Inhibition of a MicroRNA From Amelogenin Exon4 and Enamel Mineralization

Objectives: Alternative splicing of amelogenin exon4 is key to forming major functional amelogenin isoforms critical for enamel formation. When amelogenin exon4 is spliced out, a microRNA (miR-exon4) is derived from the spliced exon4. Our previous studies suggest the possible involvement of miR-exon4 in enamel mineralization and amelogenin exon4 splicing. This study aims to quantitatively assess the effect of miR-exon4 inhibition on enamel mineralization in vivo and explore the potential mechanism of miR-exon4 involved in amelogenin exon4 splicing.
Methods: A miR-exon4 inhibitor was injected into mouse pups at postnatal days 4 (P4) and P7. At P11, mandibles were collected and analyzed via microCT to examine the change in the mineralization progress within developing enamel. Immunohistochemistry was performed to investigate RUNX2, a downstream target of miR-exon4 and critical for enamel formation. For some pups, miR-exon4 inhibitor was injected at P4, and RNA was extracted from the first molar enamel organ after 24 hours of primary injection to assess exon4 splicing.
Results: The microCT analysis revealed the inhibitory effect of miR-exon4 inhibitor on enamel mineralization, seen as a significant decrease in the matured enamel portion within molars and incisors and a significantly reduced mineral density in incisor enamel in earlier stages of development. Immunohistochemical staining showed an apparent reduction in RUNX2 in both secretory and maturation ameloblasts caused by the miR-exon4 inhibitor.
Our in-silico analysis predicted some Serine/Arginine-rich splicing factors (SFSFs) as direct targets of miR-exon4. We selected four SRSFs potentially involved in amelogenin exon4 alternative splicing. When exon4 splicing was reduced after 24 hours of miR-exon4 inhibition, the mRNA expression of Srsfs 2, 3, 6, and TRA2b were significantly altered.
Conclusions: We confirmed that miR-exon4 plays a significant role in enamel mineralization in vivo, possibly associated with the reduced RUNX2 expression. Also, miR-exon4 regulates Srsf expression in vivo, which can be related to exon4 splicing regulation.