Table 1.
Characteristics of 108 bladder cancer patients treated with cisplatin-based adjuvant chemotherapy.
Fig 1.
Expression and prognosis of miR-203 for bladder cancer patients treated with cisplatin-based chemotherapy.
(A) miR-203 levels were detected by RT-qPCR method and normalized against U6 RNA in 108 cases of bladder cancer tissues. Levels of miR-203 in progression group were significantly lower than those in non-progression group (P<0.001, Mann-Whitney U test). (B) ROC curve distinguished patients with progression from those without progression using miR-203, with an area under the ROC curve value of 0.839 (95% CI, 0.756–0.903). (C, D) Kaplan-Meier PFS and OS curves based on miR-203 expression of bladder cancer patients. The optimal cut off value (0.345) calculated by ROC analysis was used to classify the patients as high and low miR-203 expression groups. Low expression of miR-203 was significantly correlated with shortened PFS or OS (Both P<0.001, log-rank test).
Table 2.
Associations between miR-203 and clinicopathological characteristics.
Table 3.
Univariate and multivariate Cox analysis of progression free survival and overall survival for bladder cancer patients.
Fig 2.
miR-203 enhances cytotoxicity of cisplatin on 5637 and T24 bladder cancer cells.
(A) miR-203 expression levels in 5637 and T24 cells transfected with miR-203 mimics/negative control. miR-203 levels were significantly increased in cells transfected with miR-203 mimics compared with cells transfected with negative control (*P<0.001, t test, n = 6). (B, C) Concentration-dependent curves for 5637 and T24 cell lines transfected with miR-203 mimics and negative control at 24h. Cell viabilities of miR-203-overexpressing cells were dramatically reduced when compared with negative control cells at 5, 10, 15, 20, 25 and 30μM cisplatin (all at P<0.05, t test). (D) Flow cytometry analysis for apoptosis via double staining of cells with Annexin V FITC and propidium iodide (PI). (E) Quantification analysis of apoptosis shown in Fig 2B. Overexpression of miR-203 significantly augmented apoptosis in 5637 and T24 cell lines (*P<0.01, t test, n = 6). (F) TUNEL assay indicated 5637 and T24 cell lines transfected with miR-203 mimics showed elevated levels of DNA cleavage compared with normal control (40×). Cells were stained with DAPI and subjected to TUNEL assay to detect DNA and apoptotic cells.
Fig 3.
Bcl-w and Survivin are direct downstream targets of miR-203.
(A) Illustration of the predicted miR-203 targeting sites in 3’UTR regions of Bcl-w and Survivin. (B) Dual-Luciferase activity assay was performed in HEK293T cells co-transfected with control/ miR-203 and pmiR-REPORT vectors with wild-type/mutant 3′-UTR of Bcl-w (above) and Survivin (below). *P<0.01, t test, n = 6. (C) Western blots showing downregulation of Bcl-w and Survivin proteins after transfection of miR-203 mimics in 5637 and T24 cell lines. β-actin was used as a control. (D) RT-qPCR showing downregulation of Bcl-w and Survivin mRNAs after transfection of miR-203 mimics in 5637 and T24 cell lines. *P<0.01, t test, n = 6. (E) Spearman’s correlation analysis showing a significant inverse association between miR-203 and Bcl-w mRNA or Survivin mRNA expression in bladder cancer tissues(r = -0.781, -0.740, both at P<0.001).