MiR-429 reverses epithelial-mesenchymal transition by restoring E-cadherin expression in bladder cancer

Abstract

Epithelial-mesenchymal transition (EMT) accompanying loss of E-cadherin is important for invasiveness and metastasis of bladder cancer. MicroRNAs (miRs) had been associated with cancer progression and differentiation in several cancers. Our goal is to find out the specific miR which modulates EMT in bladder cancer. Real-time quantitative polymerase chain reaction was used to measure the miRs expression in urothelial cell carcinoma (UCC) cell lines. MiR or siRNA mimics was used to regulate miR and mRNA level respectively. Migration and scratch assays were used to determine the migratory ability. Zymography assay was used to confirm the metalloproteinase activity. Western blotting was used to elucidate the mechanism which regulated by specific miR. MiR-429 was highly expressed in low grade UCC cell lines. Exogenous mimic of miR-429 treatment dramatically inhibited the migratory ability of T24 cells. MiR-429 downstream target ZEB1 was decreased, E-cadherin was restored, and β-catenin was contrarily decreased by exogenous mimic of miR-429 treatment in T24 cells. Cell invasive ability was also inhibited by exogenous mimic of miR-429 treatment through inactivating the MMP-2 activity in T24 cells. E-cadherin protein expression level was inhibited by E-cadherin siRNA accompanied with increasing cell migratory ability when compared with control group in low grade TSGH8301 cells. MiR-429 decreased the cell migratory and invasive abilities through reducing ZEB1 and β-catenin, restoring the E-cadherin expression and inactivation of MMP-2 of UCC cells. MiR-429 might be used as a progression marker of bladder cancer.

Keywords: E-cadherin; bladder cancer; epithelial-mesenchymal transition; microRNA-429; urothelial cell carcinoma.

Figure 1. Different miR-429 and E-cadherin expression in UCC cell lines

(A) Relative miR-429 level. Using U6 as a loading control, data was quantified with 2^−ΔΔCT method and T24 cell was used as reference group. (B) Western blotting and relative E-cadherin level (E-cadherin/GAPDH, 100X) in high grade T24 and TSGH2010 and low grade TSGH8301 and TSGH9202 UCC cell lines, statistical analysis was showed as histogram graph (Student t-test, *p < 0.05; **p < 0.01; ***p < 0.001, Error bars = SD).

Figure 2. Exogenous miR-429 regulates migratory ability of T24 cells

Migration assay of observed remained area of (A) control 0 hour, (B) control 48 hours, (C) 4 nM scrambled control 48 hours, (D) 4 nM mimic of miR-429 48 hours in T24 cells and (E) statistical analysis was showed as histogram graph (Student t-test, *p < 0.05, Error bars = SD).

Figure 3. MiR-429 modifies E-cadherin expression in UCC cell line T24 through ZEB1-β-catenin axis in T24 cells

Western blotting of E-cadherin, ZEB1, ZEB2, and beta-catenin was performed in three groups, negative control (NC), 4 nM scrambled miRNA control (SC), and 4 nM mimic of miR-429 (M) in T24 cells. GAPDH was used as loading control. Statistical analysis was showed as histogram graph histogram graph (Student t-test, *p < 0.05; ***p < 0.001, Error bars = SD).

Figure 4. Invasive ability and MMP-2 activity are inhibited by exogenous miR-429 in T24 cells

Invasion assay of (A) control 0 hour, (B) control 64 hours, (C) 4 nM scrambled control 64 hours, (D) 4 nM mimic of miR-429 64 hours in T24 cells and (E) statistical analysis was showed as histogram graph. (F) Zymography assay was performed in three groups, negative control (NC), 4 nM scrambled miRNA control (SC), and 4 nM mimic of miR-429 (M) from left to right lane in T24 cells and statistical analysis was showed as histogram graph (Student t-test, *p < 0.05; ***p < 0.001, Error bars = SD).

Figure 5. E-cadherin knockdown by siRNA in TSGH8301 cells

Western blotting and histogram graph of E-cadherin. Histogram graph was performed in five groups, negative control (NC), 5 nM scrambled control (SC5), 25 nM scrambled control (SC25), 5 nM siRNA of E-cadherin (SI5), and 25 nM siRNA of E-cadherin (SI25) and GAPDH was used as loading control (Student t-test, *p < 0.05, Error bars = SD).

Figure 6. E-cadherin knockdown reduces migratory ability of TSGH8301 cells

Scratch assay of observed remained diameter of (A) control 0 hour, (B) negative control (NC) 16 hours, (C) 5 nM scrambled control (SC5) 16 hours, (D) 25 nM scrambled control (SC25) 16 hours (E) 5 nM siRNA of E-cadherin (SI5) 16 hours (F) 25 nM siRNA of E-cadherin (SI25) 16 hours in TSGH8301 cells and (G) statistical analysis was showed as histogram graph (Student t-test, *p < 0.05, Error bars = SD).

Figure 7. Schematic diagram of miR-429 in UCC cells

Schematic diagram of miR-429 reduces ZEB1 and restores E-cadherin which results in downregulation of β-catenin and therefore inhibits cell migratory and invasive ability in bladder cancer cell.