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. 2022 Jul 11;29:296–311. doi: 10.1016/j.omtn.2022.07.008

Figure 1.

Figure 1

Effects of rAAV9 carrying miR-214-3p or miR-214-3p TuD on osteoblast or osteoclast differentiation

(A) Diagram showing AAV vector genome that contains miR-214-3p and EGFP gene (top) or miR-214-3p TuD and Guassia luciferase gene (gLuc, bottom). CBA, CMV enhancer/chicken β-actin promoter. (B) Control (ctrl) or miR-214-3p plasmid was transfected into HEK293 cells and, 48 h later, expression of miR-214-3p was measured by RT-PCR and normalized to U6. (C) HEK293 cells were transfected with ctrl-sensor or miR-214-3p-sensor plasmid in the absence or presence of miR-214-3p plasmid and, 48 h later, β-galactosidase activity was measured and normalized to firefly luciferase. (D) The miR-214-3p plasmid was transfected into HEK293 cells along with the miR-214-3p-sensor plasmid and increasing concentrations of the miR-214-3p TuD plasmid. After 48 h, β-galactosidase activity was measured and normalized to firefly luciferase. (E–G) Mouse bone marrow-derived stromal cells (BMSCs) were transduced with rAAV9 carrying ctrl, miR-214-3p, or miR-214-3p TuD for 2 days and cultured under osteogenic conditions. ALP staining and activity (E) and expression of Bglap and Ibsp (F) were assessed at day 6 of culture. (G) Computational analysis showing the complementarities of miR-214-3p to the 3′ UTR of Atf4 (left). mRNA levels of Atf4 in miR-214-3p-expressing BMSCs were assessed by RT-PCR (right). (H–K) Two days after treatment with M-CSF and RANKL, mouse bone marrow monocytes (BMMs) were transduced with rAAV9 carrying ctrl, miR-214-3p, or miR-214-3p TuD, cultured under osteoclast differentiation conditions, and stained for TRAP. Representative images of TRAP-stained osteoclasts are shown (H), and the number of TRAP-positive osteoclasts assessed (I). mRNA levels of Rank and Acp5 were measured by RT-PCR and normalized to Actb (J). (K) The predicted consequential pairing of Pten 3′ UTR with miR-214-3p is shown (top) and Pten expression as measured by RT-PCR (bottom). Scale bar, 400 μm in (H). Values represent mean ± SD: ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by an unpaired two-tailed Student’s t test (B, G, and K) and one-way ANOVA test (C–F, I, and J).