The VIM-AS1/miR-655/ZEB1 axis modulates bladder cancer cell metastasis by regulating epithelial-mesenchymal transition

Abstract

Background: Invasive bladder tumors cause a worse prognosis in patients and remain a clinical challenge. Epithelial-mesenchymal transition (EMT) is associated with bladder cancer metastasis. In the present research, we attempted to demonstrate a novel mechanism by which a long noncoding RNA (lncRNA)-miRNA-mRNA axis regulates EMT and metastasis in bladder cancer.

Methods: Immunofluorescence (IF) staining was used to detect Vimentin expression. The protein expression of ZEB1, Vimentin, E-cadherin, and Snail was investigated by using immunoblotting assays. Transwell assays were performed to detect the invasive capacity of bladder cancer cells. A wound healing assay was used to measure the migratory capacity of bladder cancer cells.

Results: Herein, we identified lncRNA VIM-AS1 as a highly- expressed lncRNA in bladder cancer, especially in metastatic bladder cancer tissues and high-metastatic bladder cancer cell lines. By acting as a ceRNA for miR-655, VIM-AS1 competed with ZEB1 for miR-655 binding, therefore eliminating the miR-655-mediated suppression of ZEB1, finally promoting EMT in both high- and low-metastatic bladder cancer cells and enhancing cancer cell metastasis.

Conclusions: In conclusion, the VIM-AS1/miR-655/ZEB1 axis might be a promising target for improving bladder cancer metastasis via an EMT-related mechanism.

Keywords: Bladder cancer; Epithelial–mesenchymal transition (EMT); VIM-AS1; ZEB1; miR-655.

Fig. 1

LncRNA VIM-AS1 is upregulated in bladder cancer and is related tobladder cancer metastasis. a, b The expression of lncRNA VIM-AS1 was determined in 35 paired bladder cancer and noncancerous tissue samples, and analyzed in 15 nonmetastatic and 20 metastatic bladder cancer tissues. c Vimentin expression was determined in 35 paired bladder cancer and noncancerous tissue samples by RT-PCR. d The correlation of VIM-AS1 and Vimentin expression in tissue samples was analyzed using Pearson’s correlation analysis. e, f The expression of Ki67 and Vimentin in tissue samples were determined by IHC staining (×200). g The expression of VIM-AS1 and Vimentin was determined in a normal cell line, SV-HUC-1, three high-metastatic bladder cancer cell line (TCCSUP, HT-1376, and T24 and a low-metastatic bladder cancer cell line RT4 using real-time PCR. h, i T24 and RT24 cells were stimulated with 0, 2, 5 and 10 ng/ml TGFβ1 and examined for the expression of VIM-AS1 and Vimentin using real-time PCR. *P < 0.05, **P < 0.01

Fig. 2

LncRNA VIM-AS1 promotes bladder cancer cell invasion and migration. a, f VIM-AS1 was knocked down in the high-metastatic bladder cancer cell line T24 by transfection of si1-VIM-AS1 or si2-VIM-AS1 and VIM-AS1 was overexpressed in the low-metastatic bladder cancer cell line RT24 by transfection of the VIM-AS1-overexpressing vector. si-NC or NC vector was used as a negative control. Transfection efficiency was confirmed by real-time PCR. si1-VIM-AS1 was selected for further experiments due to its better transfection efficiency. b, g The protein content and distribution of Vimentin in T24 and RT24 cells were detected using immunofluorescence (IF) staining (×400). c, h The protein levels of Snail, E-cadherin and Vimentin in T24 and RT24 cells were determined by immunoblotting. d, i The invasive ability of T24 and RT24 cells was determined by Transwell assay. Scale bar = 100 µm. e, j The migratory ability of T24 and RT24 cells was determined by wound healing assay. Scale bar = 100 µm. *P < 0.05, **P < 0.01

Fig. 3

LncRNA VIM-AS1 competes with ZEB1 for miR-655 binding to modulate bladder cancer metastasis. a, b T24 and RT24 cells were stimulated with 0, 2, 5 and 10 ng/ml TGFβ1 and examined for the protein (upper) and mRNA (under) expression levels of ZEB1. c T24 cells were transfected with si-VIM-AS1 and RT24 cells were transfected with VIM-AS1 vector and examined for the protein (upper) and mRNA (under) levels of ZEB1. d The expression of miR-655 was determined in 35 paired bladder cancer and noncancerous tissue samples. e The expression of miR-655 was analyzed in 15 nonmetastatic and 20 metastatic bladder cancer tissues. f miR-655 was overexpressed or inhibited in T24 and RT24 cells by transfection of miR-655 mimics or miR-655 inhibitor, as confirmed by real-time PCR. g Wild-type and mutant ZEB1 3′UTR and VIM-AS1 luciferase reporter vectors were constructed as described. h The wt-VIM-AS1 or mut-VIM-AS1 vector were co-transfected into 293T cells with miR-655 mimics or miR-655 inhibitor and the luciferase activity was determined. i The wt-ZEB1 3′UTR or mut-ZEB1 3′UTR vector was co-transfected into 293T cells with si-VIM-AS1, and the luciferase activity was determined. j–l T24 cells were co-transfected with miR-655 inhibitor and si-VIM-AS1 and examined for the level of VIM-AS1 (j), miR-655 (k) and expression of ZEB1 protein and mRNA (l). m–o T24 cells were co-transfected with miR-655 mimics and VIM-AS1 vector and examined for the level of VIM-AS1 (m), miR-655 (n) and expression of ZEB1 protein and mRNA (O). *P < 0.05, **P < 0.01 compared to control, si-NC, NC, NC mimics, NC inhibitor, inhibitor NC + si-NC or mimics NC + vector group. ^##P < 0.01 compared to miR-655 inhibitor + si-NC or miR-655 mimics + vector group

Fig. 4

Dynamic effects of VIM-AS1 and miR-655 on bladder cancer metastasis. a, d T24 cells were co-transfected with si-VIM-AS1 and miR-655 inhibitor; RT24 cells were co-transfected with VIM-AS1 and miR-655 mimics; the protein levels of Vimentin, E-cadherin, and Snail were determined by immunoblotting. b, e The invasive ability of T24 and RT24 cells was determined by Transwell assay. Scale bar = 100 µm. c, f The migratory ability of T24 and RT24 cells was determined by wound healing assay. Scale bar = 100 µm. *P < 0.05, **P < 0.01 compared to inhibitor NC + si-NC or mimics NC + vector group. ^##P < 0.01 compared to miR-655 inhibitor + si-NC or miR-655 mimics + vector group

Fig. 5

Effects of VIM-AS1 and miR-655 on the growth of xenograft formed in nude mice. The stable T24 cells of Lv-negative control (NC), Lv-VIM-AS1, Lv-miR-655 and Lv-VIM-AS1 + miR-655 were subcutaneous injected into the armpit of nude mice respectively. a At the end of the xenograft formed experiment (the 25th day), mice were euthanized and tumor tissues were excised, the tumor volumes, weight and sizes in different experiment groups were detected. b IHC staining (×200) was applied to detect the protein expression of proliferation marker Ki67 and ZEB1 in tumor tissues from nude mice. c The VIM-AS1 and miR-655 expression levels in tumor tissues from nude mice were determined by real-time PCR assay. *P < 0.05, **P < 0.01 compared to Lv-NC group; ^##P < 0.01 compared to Lv-VIM-AS1 or Lv-miR-655 group

Fig. 6

Dynamic effects of VIM-AS1 and ZEB1 on bladder cancer metastasis. a–c T24 cells were cotransfected with si-VIM-AS1 and ZEB1 vector and the expression levels of VIM-AS1 (a), ZEB1 (b) and miR-655 (c) were detected by real-time PCR analysis. d, j The protein levels of Vimentin, E-cadherin, Snail and ZEB1 were determined by immunoblotting in T24 and RT24 cells. e, k The invasive ability of T24 and RT24 cells was determined by Transwell assay. Scale bar = 100 µm. f, l The migratory ability of T24 and RT24 cells was determined by wound healing assay. Scale bar = 100 µm. g–i RT24 cells were cotransfected with VIM-AS1 vector and si-ZEB1 and the expression levels of VIM-AS1 (g), ZEB1 (h) and miR-655 (i) were detected by real-time PCR assay. **P < 0.01 compared to si-NC + vector group. ^##P < 0.01 compared to si-NC + ZEB1 or vector + si-ZEB1 group

Fig. 7

A schematic diagram of the proposed mechanisms of VIM-AS1/miR-655-3P/ZEB1 axis in bladder cancer cells