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. 2017 Jan 10;7:40487. doi: 10.1038/srep40487

Figure 4. MiR-214-3p targets Traf3 to functionally promote osteoclast differentiation in vitro.

Figure 4

(a) Sequence alignments between miR-214-3p and candidate binding sites in the 3′UTR of Traf3. (b) Schematic diagram illustrating the design of luciferase reporters with the WT Traf3 3′ UTR (WT 3′ UTR) or the site-directed mutant Traf3 3′ UTR (3′ UTR-Mut). The sequences of agomir-214-3p and a agomir-214-3p mutant (agomir-214-Mut) are also shown. Luc, luciferase. (c) The relationship between miR-214-3p level (top) and the mount of TRAF3 protein (bottom) during osteoclast differentiation in RAW264.7 cells. (d) The effects of agomir-214-3p and agomir-214-Mut on Traf3 mRNA levels (top) and the amount of TRAF3 protein (bottom) in RAW 264.7 cells. (e) The effect of agomir-214-3p and mutated agomir-214-3p on luciferase activity in RAW 264.7 cells transfected with either the WT the mutant Traf3 3′ UTR reporter (left) or Traf3 3′ UTR reporter (right). (f) The effect of antagomir-214-3p on luciferase activity in RAW 264.7 cells transfected with either the WT Traf3 3′ UTR reporter or the mutant Traf3 3′ UTR reporter. (g) Real-time PCR analysis of Trap and Ctsk mRNA levels (top) and western blot analysis of TRAF3 protein (bottom) in RAW264.7 cells after blockade of miR-214-3p binding to Traf3 by overexpression of WT Traf3 3′UTR (UTR). (h) Trap and Ctsk mRNA levels in RAW264.7 cells after the indicated treatment. The results from a scrambled control siRNA (siRNA-NC) and a mock transfection are also shown. (i) Western blot analysis of whole cell lysates of osteoclasts transfected with antagomir-214-3p and cultured with RANKL for 5 days. Note: All data are the mean ± s.d. from three independent experiments. *P < 0.05, NS, not significant.