Figure 4.

CtBP1 is a direct target of miR-485-3p. The relative miR-485-3p level in hFOB1.19, U2OS, MG63, Saos-2 and HOS cells determined by qRT-PCR. **P<0.001. (B) Schematic representation of CtBP1 3′-UTR contained a putative miR-485-3p binding site. The binding position of miR-485-3p in the 3'-UTR of CtBP1 is shown by the red arrow. The seed location of miR-485-3 is indicated with the red font. The wild-type (WT) and mutant (Mut) 3′-UTRs of CtBP1 were shown. The following combinations of vectors: (a) no transfection, (b) miR-NC (negative control), (c) miR-NC + pCDNA3-CtBP1-3′-UTR^WT, (d) miR-NC + pCDNA3-CtBP1-3′-UTR^Mut, (e) miR-485-3p-mimic, (f) miR-485-3p-mimic + pCDNA3-CtBP1-3′-UTR^WT, (g) miR-485-3p-mimic + pCDNA3-CtBP1-3′-UTR^Mut, (h) anti-miR-485-3p, (i) anti-miR-485-3p + pCDNA3-CtBP1-3′-UTR^WT, and (j) anti-miR-485-3p + pCDNA3-CtBP1-3′-UTR^Mut, were transfected into U2OS cells, respectively. Then, qRT-PCR was performed to examine the miR-485-3p level (C) and CtBP1 mRNA level (D). **P<0.001. (E) Western blot was performed to detect the protein levels of CtBP1 and CtBP2 in cells used in (C). GAPDH was used as a loading control. (F) The miR-485-3p failed to bind the mutated 3′-UTR of CtBP1. The following combinations of plasmids were transfected into U2OS cells, respectively. (a) pGL3-CtBP1-3′-UTR^WT + pRL-TK-Renilla + miR-NC; (b) pGL3-CtBP1-3′-UTR^WT + pRL-TK-Renilla + miR-485-3p-mimic; (c) pGL3-CtBP1-3′-UTR^WT + pRL-TK-Renilla + anti-miR-485-3p; (d) pGL3-CtBP1-3′-UTR^Mut + pRL-TK-Renilla + miR-NC; (e) pGL3-CtBP1-3′-UTR^Mut + pRL-TK-Renilla + miR-485-3p-mimic; and (f) pGL3-CtBP1-3′-UTR^Mut + pRL-TK-Renilla + anti-miR-485-3p. The luciferase activity was measured using a Dual-Luciferase Reporter Assay System. ** P < 0.001.