Research Paper Volume 14, Issue 10 pp 4486—4499

Figure 5. miR-128-3p regulates the migration and osteoclast differentiation of RAW 264.7 cells by targeting NFAT5. (A) StarBase database showing the putative binding sites between miR-128-3p and NFAT5. The relative luciferase activity was detected in HEK-293T cells transfected with agomiR-NC or agomiR-128-3p in WT and MUT groups, respectively. (B–D) The mRNA and protein expression of NFAT5 after transfection of miR-128-3p mimic or miR-128-3p inhibitor, were analyzed by qRT-PCR and Western blotting, respectively. (E–G) The mRNA and protein expression of NFAT5 after transfection of pcDNA-KCNQ1OT1 or si-KCNQ1OT1, were analyzed by qRT-PCR and Western blotting, respectively. (H) Relative expression of NFAT5 in non-osteoporotic and osteoporotic bone tissues was detected by qRT-PCR (N = 6, 3 osteoporotic and 3 non-osteoporotic). (I) The influence on cell migration ability of si-NFAT5 and the rescue effect of miR-128-3p inhibitor was assessed by transwell migration assay (Scale bar: 100 μm). (J) Multinucleated osteoclasts were stained by TRAP and then counted (Scale bar: 200 μm). (K–P) Five days after osteoclastic differentiation, qRT-PCR and Western blotting were performed to detect the mRNA and protein levels of NFAT5, c-Fos, NFATc1, and Ctsk in RAW 264.7 cells transfected with si-NFAT5, miR-128-3p inhibitor or si-NFAT5 + miR-128-3p inhibitor. ^*P < 0.05, ^**P < 0.01; ^***P < 0.001, ns: not significant.