Abstract
BackgroundCleft palate (CP) is the second most common congenital birth defect; however, the relationship between CP-associated genes and epigenetic regulation remains largely unknown. In this study, we investigated the contribution of microRNAs (miRNAs) to cell proliferation and regulation of genes involved in CP development.MethodsIn order to identify all genes for which mutations or association/linkage have been found in individuals with CP, we conducted a systematic literature search, followed by bioinformatics analyses for these genes. We validated the bioinformatics results experimentally by conducting cell proliferation assays and miRNA-gene regulatory analyses in cultured human palatal mesenchymal cells treated with each miRNA mimic.ResultsWe identified 131 CP-associated genes in the systematic review. The bioinformatics analysis indicated that the CP genes were associated with signaling pathways, microRNAs (miRNAs), metabolic pathways, and cell proliferation. A total 17 miRNAs were recognized as potential modifiers of human CP genes. To validate miRNA function in cell proliferation, a main cause of CP, we conducted cell proliferation/viability assays for the top 11 candidate miRNAs from our bioinformatics analysis. Overexpression of miR-133b, miR-374a-5p, and miR-4680-3p resulted in a more than 30% reduction in cell proliferation activity in human palatal mesenchymal cell cultures. We found that several downstream target CP genes predicted by the bioinformatics analyses were significantly downregulated through induction of these miRNAs (FGFR1, GCH1, PAX7, SMC2, and SUMO1 by miR-133b; ARNT, BMP2, CRISPLD1, FGFR2, JARID2, MSX1, NOG, RHPN2, RUNX2, WNT5A and ZNF236 by miR-374a-5p; and ERBB2, JADE1, MTHFD1 and WNT5A by miR-4680-3p) in cultured cells.ConclusionsOur results indicate that miR-374a-5p, miR-4680-3p, and miR-133b regulate expression of genes that are involved in the etiology of human CP, providing insight into the association between CP-associated genes and potential targets of miRNAs in palate development.
Highlights
Cleft palate (CP) is the second most common congenital birth defect; the relationship between CP-associated genes and epigenetic regulation remains largely unknown
We identified the networks and pathways of CP-associated genes and miRNAs potentially involved in the pathology of human CP, through bioinformatics analyses of CP-associated genes and subsequent experimental validation of miRNAs that regulate cell proliferation and expression of CP-associated genes in cultured human palatal mesenchymal cells
We identified 364 studies eligible to identify genetic mutations associated with CP (Fig. 1)
Summary
Cleft palate (CP) is the second most common congenital birth defect; the relationship between CP-associated genes and epigenetic regulation remains largely unknown. We investigated the contribution of microRNAs (miRNAs) to cell proliferation and regulation of genes involved in CP development. Cleft lip with/without cleft palate (CL/CP) is the second most common birth defect in humans worldwide [1]. Maternal age, smoking, alcohol consumption, obesity, and micronutrient deficiencies are known, or strongly suspected, experimental risk factors for CP. The etiology of CP is complex, and its risk factors are still being elucidated [8,9,10]. Recent studies suggest that environmental factors control gene expression at the post-transcriptional level through epigenetic factors [11], including microRNAs (miRNAs), which are short noncoding RNAs [12]
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