains largely unknown and a better understanding may require loss-of-function studies in vivo. It has been shown that intravenous administration of a novel class of chemically engineered oligonucleotides termed M-^SantagomirsM-^T resulted in a marked reduction of corresponding miRNA levels in most tissues. This resulted in upregulated expression of hundreds of genes predicted to be repressed by miR-122 because these genes had a miR-122 recognition motif in their 3M-4UTR region. Paradoxically, antagomir treatment also revealed a significant number of downregulated genes that may be activated by miR-122. The mechanism by which miRNAs may activate gene expression in vivo is unknown. However, it may involve an indirect effect (the suppression of a transcriptional repressor), or alternatively, miRNA may have a direct effect on gene activation (e.g. chromatin remodeling). Studies of microRNA expression profiles of the developing murine molar tooth germ have shown these to be highly dynamic, some miRNAs being abundantly expressed during the early development of the molar tooth germ, while others are highly expressed only at later developmental stages. A recent study also suggested that miRNAs modulate tooth morphogenesis and ameloblast differentiation, perhaps largely by fine tuning conserved signaling networks. The function of microRNA-214 (miR-214) is unknown, however, it has been related to normal growth and skeletal development in mice. In tooth germs miR-214 is more highly expressed during later developmental stages. It was selected as a target for silencing using anti-miR-214. Here we report effects from injections of 1-100 pmol of anti-miR-214 close to developing first mandibular molar in newborn mice. At 24 hrs, or more, after injection significant changes in gene expression, and in levels of several correspondingly encoded proteins, were found in the tooth germ.

ORGANISM(S): Mus musculus

SUBMITTER: Amer Sehic

PROVIDER: E-MTAB-357 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The tooth is a convenient experimental model for the study of the basic mechanisms of organ development, including differentiation, cellular interaction, morphogenesis, and production and mineralization of extracellular matrices. The rodent incisor teeth grow continuously and exhibit all stages of tooth development at any time. This characteristic makes them convenient models for the study of enamel formation, which occurs in several distinct stages along the tooth axis. The distribution and structure of mouse incisor enamel resemble that of the rat. The enamel covers only the labial aspect of the tooth, and can be divided into four layers: a thin inner prism-free layer, inner enamel with prism decussation, i.e. transverse rows of prisms with prisms inclined medially and laterally in alternate rows, outer enamel with parallel prisms inclined incisally and a thin superficial prism-free layer. Recently, we have described how this elaborate organization of the enamel is established in the initial enamel formed on the unerupted and unworn incisal tip of the incisors. The very first enamel formed, i.e. the most incisally situated enamel, is always prism-free, corresponding to the superficial layer in fully established enamel, although thicker. Going in apical direction, i.e. in the direction of initiation of new enamel formation, isolated prisms appear among the crystals continuous with the prism-free zone crystals. These initial prisms are in general inclined incisally, corresponding to the prisms in the outer enamel layer in the fully established enamel. Inner enamel with the characteristic decussation pattern is added somewhat further apically and increases in thickness with increasing enamel thickness. Based on the structure of the enamel that they produce, the ameloblasts producing the initial, thin, prism-free enamel at the incisal tip of the unworn mouse incisor are probably differently configured (e.g. lacking Tomes' processes), probably differently organized (e.g. not organized in transverse rows moving sideways in opposite directions), and probably have a shorter life-span than the more apically situated ameloblasts which produce thicker enamel with the full four-layered configuration of mouse incisor enamel. For this reason it would be of interest to compare the gene expression profile of the incisal tip segment where prism-free enamel is being formed with the profile of the immediately adjacent segment where enamel with all four layers is being formed. As long as the incisor is unworn enamel formation and gene expression in these segments will likely reflect mechanistic differences between these segments as regards enamel biosynthesis. MicroRNAs (miRNA) are a class of non-coding RNAs that regulate gene expression at a post-transcriptional level, and are considered as important regulatory molecules during foetal development. By binding to target mRNA, miRNA induce mRNA decay or translation repression. Recent bioinformatic predictions of miRNA targets in vertebrates indicate that hundreds of miRNAs are responsible for regulating the expression of up to 30% of the human protein-coding genes, and miRNAs have recently also been shown to regulate expression of hundreds of mRNAs in cultured cells. Recently, miRNA expression profiles of the developing murine molar tooth germ and submandibular salivary gland have been established. We here report expression profiling of miRNAs present in the two adjacent segments of the incisor tooth germ on which we also have carried out mRNA expression profiling. The results suggest that also miRNAs are abundantly, and differentially, expressed during development of the incisor tooth germ in mouse. By analogy with the mRNA data also miRNA expression is dynamic, with respect to both the two incisor segments investigated and to the developmental stage of the incisor.

Project description:Abstract: Background & Aims: Unusual hypervascularity is a hallmark of human hepatocellular carcinoma (HCC). Although microRNA-214 (miR-214) is upregulated in other human cancers, it is downregulated in HCC. We elucidated the biological and clinical significance of miR-214 downregulation in HCC. Methods: MicroRNAs deregulated in HCC were identified using array-based MicroRNA profiling. A luciferase reporter assay confirmed target association between miR-214 and hepatoma-derived growth factor (HDGF). Tube formation and in vivo angiogenesis assays validated the roles of miR-214/HDGF in angiogenesis. Results: MiR-214 downregulation was associated with higher tumor recurrence and worse clinical outcomes. Ectopic expression of miR-214 suppressed xenograft tumor growth and microvascularity of the tumor and its surrounding tissues. The genes downregulated by ectopic expression of miR-214 were involved in the regulation of apoptosis, cell cycle, and angiogenesis. Integrated analysis disclosed HDGF as a downstream target of miR-214. Conditioned medium of HCC cells contained bioactivity to stimulate tube formation of human umbilical vein endothelial cells, which was abolished by pretreatment of the conditioned media with HDGF antibodies, silencing of HDGF expression or ectopic expression of miR-214 in the donor HCC cells. The angiogenic activity of the conditioned media lost by ectopic expression of miR-214 in the donor cells was restored by supplementation with recombinant HDGF. In vivo tumor angiogenesis assays showed significant suppression of tumor vascularity by ectopic expression of miR-214. Conclusions: A novel role of microRNA in tumrigenesis is identified. Downregulation of miR-214 contributes to unusual hypervascularity of HCC via activation of the HDGF paracrine pathway for tumor angiogenesis. To identify miRNAs that are deregulated in human HCC, 68 HCC and 21 non-tumor liver tissues were subjected to profiling of miRNA expression using miRNA arrays containing 739 human miRNA probes. Differentially expressed microRNAs were identified.

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Effects of in vivo transfection with anti-miR-214 on gene expression in murine molar tooth germ

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