Abstract
Frontonasal malformations are caused by a failure in the growth of the frontonasal prominence during development. Although genetic studies have identified genes that are crucial for frontonasal development, it remains largely unknown how these genes are regulated during this process. Here, we show that microRNAs, which are short non-coding RNAs capable of targeting their target mRNAs for degradation or silencing their expression, play a crucial role in the regulation of genes related to frontonasal development in mice. Using the Mouse Genome Informatics (MGI) database, we curated a total of 25 mouse genes related to frontonasal malformations, including frontonasal hypoplasia, frontonasal dysplasia, and hypotelorism. MicroRNAs regulating the expression of these genes were predicted through bioinformatic analysis. We then experimentally evaluated the top three candidate miRNAs (miR-338-5p, miR-653-5p, and miR-374c-5p) for their effect on cell proliferation and target gene regulation in O9-1 cells, a neural crest cell line. Overexpression of these miRNAs significantly inhibited cell proliferation, and the genes related to frontonasal malformations (Alx1, Lrp2, and Sirt1 for miR-338-5p; Alx1, Cdc42, Sirt1, and Zic2 for miR-374c-5p; and Fgfr2, Pgap1, Rdh10, Sirt1, and Zic2 for miR-653-5p) were directly regulated by these miRNAs in a dose-dependent manner. Taken together, our results highlight miR-338-5p, miR-653-5p, and miR-374c-5p as pathogenic miRNAs related to the development of frontonasal malformations.