Figure 6.

Itgb1 is a direct target of miR-134-5p.A, diagram showing the potential targets of miR-134-5p predicted by TargetScan, miRDB, and miRWalk. Crosschecking of these data indicated six potential targets of miR-134-5p. B and C, the mRNA expression levels of Rab27a, Serpine1, Hyal1, Itgb1, Antxr1, and Golm1 in BMMs with miR-134-5p overexpression or knockdown were measured by qRT-PCR. β-Actin was used as an internal reference gene. D, bioinformatics prediction of miR-134-5p binding sites in the 3′-UTR of Itgb1 mRNA. E, the luciferase activities of a reporter containing the 3′-UTR of Itgb1 in miR-134-5p-transduced, miR-134-5p-mutant-transduced, or negative control 293T cells were determined. F, the mRNA expression level of Itgb1 was determined at 1, 3, and 7 days as BMMs were induced into osteoclasts. β-Actin was used as an internal reference gene. G and H, the protein expression levels of Itgb1 were determined by Western blot. I and J, immunofluorescence staining was performed to detect Itgb1 expression after miR-134-5p overexpression or knockdown. The relative fluorescence intensity was measured. The scale bars represent 25 μm. Κ, immunohistochemistry staining of sham and OVX mouse bone tissue was performed to evaluate Itgb1 expression in vivo. The number of Itgb1-positive cells was measured (n = 6). The scale bars for main panels represent 200 μm. The scale bars for the zoom magnification panels represent 50 μm. All the data are expressed as the means ± SDs. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. BMMs, bone marrow macrophages; OVX, ovariectomized.