Figure 3: Identification of PIAS3 as a target of miR-199a-5p and the activation of STAT3 by miR-199a-5p.

(A) TargetScan prediction software identified one seed match for miR-199a-5p in the 3′UTR of PIAS3; the predicted seed-recognition site in the PIAS3 mRNA sequence and the corresponding miR-199a-5p sequence are marked in red. (B) The relative luciferase activity of the PIAS3 3′-UTR reporter plasmid was assayed in MNNG/HOS cells after transfection with pre/anti-miR-199a-5p or the corresponding control. (C) The relative mRNA level of PIAS3 in Saos-2 (left) or MNNG/HOS cells (right) after transfection with pre/anti-miR-199a-5p or the corresponding control. (D) The protein level of PIAS3 in Saos-2 (left) or MNNG/HOS cells (right) after transfection with pre/anti-miR-199a-5p or the corresponding control. (E) The protein level of PIAS3 in the eight pairs of NAT and OS samples, as determined using western blotting (left). Pearson’s correlation scatter plot comparing the fold changes in the expression of miR-199a-5p and the PIAS3 protein in OS patients (right). (F) The relative luciferase activity of the STAT3-dependent reporter construct in MNNG/HOS cells (left) and the protein levels of p-STAT3 and total STAT3 in Saos-2 and MNNG/HOS cells (right) after transfection with pre/anti-miR-199a-5p or the corresponding control. (G) The protein levels of PIAS3, p-STAT3 and total STAT3 in MNNG/HOS cells stably over-expressing PIAS3 and the control cells after transfection with pre-miR-199a-5p or the corresponding control. One representative image of three reproducible results is shown for the western blot assay. The data are expressed as the means ± SEM, **P < 0.01.