miR-150 modulates cisplatin chemosensitivity and invasiveness of muscle-invasive bladder cancer cells via targeting PDCD4 in vitro

Abstract

Background: Chemotherapeutic insensitivity and tumor cell invasiveness are major obstacles to effectively treating muscle-invasive bladder cancer (MIBC). Recent reports show that microRNAs (miRNAs) play an important role in the chemotherapeutic response and disease progression of MIBC. Therefore, here we investigated the role of miR-150 in MIBC cells in vitro.

Material and methods: miR-150 expression was quantified by qRT-PCR in two MIBC cell lines (5637 and T24). After successful miR-150 inhibition by transfection, MTS and transwell assays were used to assess the MIBC's cisplatin sensitivity and cell invasiveness, respectively. The TargetScan database and a luciferase reporter system were used to identify whether the programmed cell death 4 protein (PDCD4) is a direct target of miR-150 in MIBC cells.

Results: miR-150 expression was found to be significantly increased in both MIBC cell lines, and treatment with a miR-150 inhibitor significantly sensitized MIBC cells to cisplatin and inhibited MIBC cell invasiveness. PDCD4 was identified as a direct target of miR-150 in MIBC cells, and increased PDCD4 expression via transfection with the pLEX-PDCD4 plasmid efficiently sensitized MIBC cells to cisplatin chemotherapy and inhibited MIBC cell invasiveness.

Conclusions: This study provides novel evidence that miR-150 functions as a tumor promoter in reducing chemosensitivity and promoting invasiveness of MIBC cells via targeting PDCD4. Thus, modulation of the miR-150-PDCD4 axis shows promise as a therapeutic strategy for MIBC.

Figure 1

miR-150 inhibition enhances chemosensitivity to Cisplatin and suppresses invasiveness of MIBC cells. (A) miR-150 expression in MIBC cell lines tested by qRT-PCR. (B) Dose-inhibition rate curves plotted from three independent MTS assays showing the effect of miR-150 inhibition on MIBC cells’ chemosensitivity to cisplatin. (C) IC50 values affected by miR-150 inhibition. (D) Transwell assay performed on the control cell lines and miR-150-inhibited cell lines. * p<0.01.

Figure 2

miR-150 targets PDCD4 in MIBC cells. (A) Schematic of the broadly-conserved miR-150 target site in the human PDCD4 3′UTR. (B) Inverse relationship between miR-150 expression and PDCD4 protein levels. (C) miR-150 inhibitor increases PDCD4 protein expression in MIBC cells. (D, E) miR-150 suppresses the luciferase activity linked to the 3′UTR of PDCD4 with a Renilla luciferase reporter used for normalization. Data were obtained from three independent experiments, and the mean of the results from (D) pMir-control-transfected SV-HUC-1 cells and (E) pMir-control-transfected HEK293T cells were set at 100%. * p<0.01.

Figure 3

PDCD4 enhances chemosensitivity to Cisplatin and suppresses invasiveness of MIBC cells. (A) PDCD4 expression increased after pLEX-PDCD4 transfection. (B) Dose-inhibition rate curves were plotted from three independent MTS assays, showing that restoring PDCD4 expression significantly decreased cell viability upon cisplatin treatment. (C) IC50 values decreased by restoring PDCD4 expression. (D) Transwell assay performed on the control cell lines and PDCD4-restored cell lines. * p<0.01.