Researchers at Dalian Medical University used C57BL/6J mice to establish the RA induced cleft palate mouse model. Nine pairs of tissues were obtained on embryonic day 15.5 (E15.5). Total RNA was extracted and miRNA microarray was used to screen the miRNAs. Real-time quantitative PCR (RT-qPCR) was used to verify the differential miRNA results obtained from microarray. Cleft palate related genes targeted by the miRNAs were predicted by TargetScan and miRTarBase, while functional annotation clustering of Gene Ontology (GO) terms and KEGG signaling pathways in DAVID were used to analyze these target genes.
1265 miRNAs were identified in cleft palate, and among them 31 were differentially expressed (p < 0.01, fold change > 1.5), including 17 up-regulated and 14 down-regulated miRNAs in cleft palate. 7 up-regulated miRNAs (mmu-miR- 181a-5p, mmu-miR-410-3p, mmu-miR-3960, mmu-miR-1224-5p, mmu-miR-3970, mmu-let-7e-5p and mmu-miR-1907) and 3 down-regulated miRNAs (mmu-miR-140-3p, mmu-miR-351-5p and mmu-miR-503-5p) were validated by RT-qPCR, and mmumiR-181a-5p and mmu-miR-410-3p were in concordance with those of miRNA microarray chip detection. 484 target genes were predicted and proven by TargetScan and miRTarBase. GO terms showed that RA-induced cleft palate was associated with enrichments in miRNAs involved in embryo development, osteoblast differentiation and so on. KEGG signaling pathway analysis indicated that the differentially expressed miRNAs were involved in MAPK, TGFβ and WNT signaling pathways.
This study has identified 10 differentially expressed miRNAs in cleft palate. These miRNAs and their target genes may become new therapeutic targets for cleft palate.