Background The etiology of cleft lip with or without cleft palate (CL/P), a common congenital birth defect, is complex and involves the contribution of genetic and environmental factors. Although many candidate genes have been identified, the regulation and interaction of these genes in CL/P remain unclear. In addition, the contribution of microRNAs (miRNAs), non-coding RNAs that regulate the expression of multiple genes, to the etiology of CL/P is largely unknown. Methods To identify the signatures of causative biological pathways for human CL/P, we conducted a systematic literature review for human CL/P candidate genes and subsequent bioinformatics analyses. Functional enrichment analyses of the candidate CL/P genes were conducted using the pathway databases GO and KEGG. The miRNA-mediated post-transcriptional regulation of the CL/P candidate genes was analyzed with miRanda, PITA, and TargetScan, and miRTarbase. Genotype-phenotype association analysis was conducted using GWAS. The functional significance of the candidate miRNAs was evaluated experimentally in cell proliferation and target gene regulation assays in human lip fibroblasts. Results Through an extensive search of the main biomedical databases, we mined 177 genes with mutations or association/linkage reported in individuals with CL/P, and considered them as candidate genes for human CL/P. The genotype-phenotype association study revealed that mutations in 12 genes ( ABCA4 , ADAM3A , FOXE1 , IRF6 , MSX2 , MTHFR , NTN1 , PAX7 , TP63 , TPM1 , VAX1 , and WNT9B ) were significantly associated with CL/P. In addition, our bioinformatics analysis predicted 16 microRNAs (miRNAs) to be post-transcriptional regulators of CL/P genes. To validate the bioinformatics results, the top six candidate miRNAs (miR-124-3p, miR-369-3p, miR-374a-5p, miR-374b-5p, miR-497-5p, and miR-655-3p) were evaluated by cell proliferation/survival assays and miRNA-gene regulation assays in cultured human lip fibroblasts. We found that miR-497-5p and miR-655-3p significantly suppressed cell proliferation in these cells. Furthermore, the expression of the predicted miRNA-target genes was significantly downregulated by either miR-497-5p or miR-655-3p mimic. Conclusion Expression of miR-497-5p and miR-655-3p suppresses cell proliferation through the regulation of human CL/P-candidate genes. This study provides insights into the role of miRNAs in the etiology of CL/P and suggests possible strategies for the diagnosis of CL/P. Electronic supplementary material The online version of this article (10.1186/s12920-019-0535-2) contains supplementar...
Background Cleft 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. Methods In 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. Results We 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. Conclusions Our 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. Electronic supplementary material The online version of this article (10.1186/s12920-019-0546-z) contains supplementary material, which is available to authorized users.
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