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

Abstract

MIBC (muscle invasive bladder cancer) only accounts for only a minority of bladder cancers, however, the disease-specific and overall survival rates of patients with MIBC are low. Macrophage M2 polarization has been reported to be associated with poorer prognosis in bladder cancer. Through cancer bioinformatics and experimental analyses, FGF9 was found to be upregulated in MIBC tissues. FGF9 knockdown in T24 cells strongly suppressed the viability, migratory capacity, and invasive capacity of cells; culture with medium from FGF9 knockdown T24 cells (si-FGF9-CM) significantly inhibited macrophage M2 polarization, while promoting M1 polarization. The long noncoding RNA (lncRNA) LINC01140 was positively correlated with FGF9 and was significantly upregulated in MIBC tissues. LINC01140 knockdown inhibited the viability, migratory capacity and invasive capacity of T24 cells; culture in si-LINC01140-CM also inhibited macrophage M2 polarization, while promoting M1 polarization. LINC01140 targeted miR-140-5p, while miR-140-5p targeted FGF9 to form a lncRNA-miRNA-mRNA axis. The effects of miR-140-5p inhibition on bladder cancer aggressiveness and macrophage M2 polarization were opposite to those of LINC01140 or FGF9 knockdown; additionally, miR-140-5p inhibition partially reversed the effects of LINC01140 knockdown on FGF9 protein levels, bladder cancer phenotype, and macrophage M2 polarization. In conclusion, LINC01140, miR-140-5p, and FGF9 form a lncRNA-miRNA-mRNA axis that modulates the bladder cancer phenotype, affects macrophage M2 polarization through the tumor microenvironment, and in turn affects bladder cancer cell aggressiveness.

Keywords: FGF9; MIBC (muscle invasive bladder cancer); long non-coding RNA LINC01140; macrophage M2 polarization; miR-140-5p.

Figure 1

Expression of FGF9 in muscular invasive bladder cancer (MIBC) and non-muscular invasive bladder cancer (NMIBC) tissues. (A) The expression of FGF9 in MIBC and NMIBC tissues based on the data from GSE77952. P=0.007, student’s T test. (B) The overall survival of patients with bladder cancer was analyzed by grouping the samples based on FGF9 expression using log-rank test. Data are based on TCGA database. (C) The pathological changes in MIBC and NMIBC tissues was shown by H&E staining. (D) The expression of FGF9 in tissues of 12 cases of MIBC and 12 cases of NMIBC was determined using real-time PCR. P<0.01, paired student’s T test. (E) The protein levels of FGF9 in tissues of 12 cases of MIBC and 12 cases of NMIBC were determined using immunoblotting. P<0.01, paired student’s T test. (F) The protein distribution of CD163 (M2 macrophage marker) was determined in MIBC and NMIBC tissues using immunofluorescence (IF) staining.

Figure 2

Effects of FGF9 on bladder cancer cell aggressiveness and macrophage M2 polarization. (A) FGF9 knockdown was generated in the T24 bladder cancer cell line by transfection of si-FGF9. The transfection efficiency was validated by real-time PCR. P<0.01, student’s T test. Next, T24 cells were transfected with si-FGF9 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) the 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. (F) Monocytes were isolated from peripheral blood (PBMCs) and treated with 50 ng/ml M-CSF to stimulate the monocyte differentiation into macrophages (M0). The M0 macrophages were identified as CD11b positive by flow cytometry. (G) M0 macrophages were then stimulated with 20 ng/ml IL-4 (eBioscience) for two days to induce M2 polarization and authenticated using IF staining with anti-CD11b and anti-CD206 antibodies. The inflorescence intensity is shown in the right panel. P<0.01, student’s T test. (H–K) Next, T24 cells were transfected with si-FGF9 or si-NC (negative control) and the culture medium (shown in the figures as conditioned medium, si-NC-CM and si-FGF9-CM) was collected for macrophage incubation. M0 macrophages were divided into four groups: IL-4 (M2 polarization inducing) + si-NC-CM, IL-4 (M2 polarization inducing) + si-FGF9-CM, LPS + IFNγ (M1 polarization inducing) + si-NC-CM, and LPS + IFNγ (M1 polarization inducing) + si-FGF9-CM, and examined for (H) the protein levels of CD206 and CD16 by immunoblotting. P<0.05 or P<0.01, student’s T test.; (I) the percentage of CD206 and CD16 positive cells was determined by flow cytometry; (J) the inflorescence intensity of CD206 and CD16 was measured by IF staining. The inflorescence intensity is shown in the right panel. P<0.01, student’s T test. (K) The concentrations of IL-10, Arg1, iNOS, and TNF-α in the culture medium was determined by ELISA. **P<0.01, student’s T test.

Figure 3

Expression of lncRNA LINC01140 in NMIBC and MIBC tissues. (A) The expression of lncRNA LINC01140 in MIBC and NMIBC tissues based on the data from GSE77952. P=0.0295, student’s T test. (B) The overall survival of patients with bladder cancer was analyzed by grouping the samples based on LINC01140 expression using log-rank test. Data are based on TCGA database. (C) The expression of LINC01140 in tissues of 12 cases of MIBC and 12 cases of NMIBC was determined using real-time PCR. P<0.01, paired student’s T test. (D) The correlation between LINC01140 and FGF9 expression in tissue samples was analyzed using Pearson’s correlation analysis. **P<0.01.

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.

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.

Figure 6

Dynamic effects of LINC01140 and miR-140-5p on bladder cancer cell aggressiveness and macrophage M2 polarization T24 cells were cotransfected with si-LINC01140 and anti-miR-140-5p and examined for (A) the cell viability by MTT assay; (B) protein levels of FGF9, ki-67, MMP-2, and MMP-9 by immunoblotting; (C) migration capacity by wound healing assay; and (D) invasive capacity by Transwell assay. (E, F) T24 cells were cotransfected with si-LINC01140 and anti-miR-140-5p and the culture medium (shown in the figures as conditioned medium (CM) si-NC + anti-NC, si-LINC01140 + anti-NC, si-NC + anti-miR-140-5p, and si-LINC01140 + anti-miR-140-5p) was collected for macrophage incubation. M0 macrophages were cultured in the above-described four kinds of CMs and polarized to M1 or M2, respectively, and examined for (E) the protein levels of CD206 and CD16 by immunoblotting. (F) The concentrations of IL-10, Arg1, iNOS, and TNF-α in the macrophage culture medium was determined by ELISA. *P<0.05, **P<0.01, compared to control group; #P<0.05, ##P<0.01, compared to si-NC + anti-miR-140-5p group. One way ANOVA test was used for statistical analysis.

Figure 7

Schematic diagram of the proposed mechanism. LINC01140/miR-140-5p/FGF9 axis modulates bladder cancer phenotype and promotes macrophage M2 polarization through tumor microenvironment.