Figure 4.

Inhibition of miR-208a-3p Resumes Mechanical Unloading Reduced ACVR1 and Osteoblast Differentiation

(A) qPCR analysis of miR-208a-3p level in MC3T3-E1 cells after treatment with mock, antagomir-NC, and antagomir-208a-3p for 48 hr under RPM unloading condition. (B) Effect of mock, negative control, and antagomiR-208a-3p transfection for 48 hr on ACVR1 mRNA levels in osteoblast cells under RPM unloading conditions. (C) miR-208a-3p expression in osteoblasts for 48 hr under RPM unloading conditions without antagomiR-208a-3p transfection. (D) ACVR1 expression in osteoblasts for 48 hr under RPM unloading conditions without antagomiR-208a-3p transfection. (E) Effect of antagomir-208a-3p on ALP, Col Ia1 Ocn, and Runx2 mRNA levels in MC3T3-E1 cells for 48 hr under RPM unloading conditions. (F) Western blot analysis of ACVR1 protein in MC3T3-E1 cells after transfection with mock, negative control, and antagomiR-208a-3p for 48 hr under RPM unloading conditions. (G) Calcium deposition staining and area of mineralization with alizarin red in MC3T3-E1 cells under RPM for different time periods (the cells were seeded on coverslips). All data were expressed as means ± SD. Significance is noted at these thresholds: *p < 0.05, **p < 0.01, ***p < 0.001. One-way ANOVA with a post hoc test was performed. Statistical differences between two groups were determined by Student’s t test.