Figure 5.

miR-134-5p overexpression suppress the osteoclastogenesis process in BMMs.A, miR-134-5p agomir or its negative control agomir NC was transfected into BMMs, and the BMMs were then cultured in the presence of 30 ng/ml M-CSF and 50 mg/ml RANKL for 7 days. The transfection efficiency was analyzed by qRT-PCR. B, the cell viability of BMMs transfected with agomir or its negative control was detected by CCK-8 assay and measured at 450 nm under a microplate reader. C and D, cell apoptosis was determined with a TUNEL kit, and images were observed under a fluorescence microscope. The scale bars represent 100 μm. E and F, TRAP staining was used for the analysis of osteoclast activity. The number of TRAP-positive multinuclear osteoclasts was counted. The scale bars represent 100 μm. G and H, bone resorption ability was screened on bone slices through scanning electron microscope and bone resorption area was quantified with ImageJ software. The scale bars represent 50 μm. I and J, BMMs were induced to differentiate into osteoclasts and stained with phalloidin for the evaluation of actin ring formation. The number of F-actin rings per field was counted. The scale bars represent 100 μm. K and L, the protein expression of TRAP, CTSK, and NFATc1 was analyzed by Western blot. Semiquantitative analysis was conducted using ImageJ software. GAPDH was used as an internal reference control. M, the mRNA expression levels of TRAP, CTSK, and NFATc1 were analyzed by quantitative RT-PCR. β-Actin was used as an internal reference gene. All the data were expressed as the means ± SDs. ∗∗p < 0.01, ∗∗∗p < 0.001. BMMs, bone marrow macrophages; CCK-8, cell counting kit-8.