Silencing Trim59 inhibits invasion/migration and epithelial-to-mesenchymal transition via TGF-β/Smad2/3 signaling pathway in bladder cancer cells

Figures & data

Table 1 Patients’ characteristics

Characteristics	Bca (n=15)
Age (years)
Median (range)	60 (56–77)
Sex
Male	10
Female	5
Tumor stage
Ta–T1	6
T2–T4	9
Tumor grade
G1	7
G2–G3	8

Abbreviation: Bca, bladder cancer.

Figure 1 Trim59 is upregulated in Bca cell lines and tissues.

Notes: (A) The mRNA expression of Trim59 in Bca samples was highly regulated than the paired adjacent normal tissues. (B) Trim59 level in 5637 and T24 cell lines was relatively high compared with TCCSUP cells. (C) Trim59 protein level in Bca tissues was upregulated than that in the adjacent normal tissues. (D) Trim59 mRNA expression of T24 and 5637 cells with si-Trim59 transfection decreased significantly compared to NC or blank. The protein level of Trim59 in T24 and 5637 cells reduced significantly compared with NC or blank after si-Trim59 transfection. Data are presented as mean ± SD. *P<0.05 by independent-sample t-test.
Abbreviations: Bca, bladder cancer; NC, negative control; SD, standard deviation; si-Trim59, small interfering-Trim59.

Figure 2 Knockdown of Trim59 inhibits migration and invasion of Bca cell lines.

Notes: (A) Effect of Trim59 on wound healing of T24 and 5637 cells. The invasion cells in which Trim59 was downregulated were analyzed by a wound healing assay. (B) The decreased Trim59 expression suppressed the invasion and migration of T24 and 5637. The invasion of the treated cells was evaluated in an invasion assay using a transwell insert coated with Matrigel. Data are presented as mean ± SD. *P<0.05 compared with NC.
Abbreviations: Bca, bladder cancer; NC, negative control; SD, standard deviation; si-Trim59, small interfering-Trim59.

Figure 3 Trim59 inhibits cell apoptosis and promotes cell proliferation of Bca cell lines.

Notes: (A) Flow cytometry analysis showed that cell apoptosis rates increased significantly after the transfection of si-Trim59. (B) si-Trim59 transfection of T24 and NC cells did not contribute to the cell cycle arrest compared with NC. Knockdown of Trim59 did not increased G1 phase percentage of T24 and 5637 cells. (C) Cell proliferation by CCK-8 assay and colony formation experiments. The proliferation capacity of T24 and 5637 cells was significantly inhibited by the downregulation of Trim59. The colony number of T24 and 5637 cells with si-Trim59 transfection decreased significantly compared with NC.
Abbreviations: Bca, bladder cancer; CCK-8, cell counting kit-8; NC, negative control; SD, standard deviation; si-Trim59, small interfering-Trim59.

Figure 4 Effect of si-Trim59 on EMT and TGF-β/Smad2/3 signaling pathway of Bca cell lines.

Notes: (A) Knockdown of Trim59 blocks EMT and MMP-9 protein levels in Bca cells. Western blot analysis was used to detect the changes in EMT markers in NC group and si-Trim59 group cells. The reduction of N-cadherin, vimentin, and MMP-9 was observed in si-Trim59 group cells. Data are presented as mean ± SD. (B) Knockdown of Trim59 inhibits TGF-β/Smad2/3 signaling pathways. Western blot analysis was used to detect the changes of TGF-β signaling markers in NC and si-Trim59 group. The reduction of p-Smad2 and p-Smad3 was observed in si-Trim59 group cell markers in NC group and si-Trim59 group cells. The reduction of n-cadherin, vimentin, and MMP-9 was observed in si-Trim59 group cells.
Abbreviations: Bca, bladder cancer; EMT, epithelial-mesenchymal transition; MMP-9, matrix metallopeptidase-9; NC, negative control; p-Smad2, phosphorylated Smad2; SD, standard deviation; si-Trim59, small interfering-Trim59; TGF-β, transforming growth factor beta.

Figure 5 TGF-β promotes cell migration and invasion in Bca cell lines.

Notes: (A) TGF-β promotes migration and invasion of T24 cells. (B) TGF-β promotes migration and invasion of 5637 cells. Data are expressed as the mean ± SD.
Abbreviations: Bca, bladder cancer; NC, negative control; SD, standard deviation; si-Trim59, small interfering-Trim59; TGF-β, transforming growth factor beta.