MicroRNA-dependent Posttranscriptional Regulation in Autosomal Recessive Radicular Dentin Dysplasia

MicroRNAs (miRNAs) are small noncoding RNAs that bind to specific mRNA 3’ untranslated regions (UTR) and function as posttranscriptional regulators in development, differentiation and disease. We have identified a 3’UTR alteration in the tooth root master gene NFI-C associated with a novel rare autosomal recessive (AR) form of Radicular Dentin Dysplasia (RDD), a disease characterized by missing roots.  Objectives: To characterize the NFI-C 3’UTR and determine the functional role of the AR-RDD alteration on mRNA stability, miRNA regulation and protein level. Methods: AR-RDD and unaffected control dental pulp (DP) cells were grown and harvested for qRT-PCR, immunocytochemistry (ICC), Western blotting (WB), mRNA stability assay, and miRNA PCR array. The 3’UTR region of NFI-C from multiple species was identified (Ensembl) and the degree of conservation determined (ClustalW). Target prediction algorithms (TargetScan, PITA, miRanda, & Micro Inspector) were used to identify potential miRNA binding sites. ICC and WB were performed for NFI-C cellular localization and relative protein levels, respectively.  Results: AD-RDD-DP cells were morphologically normal with no alterations in NFI-C cellular localization or isoform expression profile. The identified NFI-C 3’UTR alteration is within a highly conserved element and results in decreased mRNA stability (> 8 hrs) and protein levels (2.2 fold lower).  Furthermore, miRNA PCR array data showed differentially expressed miRNAs levels in the AD-RDD-DP cell populations as compared to the control DP. Of the 88 development and differentiation associated miRNAs tested, 15 were downregulated (>2 fold) and 8 were upregulated (> 2 fold) in AR-RDD cells. These miRNAs have targets in genes known to be dysregulated in AR-RDD.  Conclusions: Our studies shows for the first time NFI-C is potentially posttranscriptionally regulated by a unique conserved 3’UTR element and that an AR-RDD alteration results in decrease mRNA stability and protein levels resulting in altered signaling cascades critical for root formation. Support: UAB-IOHR/NIDCR-F30DE0180803.