Downregulation of long noncoding RNA TUG1 inhibits proliferation and induces apoptosis through the TUG1/miR-142/ZEB2 axis in bladder cancer cells

Figures & data

Figure 1 Expression levels of TUG1 and ZEB2 in bladder cancer tissues (n=36) and corresponding nontumor tissues (n=36).

Notes: (A) TUG1 was significantly upregulated in bladder cancer tissues. (B and C) The mRNA and protein expression levels of ZEB2 were significantly upregulated in bladder cancer tissues. (D) The positive correlation between TUG1 and ZEB2 mRNA in bladder cancer tissues (r=0.5707, P=0.0029). *P<0.05.
Abbreviations: mRNA, messenger RNA; N, adjacent normal tissues; T, tumor tissues; TUG1, taurine upregulated gene 1; ZEB2, zinc finger E-box binding homeobox 2.

Figure 2 Knockdown of TUG1 suppressed bladder cancer cell proliferation and induced apoptosis in vitro.

Notes: (A) The expression levels of TUG1 in HCV-29, T24, and BIU-87 cells were detected by qRT-PCR. (B and C) MTT assay was used to detected cell viability of T24 and BIU-87 cells transfected with si-TUG1 at the indicated time points. (D–G) Flow cytometry was used to detect apoptosis of T24 and BIU-87 cells transfected with si-TUG1 at 48 h. *P<0.05.
Abbreviations: FITC, fluorescein isothiocyanate; NC, negative control; OD, optical density; PI, propidium iodide; si, small interfering; TUG1, taurine upregulated gene 1.

Figure 3 Knockdown of ZEB2 suppressed bladder cancer cell proliferation and induced apoptosis in vitro.

Notes: (A, B) The mRNA and protein expression levels of ZEB2 in HCV-29, T24, and BIU-87 cells were detected by qRT-PCR and Western blot, respectively. (C) The expression levels of ZEB2 in T24 and BIU-87 cells 48 h after transfection with si-ZEB2 or pcDNA-ZEB2. (D–G)1 MTT assay was used to detect cell viability of T24 and BIU-87 cells transfected with si-ZEB2 or pcDNA-ZEB2 at the indicated time points. (H and I) Flow cytometry was used to evaluate apoptosis of T24 and BIU-87 cells transfected with si-ZEB2 at 48 h. *P<0.05.
Abbreviations: mRNA, messenger RNA; NC, negative control; OD, optical density; si, small interfering; ZEB2, zinc finger E-box binding homeobox 2.

Figure 4 ZEB2 overexpression reversed effects of TUG1 knockdown on proliferation and apoptosis of bladder cancer cells.

Notes: (A and C) Western blot analyses showed the expression of ZEB2 was effectively upregulated by pcDNA-TUG1 in T24 and BIU-87 cells. (B and D) Western blot analyses indicated that the expression of ZEB2 was effectively downregulated by si-TUG1 in T24 and BIU-87 cells. (E and F) The viability of T24 and BIU-87 cells transfected with si-TUG1 or si-TUG1 + pcDNA-ZEB2 was detected at the indicated time points by MTT. (G and H) Flow cytometry was used to measure apoptosis of T24 and BIU-87 cells transfected with si-TUG1 or si-TUG1 + pcDNA-ZEB2 at 48 h. *P<0.05.
Abbreviations: NC, negative control; OD, optical density; si, small interfering; TUG1, taurine upregulated gene 1; ZEB2, zinc finger E-box binding homeobox 2.

Figure 5 TUG1 targeted miR-142 to regulate ZEB2 expression.

Notes: (A) The predicted binding sites of miR-142 in TUG1. (B and C) The luciferase activity in T24 and BIU-87 cells simultaneously transfected with miR-142 and TUG1-WT was significantly reduced, whereas cotransfection of miR-142 and TUG1-MUT did not change the luciferase activity. (D and E) The expression of miR-142 in T24 and BIU-87 cells treated with si-TUG1 or pcDNA-TUG1 was detected by qRT-PCR. (F) The predicted binding sites of miR-142 in 3′-UTR of ZEB2. (G and H) The 3′-UTR of wild type ZEB2 (WT) or 3′-UTR of mutant ZEB2 (MUT) reporter plasmids was co-transfected into T24 or BIU-87 cells with miR-control, miR-142, miR-142 + pcDNA-control, or miR-142 + pcDNA-TUG1. The luciferase activity was detected 36 h posttransfection. (I and J) The expression of ZEB2 in T24 and BIU-87 cells treated with miR-142 or miR-142 + pcDNA-TUG1 was detected by Western blot analysis. *P<0.05.
Abbreviations: MUT, mutant; NC, negative control; qRT-PCR, quantitative real time polymerase chain reaction; si, small interfering; TUG1, taurine upregulated gene 1; ZEB2, zinc finger E-box binding homeobox 2; WT, wild type.

Figure 6 TUG1 regulated the Wnt/β-catenin pathway by affecting ZEB2.

Notes: (A and B) Protein expressions of β-catenin, c-Myc, and cyclinD1 were evaluated at 48 h in T24 cells transfected with pcDNA-TUG1 or pcDNA-TUG1 + si-ZEB2 by Western blot analysis. (C and D) Protein expressions of β-catenin, c-myc, and cyclinD1 were measured at 48 h in T24 cells transfected with si-TUG1 and si-TUG1 + pcDNA-ZEB2 by Western blot analysis. *P<0.05.
Abbreviations: si, small interfering; TUG1, taurine upregulated gene 1; ZEB2, zinc finger E-box binding homeobox 2.