Involvement of microRNA-4680-3p against phenytoin-induced cell proliferation inhibition in human palate cells.

Cleft palate (CP) is one of the most common birth defects and is caused by a combination of genetic and/or environmental factors. Environmental factors such as pharmaceutical exposure in pregnant women are known to induce CP. Recently, microRNA (miRNA) was found to be affected by environmental factors. The aim of the present study was to investigate the involvement of miRNA against phenytoin (PHE)-induced inhibition of proliferation in human embryonic palatal mesenchymal (HEPM) cells. We demonstrated that PHE inhibited HEPM cell proliferation in a dose-dependent manner. We found that treatment with PHE downregulated cyclin-D1 and cyclin-E expressions in HEPM cells. Furthermore, PHE increased miR-4680-3p expression and decreased two downstream genes (ERBB2 and JADE1). Importantly, an miR-4680-3p-specific inhibitor restored HEPM cell proliferation and altered expression of ERBB2 and JADE1 in cells treated with PHE. These results suggest that PHE suppresses cell proliferation via modulation of miR-4680-3p expression.

Let-7c-5p associate with inhibition of phenobarbital-induced cell proliferation in human palate cells.

Cleft palate (CP) is one of the most common congenital diseases, and is accompanied by a complicated etiology. Medical exposure in women is among one of the reasons leading to CP. Recently, it has been reported that microRNA (miRNA) plays a crucial role in palate formation and the disruption of miRNA that influence the development of CP. Although association with pharmaceuticals and miRNAs were suggested, it has remained largely unknow. The aim of the current investigation is to elucidate upon the miRNA associated with the inhibition of phenobarbital (PB)-induced cell proliferation in human embryonic palatal mesenchymal (HEPM) cells. We showed that PB inhibited HEPM cell viability in a dose-dependent manner. We demonstrated that PB treatment suppressed cyclin-D1 expression in HEPM cells. Furthermore, PB upregulated let-7c-5p expression and downregulated the expression of two downstream genes (BACH1 and PAX3). Finally, we demonstrated that the let-7c-5p inhibitor alleviated PB-induced inhibition of cell proliferation and altered BACH1 and PAX3 expression levels. These results suggest that PB suppresses cell viability by modulating let-7c-5p expression.

Multi-omics analysis in developmental bone biology.

Single-cell omics and multi-omics have revolutionized our understanding of molecular and cellular biological processes at a single-cell level. In bone biology, the combination of single-cell RNA-sequencing analyses and in vivo lineage-tracing approaches has successfully identified multi-cellular diversity and dynamics of skeletal cells. This established a new concept that bone growth and regeneration are regulated by concerted actions of multiple types of skeletal stem cells, which reside in spatiotemporally distinct niches. One important subtype is endosteal stem cells that are particularly abundant in young bone marrow. The discovery of this new skeletal stem cell type has been facilitated by single-cell multi-omics, which simultaneously measures gene expression and chromatin accessibility. Using single-cell omics, it is now possible to computationally predict the immediate future state of individual cells and their differentiation potential. In vivo validation using histological approaches is the key to interpret the computational prediction. The emerging spatial omics, such as spatial transcriptomics and epigenomics, have major advantage in retaining the location of individual cells within highly complex tissue architecture. Spatial omics can be integrated with other omics to further obtain in-depth insights. Single-cell multi-omics are now becoming an essential tool to unravel intricate multicellular dynamics and intercellular interactions of skeletal cells.

Sasa veitchii extracts protect phenytoin-induced cell proliferation inhibition in human lip mesenchymal cells through modulation of miR-27b-5p.

A cleft lip, with or without a cleft palate, is a common birth defect caused by environmental factors or genetic mutations. Environmental factors, such as pharmaceutical exposure in pregnant women, are known to induce cleft lip, with or without cleft palate in the child. This study aimed to investigate the protective effect of Sasa veitchii extract (SE) on phenytoin-induced inhibition of cell proliferation in human lip mesenchymal cells (KD cells) and human embryonic palatal mesenchymal cells (HEPM cells). We demonstrated that cell proliferation was inhibited by phenytoin in a dose-dependent manner in both KD and HEPM cells. Co-treatment with SE restored phenytoin-induced toxicity in KD cells but did not protect HEPM cells against phenytoin-induced toxicity. Several microRNAs (miR-27b, miR-133b, miR-205, miR-497-5p, and miR-655-3p) is reported to associate with cell proliferation in KD cells. We measured the seven kinds of microRNAs (miR27b-3p, miR-27b-5p, miR-133b, miR-205-3p, miR-205-5p, miR-497-5p, and miR-655-3p) and found that SE suppressed miR-27b-5p induced by phenytoin in KD cells. Furthermore, co-treatment with SE enhanced the expression of miR-27b-5p downstream genes (PAX9, RARA, and SUMO1). These results suggest that SE protects phenytoin-induced cell proliferation inhibition by modulating miR-27b-5p.

Decreased Lamin B1 Levels Affect Gene Positioning and Expression in Postmitotic Neurons.

Gene expression programs and concomitant chromatin regulation change dramatically during the maturation of postmitotic neurons. Subnuclear positioning of gene loci is relevant to transcriptional regulation. However, little is known about subnuclear genome positioning in neuronal maturation. Using cultured murine hippocampal neurons, we found genomic locus 14qD2 to be enriched with genes that are upregulated during neuronal maturation. Reportedly, the locus is homologous to human 8p21.3, which has been extensively studied in neuropsychiatry and neurodegenerative diseases. Mapping of the 14qD2 locus in the nucleus revealed that it was relocated from the nuclear periphery to the interior. Moreover, we found a concomitant decrease in lamin B1 expression. Overexpression of lamin B1 in neurons using a lentiviral vector prevented the relocation of the 14qD2 locus and repressed the transcription of the Egr3 gene on this locus. Taken together, our results suggest that reduced lamin B1 expression during the maturation of neurons is important for appropriate subnuclear positioning of the genome and transcriptional programs.

Gfap and Osmr regulation by BRG1 and STAT3 via interchromosomal gene clustering in astrocytes.

Long-range chromatin interactions between gene loci in the cell nucleus are important for many biological processes, including transcriptional regulation. Previously, we demonstrated that several genes specifically cluster with the astrocyte-specific gene for glial fibrillary acidic protein (Gfap) during astrocyte differentiation; however, the molecular mechanisms for gene clustering remain largely unknown. Here we show that brahma-related gene 1 (BRG1), an ATP-dependent chromatin remodeling factor, and the transcription factor STAT3 are required for Gfap and oncostatin M receptor (Osmr) clustering and enhanced expression through recruitment to STAT3 recognition sequences and that gene clustering occurs prior to transcriptional up-regulation. BRG1 knockdown and JAK-STAT signaling inhibition impaired clustering, leading to transcriptional down-regulation of both genes. BRG1 and STAT3 were recruited to the same Gfap fragment; JAK-STAT signaling inhibition impaired BRG1 recruitment. Our results suggest that BRG1 and STAT3 coordinately regulate gene clustering and up-regulate Gfap and Osmr transcription.

Identification of genes associated with the astrocyte-specific gene Gfap during astrocyte differentiation.

Chromosomes and genes are non-randomly arranged within the mammalian cell nucleus, and gene clustering is of great significance in transcriptional regulation. However, the relevance of gene clustering and their expression during the differentiation of neural precursor cells (NPCs) into astrocytes remains unclear. We performed a genome-wide enhanced circular chromosomal conformation capture (e4C) to screen for genes associated with the astrocyte-specific gene glial fibrillary acidic protein (Gfap) during astrocyte differentiation. We identified 18 genes that were specifically associated with Gfap and expressed in NPC-derived astrocytes. Our results provide additional evidence for the functional significance of gene clustering in transcriptional regulation during NPC differentiation.