Fig 2: Summary of miR-exon4, Runx2, Nfia, and Prkch expression and molecular cascade.A, changes in miR-exon4 and mRNA expression, which were observed under the different genetic and experimental conditions in osteoblasts and ameloblasts in vivo and in vitro, are summarized. In all experimental models, when miR-exon4 expression changes, Runx2, Nfia, and Prkch also change accordingly. *Previously published data (11). B, the four molecule's expression changes are summarized in the miR-exon4-Runx2 axis (top). It supports the suggested regulatory relationship of the molecules in the axis confirmed through the experiments (bottom). Solid line: direct regulation; dashed line: indirect regulation.

Fig 3: Protein and mRNA levels of NFI/A in subclone mi2. Western blotting for NFI/A at day 3 (A and B) and 9 (C and D). The intensity of each band for NFI/A was normalized by the corresponding lane's total protein (A and C). The normalized intensity compared between nontransduced and mi2 (B and D) demonstrates significant upregulation of NFI/A in mi2 at day 9 (*p < 0.05) but not at day 3. Statistical significance was analyzed by independent Student's t tests following F test. Consistent with protein, Nfia mRNA was also upregulated in the subclone mi2 at day 9 compared with nontransduced control (**p < 0.01) and negative control (##p < 0.01) (F), whereas there is no change in the samples of day 3 (E). Statistical significance was analyzed by the two-tailed multiple t tests with Bonferroni correction following ANOVA. The biological replication of each experimental group is shown as the points in each plot.

Fig 4: miR-exon4 targets Nfia. A and B, immunohistochemical staining on mixed (C57BL/6J × SJL) mouse bone (A) and enamel organ (B) shows apparent positive signals on the nucleus of osteoblasts (A, arrows) and ameloblasts (B, Am). Bars represent 25 µm. C, incubation of 3'UTR Nfia luciferase reporter and miR-exon4 mimic significantly reduced the luciferase activity compared with a reporter only (*p < 0.05) and reporter plus negative siRNA (#p < 0.05) controls. D, when transfection of siRNA for Nfia in MC3T3-E1 osteoblasts significantly reduced Nfia compared with nontransfection control (**p < 0.01) and negative control (##p < 0.01). Runx2 expression is significantly upregulated compared with nontransfection control (*p < 0.05) and negative control (#p < 0.05). The biological replication of each experimental group is shown as the points in each plot. Statistical significance was analyzed by the two-tailed multiple t tests with Bonferroni correction following ANOVA.