Research Paper Volume 12, Issue 24 pp 25845—25864

The long noncoding RNA LINC01140/miR-140-5p/FGF9 axis modulates bladder cancer cell aggressiveness and macrophage M2 polarization

Figure 5. miR-140-5p directly targets LINC01140 and the FGF9 3'UTR. (A) miRNAs that might simultaneously target both LINC01140 and FGF9 3'UTR were predicted and selected by the online tools, mirDIP, TargetScan, starBase V3, microT-CDS, miRDB, and LncTar. Two miRNAs, miR-140-5p and miR-580-3p were selected. (B) The expression of miR-140-5p and miR-580-3p was determined in SV-HUC-1 and T24 cells by real-time PCR. P<0.01, student’s T test. (C) miR-140-5p was overexpressed or inhibited in T24 cells by the transfection of miR-140-5p or anti-miR-140-5p. The transfection efficiency was validated by real-time PCR. P<0.01, one way ANOVA test. (D) T24 cells were transfected with miR-140-5p or anti-miR-140-5p and examined for the protein levels of FGF9 by immunoblotting, P<0.01, one way ANOVA test. (E, F) Wild-type and mutant LINC01140 and FGF9 3'UTR luciferase reporter vectors were constructed as described in the Materials and methods section. These vectors were cotransfected with miR-140-5p or anti-miR-140-5p in 293T cells. The changes in luciferase activity were determined. P<0.01, one way ANOVA test. **P<0.01, #P<0.05, ##P<0.01.