Fig 2: miR-542-3p directly targets sFRP-1 and Smurf2. (A) RT-qPCR analyses of sFRP-1 mRNA expression in bone samples from the MTX time course study (n = 6/time point). The expression of sFRP-1 was elevated on day 3 and then declined to the near normal level by day 6. (B) RT-qPCR analyses of Smurf2 mRNA expression in bone samples. The expression of Smurf2 was pronouncedly downregulated throughout the MTX time-course study. (C) (left) Schematic diagram of the putative miR-542-3p seed sequence in the 3′ UTR of the sFRP-1; (right) relative luminescence activity in the dual luciferase reporter assay. Cells co-transfected with miR-542-3p agomir and pmirGLO-sFRP-1 wild-type vector have shown considerably decreased luciferase activity. (D) (left) Schematic diagram of the putative miR-542-3p seed sequence in the 3′ UTR of the Smurf2; (right) relative luminescence activity in the dual luciferase reporter assay. Cells co-transfected with miR-542-3p agomir and pmirGLO-Smurf2 wild-type vector have shown considerably decreased luciferase activity. Statistical significance analysis was performed via one-way ANOVA followed by Dunnett’s post-test. Significant values are marked * p < 0.05, ** p < 0.01, and **** p < 0.0005. WT: vector with wild-type sFRP-1/Smurf2 seed region; NC: negative control; Ago: miR-542-3p agomir; MUT: vector with mutated sFRP-1/Smurf2 seed region.

Fig 3: Schematic representation of the role and as a potential therapeutic target for miR-542-3p in MTX treatment-induced bone/fat switch. Methotrexate (MTX) chemotherapy decreases osteogenesis but increases adipogenesis (bone/fat switch) in the bone marrow, which may be associated with reduction in miR-542-3p expression. The secreted frizzled related protein 1 (sFPR-1) and SMAD specific E3 ubiquitin protein ligase 2 (Smurf2) as direct targets of miR-542-3p identified in the current study are known as antagonists for the Wnt/β-catenin signalling and TGF-β signalling. Treatment of miR-542-3p in preosteoblasts and preadipocytes after MTX treatment can attenuate MTX-induced bone/fat switch, which might be linked with preservation of Wnt/β-catenin signalling and suppression of TGF-β signalling.