Fig 2: E2 stimulates OPG expression through its 3'-UTR. MG-63 cells were transfected with the control vector, pmirGLO, or the pmirGLO vector harboring the 3'-UTR of OPG. A total of 24 h post-transfection, the cells were treated with the indicated concentrations of E2 for 48 h, subsequently the luciferase activities were examined by dual luciferase assay. The results were normalized to Renilla activity, and the untreated group was normalized as 1. **P<0.01 vs. control. E2, 17ß-estradiol; mut, mutant; OPG, osteoprotegerin; UTR, untranslated region; wt, wild type.

Fig 3: miR-145 targets the 3'-UTR of OPG and suppresses its expression. (A-C) miR-145 mimics or ASO-miR-145 were co-transfected with pmirGLO or pmirGLO vectors containing wt or mut 3'-UTRs of OPG into MG-63 cells. Following 48 h, cells were harvested and assayed for dual luciferase activity. The results were normalized to Renilla activity. The effects of miR-145 on endogenous OPG expression were determined by (D) reverse transcription-quantitative polymerase chain reaction and (E and F) western blotting. The mRNA and protein expression levels of OPG were normalized to the levels of ß-actin mRNA and GAPDH protein, respectively. **P<0.01 vs. control. ASO, antisense oligonucleotide; miR, microRNA; NC, negative control; OPG, osteoprotegerin; UTR, untranslated region.

Fig 4: Chemerin suppressed breast cancer cell-induced increase in the RANKL/OPG ratio in osteoblastic cells. (A) The viability of hFOB1.19 osteoblastic cells treated with chemerin for 6 and 24 h; (B) Protein expression and (C) secretion levels of RANKL and OPG in hFOB1.10 osteoblastic cells treated with chemerin in the presence of the conditioned medium (CM) of MDA-MB-231 cells for 6 h and 24 h, respectively. The expression and secretion levels of RANKL and OPG proteins were detected by Western blotting and ELISA, respectively. # p < 0.05, ## p < 0.01 versus cells without CM and chemerin, * p < 0.05, ** p < 0.01 versus cells treated with CM alone.

Fig 5: E2 decreases mature miR-145 levels. (A) miRNA target sites in the OPG 3'-UTR were analyzed by TargetScan. The potential miR-145 site is presented. (B) MG-63 cells were treated with the indicated concentrations of E2 for 48 h, miR-145 levels were determined by reverse transcription-quantitative polymerase chain reaction. U6 was used as an internal control. **P<0.01 vs. control. E2, 17ß-estradiol; miR, microRNA; OPG, osteoprotegerin; TNFRSF11B, TNF receptor superfamily member 11b; UTR, untranslated region.