Computational Identification and Experimental Validation of MicroRNAs Binding to the Fragile X Syndrome Gene Fmr1

MicroRNAs (miRNAs) usually bind to their target mRNAs through imperfect base pairing in the 3′-untranslated regions (3′ UTRs) and regulate target gene expression via post-transcriptional suppression. In recent years, computational approaches to predict miRNA targets have facilitated the identification of potential target sites. In this study, we used three programs TargetScan, miRDB and miRanda to predict potential miRNA binding sites to the fragile X gene Fmr1 and picked out 61 miRNAs which were predicted by all three programs for further investigation. Excitingly, 5 out of these miRNAs, miR-23a, miR-32, miR-124, miR-335-5p and miR-350, were experimentally verified by luciferase reporter assays. Furthermore, overexpression of miR-124 in mouse embryonic neural progenitor cells (eNPC) could not only significantly reduce Fmr1 level, but also increase Cdk4 and cyclin D1 levels which coincidently promoted eNPC proliferation. Our results imply that miR-124 plays an important role in the proliferation of mouse embryonic stem cells by promoting Cdk4 and cyclin D1 expression through directly inhibiting Fmr1 expression.