5-Aza-2'-deoxycytidine (AzaD), also known as Decitabine, is a deoxycytidine analog that is typically used to activate methylated and silenced genes by promoter demethylation. However, a survey of the scientific literature indicates that promoter demethylation may not be the only (or, indeed, the major) mechanism by which AzaD affects gene expression. Regulation of gene expression by AzaD can occur in several ways, including some that are independent of DNA demethylation. Results from several studies indicate that the effect of AzaD on gene expression is highly context-dependent and can differ for the same gene under different environmental settings. This may, in part, be due to the nature of the silencing mechanism(s) involved - DNA methylation, repressive histone modifications, or a combination of both. The varied effects of AzaD on such context-dependent regulation of gene expression may underlie some of the diverse responses exhibited by patients undergoing AzaD therapy. In this review, we describe the salient properties of AzaD with particular emphasis on its diverse effects on gene expression, aspects that have barely been discussed in most reviews of this interesting drug.
BACKGROUND Orofacial development is a multifaceted process involving precise, spatio-temporal expression of a panoply of genes. MicroRNAs (miRNAs), the largest family of noncoding RNAs involved in gene silencing, represent critical regulators of cell and tissue differentiation. MicroRNA gene expression profiling is an effective means of acquiring novel and valuable information regarding the expression and regulation of genes, under the control of miRNA, involved in mammalian orofacial development. METHODS To identify differentially expressed miRNAs during mammalian orofacial ontogenesis, miRNA expression profiles from gestation day (GD) -12, -13 and -14 murine orofacial tissue were compared utilizing miRXplore microarrays from Miltenyi Biotech. Quantitative real-time PCR was utilized for validation of gene expression changes. Cluster analysis of the microarray data was conducted with the clValid R package and the UPGMA clustering method. Functional relationships between selected miRNAs were investigated using Ingenuity Pathway Analysis. RESULTS Expression of over 26% of the 588 murine miRNA genes examined was detected in murine orofacial tissues from GD-12–GD-14. Among these expressed genes, several clusters were seen to be developmentally regulated. Differential expression of miRNAs within such clusters were shown to target genes encoding proteins involved in cell proliferation, cell adhesion, differentiation, apoptosis and epithelial-mesenchymal transformation, all processes critical for normal orofacial development. CONCLUSIONS Using miRNA microarray technology, unique gene expression signatures of hundreds of miRNAs in embryonic orofacial tissue were defined. Gene targeting and functional analysis revealed that the expression of numerous protein-encoding genes, crucial to normal orofacial ontogeny, may be regulated by specific miRNAs.
The transforming growth factor-beta (TGFbeta) family represents a class of signaling molecules that plays a central role in morphogenesis, growth, and cell differentiation during normal embryonic development. Members of this growth factor family are particularly vital to development of the mammalian secondary palate where they regulate palate mesenchymal cell proliferation and extracellular matrix synthesis. Such regulation is particularly critical since perturbation of either cellular process results in a cleft of the palate. While the cellular and phenotypic effects of TGFbeta on embryonic craniofacial tissue have been extensively catalogued, the specific genes that function as downstream mediators of TGFbeta action in the embryo during palatal ontogenesis are poorly defined. Embryonic palatal tissue in vivo and murine embryonic palate mesenchymal (MEPM) cells in vitro secrete and respond to TGFbeta. In the current study, elements of the Smad component of the TGFbeta intracellular signaling system were identified and characterized in cells of the embryonic palate and functional activation of the Smad pathway by TGFbeta1, TGFbeta2, and TGFbeta3 was demonstrated. TGFbeta-initiated Smad signaling in cells of the embryonic palate was found to result in: (1) phosphorylation of Smad 2; (2) nuclear translocation of the Smads 2, 3, and 4 protein complex; (3) binding of Smads 3 and 4 to a consensus Smad binding element (SBE) oligonucleotide; (4) transactivation of transfected reporter constructs, containing TGFbeta-inducible Smad response elements; and (4) increased expression of gelatinases A and B (endogenous genes containing Smad response elements) whose expression is critical to matrix remodeling during palatal ontogenesis. Collectively, these data point to the presence of a functional Smad-mediated TGFbeta signaling system in cells of the developing murine palate.
BACKGROUND Neurulation requires precise, spatio-temporal expression of numerous genes and coordinated interaction of signal transduction and gene regulatory networks, disruption of which may contribute to the etiology of neural tube (NT) defects. MicroRNAs are key modulators of cell and tissue differentiation. In order to define potential roles of miRNAs in development of the murine NT, miRNA microarray analysis was conducted to establish expression profiles, and identify miRNA target genes and functional gene networks. METHODS miRNA expression profiles in murine embryonic NTs derived from gestational days 8.5, 9.0 and 9.5 were defined and compared utilizing miRXplore™ microarrays from Miltenyi Biotech GmbH. Gene expression changes were verified by TaqMan™ quantitative Real-Time PCR. clValid R package and the UPGMA (hierarchical) clustering method were utilized for cluster analysis of the microarray data. Functional associations among selected miRNAs were examined via Ingenuity Pathway Analysis. RESULTS miRXplore™ chips enabled examination of 609 murine miRNAs. Expression of approximately 12% of these was detected in murine embryonic NTs. Clustering analysis revealed several developmentally regulated expression clusters among these expressed genes. Target analysis of differentially expressed miRNAs enabled identification of numerous target genes associated with cellular processes essential for normal NT development. Utilization of Ingenuity Pathway Analysis revealed interactive biological networks which connected differentially expressed miRNAs with their target genes, and highlighted functional relationships. CONCLUSIONS The present study defined unique gene expression signatures of a range of miRNAs in the developing NT during the critical period of NT morphogenesis. Analysis of miRNA target genes and gene interaction pathways revealed that specific miRNAs may direct expression of numerous genes encoding proteins which have been shown to be indispensable for normal neurulation. This study is the first to identify miRNA expression profiles and their potential regulatory networks in the developing mammalian NT.
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