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Deregulated Adhesion Program in Palatal Keratinocytes of Orofacial Cleft Patients

ABSTRACT: Orofacial clefts (OFCs) are the most frequent craniofacial birth defects. An orofacial cleft (OFC) occurs as a result of deviations in palatogenesis. Cell proliferation, differentiation, adhesion, migration and apoptosis are crucial in palatogenesis. We hypothesized that deregulation of these processes in oral keratinocytes contributes to OFC. We performed microarray expression analysis on palatal keratinocytes from OFC and non-OFC individuals. Principal component analysis showed a clear difference in gene expression with 24 and 17% for the first and second component respectively. In OFC cells, 228 genes were differentially expressed (p<0.001). Gene ontology analysis showed enrichment of genes involved in β1 integrin-mediated adhesion and migration, as well as in P-cadherin expression. A scratch assay demonstrated reduced migration of OFC keratinocytes (343.6 ± 29.62 μm) vs. non-OFC keratinocytes (503.4 ± 41.81 μm, p<0.05). Our results indicate that adhesion and migration are deregulated in OFC keratinocytes, which might contribute to OFC pathogenesis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE139222 | GEO | 2019/10/22

REPOSITORIES: GEO

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Project description:BACKGROUND: Orofacial development is a multifaceted process involving precise, spatio-temporal expression of a panoply of genes. MicroRNAs (miRNAs) constitute the largest family of noncoding RNAs involved in gene silencing, and 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. RESULTS: 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 GmbH. TaqManTM 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 (hierarchical) clustering method. Functional relationships between selected miRNAs were investigated using Ingenuity Pathway Analysis. Expression of over 26% of the approximately 588 murine miRNA genes examined was detected in murine orofacial tissues from GD 12, 13 and 14. Among these expressed genes several clusters were seen to be developmentally regulated. Differential expression of genes encoding 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. Functional relationships between miRNAs differentially expressed were investigated using Ingenuity Pathway Analysis (IPA; Ingenuity Systems). 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. Time-course experiment (Developmental Stages), ICR mice embryos on gestational days (GD) 12, 13 and 14. Biological replicates: For each day of gestation, 3 independent pools of 15 to 20 staged embryos were used to procure embryonic orofacial tissues for preparation of 3 distinct pools of RNA that were independently processed and applied to individual miRXplore™ microRNA Microarray chips (Miltenyi Biotec GmbH). Technology: 2-color spotted cDNA, Hy5 (experimental sample) vs. Hy3 (control - miRXplore Universal Reference).

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Deregulated Adhesion Program in Palatal Keratinocytes of Orofacial Cleft Patients

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