LIMA1 inhibits cisplatin resistance and malignant biological behavior of bladder cancer cells by suppressing the Wnt/β-catenin pathway

Abstract

Objective: This study aimed to explore the effect of LIM domain and actin binding 1 (LIMA1) on bladder cancer (BCa) cells and to investigate its underlying molecular mechanisms.

Methods: The expression of LIMA1 gene in clinical BCa tissue samples and BCa cell models was detected using real-time quantitative PCR and western blot. Subsequently, LIMA1 knockdown experiments were performed exclusively in the BCa J82 cell line, while LIMA1 overexpression was conducted only in the cisplatin-resistant J82/CR cell line. The proliferation of the cells was assessed by colony formation assay. Cisplatin resistance was evaluated by MTT assay. Migration and invasion of the cells were tested by Transwell assay. Additionally, the levels of key proteins in the Wnt/β-catenin signaling pathway were examined by western blotting.

Results: We found that LIMA1 was underexpressed in BCa tissues and cells (P < 0.01). Overexpression of LIMA1 inhibited the proliferation, migration, invasion, and epithelial-mesenchymal transition of BCa cells (P < 0.01) and improved their cisplatin resistance (P < 0.01), whereas knocking down LIMA1 produced opposite results (P < 0.01). Furthermore, overexpression of LIMA1 could suppress the Wnt/β-catenin signaling pathway in BCa cells (P < 0.01), and activation of this pathway partially reversed the anti-tumor effects produced by overexpression of LIMA1 (P < 0.01).

Conclusion: LIMA1 could inhibit the malignant biological behavior of BCa cells and weaken their cisplatin resistance by negatively regulating the Wnt/β-catenin signaling pathway. Our findings provide new insights for the clinical treatment of BCa.

Keywords: Bladder cancer; Cisplatin resistance; LIMA1; Wnt/β-catenin signaling pathway.

Fig. 1

LIMA1 expression is downregulated in BCa tissues and cells. (A) MTT assay was used to detect cell survival of J82 and J82/CR cells treated with different concentration gradients of cisplatin solutions (0, 1, 2, 4, 8, 16 μM), and IC50 was calculated, **P < 0.01; (B) RT-qPCR was used to detect LIMA1 mRNA levels in tumour tissues and adjacent normal tissues, **P < 0.01; (C) RT-qPCR was used to detect LIMA1 mRNA levels in SV-HUC-1, J82 and J82/CR cells; (D) Western blot was used to detect LIMA1 protein levels in SV-HUC-1, J82 and J82/CR cells; N = 3; *P < 0.05 and **P < 0.01 vs. SV-HUC-1 group; #P < 0.05 and ##P < 0.01, vs. J82 group

Fig. 2

LIMA1 overexpression inhibits proliferation and improves cisplatin resistance in BCa cells. A. RT-qPCR was used to detect LIMA1 mRNA levels in J82 cells of siRNA and si-LIMA1 groups; B. Western blot was used to detect LIMA1 protein levels in J82 cells of siRNA and si-LIMA1 groups; C. Colony formation assay was used to measure the proliferation of J82 cells of siRNA and si-LIMA1 groups; D. Colony formation assay was used to measure the proliferation ability of J82/CR cells in vector and LIMA1 groups; E. MTT assay was used to detect the cell survival rate of J82 cells in siRNA and si-LIMA1 groups treated with different concentration gradients of cisplatin solutions (0, 1, 2, 4, 8, and 16 μM), and the IC50 was calculated; F. MTT assay was used to detect the cell survival rate of J82/CR cells in vector and LIMA1 groups treated with different concentration gradients of cisplatin solutions (0, 1, 2, 4, 8, 16 μM), and IC50 was calculated. N = 3; **P < 0.01 vs. siRNA group; ##P < 0.01 vs. vector group

Fig. 3

LIMA1 overexpression inhibits the migration and invasion ability of BCa cells. (A) Transwell assay was used to detect the migration ability of J82 cells in siRNA and si-LIMA1 groups; (B) Transwell assay was used to detect the migration ability of J82/CR cells in vector and LIMA1 groups; (C) Transwell assay was used to detect the invasion ability of J82 cells in siRNA and si-LIMA1 groups; (D) Transwell assay was used to detect the invasion ability of J82/CR cells in vector and LIMA1 groups; (E) Western blot assay was used to detect protein expression levels of E-cadherin, N-cadherin, SNail in J82 cells of siRNA and si-LIMA1 groups, and quantitative analysis of the related protein levels was conducted; (F) Western blot assay was used to detect protein expression levels of E-cadherin, N-cadherin, Snail in J82/CR cells of vector and LIMA1 groups, and quantitative analysis of related protein levels was conducted. N = 3; **P < 0.01 vs. siRNA group; ##P < 0.01 vs. vector group

Fig. 4

LIMA1 overexpression downregulates the Wnt/β-catenin signalling pathway in BCa cells. A. Western blot was used to detect protein expression levels of Wnt3a, β-catenin, and C-myc in J82 cells of siRNA and si-LIMA1 groups, with representative bands provided; B. Results of statistical analysis of Wnt3a, β-catenin, and C-myc protein levels in J82 cells of siRNA and si-LIMA1 groups; C. Western blot was used to detect protein expression levels of Wnt3a, β-catenin, and C-myc in J82/CR cells of vector and LIMA1 groups, with representative bands provided; D. Results of statistical analysis of Wnt3a, β-catenin, and C-myc protein levels in J82/CR cells of vectora and LIMA1 groups. N = 3; **P < 0.01 vs. siRNA group; ##P < 0.01 vs. vector group

Fig. 5

Activation of Wnt/β-catenin pathway partially counteracts the effect of LIMA1 on BCa cells. (A) MTT assay was used to detect the cell survival rate of J82/CR cells in vector group, LIMA1 group, LiCl group, and LIMA1 + LiCl group under cisplatin treatment, and IC50 was calculated; (B) Colony formation assay was used to detect the proliferation ability of J82/CR cells in vector group, LIMA1 group, LiCl group, and LIMA1 + LiCl group; (C) Transwell assay was used to detect the migration ability of J82/CR cells in vector group, LIMA1 group, LiCl group, and LIMA1 + LiCl group; (D) Transwell assay was used to detect the invasion ability of J82/CR cells in vector group, LIMA1 group, LiCl group, and LIMA1 + LiCl group; (E) Western blot assay was used to detect protein levels of E-cadherin, N-cadherin, and Snail in J82/CR cells of vector group, LIMA1 group, LiCl group, and LIMA1 + LiCl group, with representative bands provided; (F) Results of statistical analysis of protein levels of E-cadherin, N-cadherin, and Snail in J82/CR cells of vector group, LIMA1 group, LiCl group, and LIMA1 + LiCl group. N = 3; **P < 0.01 vs. vector group; ##P < 0.01 vs. LIMA1 group