Abstract
BackgroundThe 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.MethodsTo 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.ResultsThrough 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.ConclusionExpression 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.
Highlights
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
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
We investigated whether single nucleotide polymorphisms (SNPs) mapped to the CL/ P-candidate genes are associated with human CL/P phenotypes using Transmission disequilibrium test (TDT) of The Genetics of Orofacial Clefts and Related Phenotypes Genome-wide association studies (GWAS) dataset
Summary
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. The contribution of microRNAs (miRNAs), non-coding RNAs that regulate the expression of multiple genes, to the etiology of CL/P is largely unknown. Much progress has been made in identifying genes whose mutations are associated with CL/P, little is known about the mechanisms by which environmental and epigenetic factors adversely influence gene expression during lip development. Recent studies indicate that environmental factors contribute to changes in phenotype or gene expression at post-transcriptional level through the regulation of noncoding RNAs, including microRNAs (miRNAs) [9]. An investigation of the functional regulation, at the level of biological pathways and posttranscriptional regulation mechanism, will improve our understanding of genetic susceptibility to CL/P [7, 15]
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