MicroRNA-1826 targets VEGFC, beta-catenin (CTNNB1) and MEK1 (MAP2K1) in human bladder cancer

Abstract

The Wnt/beta-catenin (CTNNB1) and Ras-Raf-MEK-ERK signaling pathway play an important role in bladder cancer (BC) progression. Tumor-suppressive microRNAs (miRNAs) targeting these cancer pathways may provide a new therapeutic approach for BC. We initially identified miRNA-1826 potentially targeting CTNNB1, VEGFC and MEK1 using several target scan algorithms. Also 3' untranslated region luciferase activity and protein expression of these target genes were significantly downregulated in miR-1826-transfected BC cells (J82 and T24). The expression of miR-1826 was lower in BC tissues and inverse correlation of miR-1826 with several clinical parameters (pT, grade) was observed. Also the expression of miR-1826 was much lower in three BC cell lines (J82, T24 and TCCSUP) compared with a normal bladder cell line (SV-HUC-1). We then performed analyses to look at miR-1826 function and found that miR-1826 inhibited BC cell viability, invasion and migration. We also found increased apoptosis and G(1) cell cycle arrest in miR-1826-transfected BC cells. To examine whether the effect of miR-1826 was through CTNNB1 (beta-catenin) or MEK1 knockdown, we knocked down CTNNB1/MEK1 messenger RNA using a small interfering RNA (siRNA) technique. We observed that CTNNB1 or MEK1 siRNA knockdown resulted in effects similar to those with miR-1826 in BC cells. In conclusion, our data suggest that the miR-1826 plays an important role as tumor suppressor via CTNNB1/MEK1/VEGFC downregulation in BC.

Fig. 1.

Role of beta-catenin and the Ras-Raf-MEK-ERK cascade in BC cells and potential miRNAs targeting beta-catenin and MEK-ERK. (A) Signal transduction of beta-catenin and the MEK-ERK cascade (B) Based on miRDB and microRNA.org, several miRNAs targeting beta-catenin and the MEK-ERK cascade genes (MEK1/2, ERK1/2) were identified.

Fig. 2.

3′ UTR luciferase assay of MEK1, CTNNB1, VEGFC and correlation between miR-1826 and target protein expression in BC tissues. (A) MEK1, CTNNB1 and VEGFC 3′ UTR positions and complementary miR-1826-binding sequences. (B) 3′ UTR luciferase assay (miR-NC and miR-1826). (C) VEGFC, MEK1, CTNNB1, survivin and beta-tubulin protein expression in miR-NC-transfected or miR-1826-transfected BC cells (T24 and J82). (D) Inverse correlation between miR-1826 and MEK1/CTNNB1/VEGFC protein expression in BC tissues.

Fig. 3.

miR-1826 expression in bladder tissues and correlation with clinical parameters. (A) The percentage of positive miRNA-1826 expression was significantly lower in BC tissues. (B) Inverse correlation between miR-1826 expression and pathological T or tumor grade was found. (C) The miRNA-1826 expression level (normalized to RNU48) was significantly lower in BC tissues compared with normal tissues (P value = 0.02, n = 19) based on real-time PCR.

Fig. 4.

Effect of miR-1826 overexpression on BC cell functions (T24 and J82). (A) The miR-1826 expression level was significantly lower in BC cell lines (J82, T24 and TCCSUP) than in normal bladder cell lines (SV-HUC-1). (B) At 48 h after transfection of miR-NC or miR-1826 into BC cells (J82 and T24), the miR-1826 expression level was verified by real-time RT–PCR (fold change; 8265 and 5196, respectively). (C) Cell viability assay (miR-NC-transfected or miR-1826-transfected J82/T24 cells). (D) Invasion assay (miR-NC-transfected or miR-1826-transfected J82/T24 cells). (E) Wound healing assay (miR-NC-transfected or miR-1826-transfected J82/T24 cells). (F) Flow cytometry analysis of apoptosis in miR-NC-transfected or miR-1826-transfected J82/T24 cells. (G) Flow cytometry analysis of the cell cycle. Data are the mean ± SD of four independent experiments.

Fig. 5.

Effect of CTNNB1 and MEK1 siRNA knockdown on BC cell function (T24). (A) Protein expression of CTNNB1 and MEK1 in si-CTNNB1-transfected, si-MEK1-transfected or control (si-NC)-transfected T24 cells. (B) MTS assay (si-NC-transfected, si-CTNNB1-transfected or si-MEK1-transfected T24 cells). (C) Invasion assay. (D) Flow cytometry analysis of apoptosis in si-NC-transfected, si-CTNNB1-transfected or si-MEK1-transfected T24 cells. (E) Flow cytometry analysis of the cell cycle. Data are the mean ± SD of four independent experiments.