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 4. Effects of LINC01140 on bladder cancer cell aggressiveness and macrophage M2 polarization. (A) LINC01140 knockdown was generated in the T24 bladder cancer cell line by transfection with si-LINC01140. The transfection efficiency was validated by real-time PCR (P<0.01, student’s T test). Next, T24 cells were transfected with si-LINC01140 and examined for (B) cell viability by MTT assay, **P<0.01, one-way ANOVA test.; (C) migration capacity by wound healing assay, P<0.05, student’s T test; (D) invasive capacity by Transwell assay, P<0.05, student’s T test; and (E) protein levels of FGF9, ki-67, MMP-2, and MMP-9 by immunoblotting, P<0.01, student’s T test. T24 cells were transfected with si-LINC01140 or si-NC (negative control) and the culture medium (shown in the figures as conditioned medium, si-NC-CM and si-LINC01140-CM) was collected for macrophage incubation. Monocytes were treated with 50 ng/ml M-CSF to stimulate monocyte differentiation into M0 macrophages. M0 macrophages were divided into four groups: IL-4 (M2 polarization inducing) + si-NC-CM, IL-4 (M2 polarization inducing) + si-LINC01140-CM, LPS + IFNγ (M1 polarization inducing) + si-NC-CM, and LPS + IFNγ (M1 polarization inducing) + si-LINC01140-CM, and examined for (F) the protein levels of CD206 and CD16 by immunoblotting, P<0.01, student’s T test; (G) the percentage of CD206 and CD16-positive cells was determined by flow cytometry; (H) the inflorescence intensity of CD206 and CD16 were measured by IF staining, The inflorescence intensity is shown in the right panel. P<0.01, student’s T test. (I) The concentrations of IL-10, Arg1, iNOS, and TNF-α in the macrophage culture medium was determined by ELISA. **P<0.01, student’s T test.