Inhibition of SND1 overcomes chemoresistance in bladder cancer cells by promoting ferroptosis

Abstract

Chemotherapy remains one of the most important adjuvant treatments for bladder cancer (BC). However, similar to other malignancies, BC is prone to chemotherapy resistance and only approximately half of muscle‑invasive patients with BC respond to chemotherapy. The present study aimed to reveal the mechanisms underlying chemoresistance in BC cells. Cell viabilities were assessed by CCK‑8 assay. The differentiated expression of genes in chemoresistant and their parental BC cells were examined by RNA sequencing. Cell death was determined by flow cytometry. Different cell death inhibitors were used to determine the types of cell death. Levels of reactive oxygen species, iron, glutathione and malondialdehyde were assessed using the corresponding commercial kits. ChIP and dual luciferase activity assays were performed to investigate the interaction between staphylococcal nuclease and tumour domain containing 1 (SND1) and glutathione peroxidase 4 (GPX4) mRNA. RNAi was used to knockdown SND1 or GPX4. The results revealed that SND1 in BC cells were insensitive to cisplatin, and inhibition of SND1 overcame this resistance. Silencing of SND1 enhanced cell death induced by cisplatin by promoting ferroptosis in BC cells. Mechanistically, SND1 was revealed to bind to the 3'UTR region of GPX4 mRNA and stabilise it. Knockdown of GPX4 could also overcome chemoresistance, and overexpressing GPX4 reversed the effects of silencing of GPX4 on the chemosensitivity of BC cells. Thus, targeting the SND1‑GPX4 axis may be a potential strategy to overcome chemoresistance in BC cells.

Keywords: bladder cancer; chemoresistance; ferroptosis; glutathione peroxidase 4; staphylococcal nuclease and tumour domain containing 1.

Figure 1.

Silencing of SND1 increases the sensitivity of BC cells to cisplatin. (A) The IC₅₀ values of the response of BC cells to cisplatin. (B) Treatment of T24/R, T24, 5637/R and 5637 cells with various concentrations of cisplatin for 24 h, and assessment of their cellular viabilities. (C) RNA sequencing analysis of gene expression in T24/R and T24 cells. (D) mRNA levels of SND1 were assessed by reverse transcription-quantitative polymerase chain reaction in T24, T24/R, 5637 and 5637/R cells. (E) Western blotting was used to assess the protein levels of SND1. (F) mRNA levels of SND1 in various BC cells. (G) Protein levels of SND1 in various BC cells. (H) T24/R and 5637/R cells were transfected as indicated, and the mRNA levels of SND1 were assessed. (I) T24/R and 5637/R cells were transfected as indicated, and protein levels of SND1 were determined. (J) T24/R and 5637/R cells were transfected as indicated, and treated with various concentrations of cisplatin for 24 h, and their cellular viabilities were assessed. Data are presented as the mean ± standard deviation. **P<0.01. SND1, staphylococcal nuclease and tudor domain containing 1; BC, bladder cancer; sh, short hairpin RNA; NC, negative control.

Figure 2.

Silencing of SND1 overcomes the resistance to cisplatin by promoting ferroptosis. (A) T24/R and 5637/R cells were transfected as indicated, and treated with or without cisplatin for 24 h, and cellular death was then assessed. (B) T24/R and 5637/R cells were treated as indicated for 24 h, and the cellular death was assessed. (C) T24/R and 5637/R cells were treated as indicated for 24 h, and the iron levels were determined. (D) T24/R and 5637/R cells were treated as indicated for 12 h, and the reactive oxygen species levels were assessed. (E) T24/R and 5637/R cells were treated as indicated for 24 h, and the glutathione levels were determined. (F) T24/R and 5637/R cells were treated as indicated for 24 h, and the MDA levels were assessed. Data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01. SND1, staphylococcal nuclease and tudor domain containing 1; sh, short hairpin RNA; NC, negative control; ROS, reactive oxygen species; GSH, glutathione; MDA, malondialdehyde.

Figure 3.

SND1 directly binds to the 3′UTR of GPX4 mRNA and stabilises it. (A) T24/R and 5637/R cells were transfected as indicated, and the mRNA levels of indicated genes were determined. (B) The protein levels of GPX4 were assessed in T24, T24/R, 5637 and 5637/R cells. (C) T24/R and 5637R cells were transfected as indicated, and the protein levels of GPX4 were assessed. (D) T24/R and 5637/R cells were subjected to immunoprecipitation with SND1 antibody or control IgG and GAPDH followed by immunoblotting analysis (left). Reverse transcription-quantitative polymerase chain reaction analysis of the relative enrichment of GPX4 mRNA in SND1-RNA binding complexes, using anti-IgG as a negative control (right). (E) T24 and 5637 cells were transfected as indicated, and the protein levels of SND1 were measured. (F) 5′UTR, 3′UTR and CDS of GPX4 were cloned into a luciferase reporter vector and co-transfected with a vector that expressed SND1 in the T24 and 5637 cells, and the relative luciferase activities were measured. (G) Wild-type or truncated 3′UTR sequences (Mut 1, Mut 2) of GPX4 3′UTR were co-transfected with SND1-expressing vector into T24 and 5637 cells, and the relative luciferase activities were measured. (H) GPX4 mRNA abundance in shNC or shSND1_1 or shSND1_2 transfected cells after actinomycin D (2.5 µg/ml) administration at different time-points (10 and 24 h). Data are presented as the mean ± standard deviation. **P<0.01. SND1, staphylococcal nuclease and tudor domain containing 1; GPX4, glutathione peroxidase 4; CDS, coding sequence; sh, short hairpin RNA; NC, negative control; Ctrl, control; EV, empty vector; OV, overexpression.

Figure 4.

Knockdown of GPX4 overcomes the resistance to cisplatin in BC cells. (A) T24/R and 5637/R cells were transfected as indicated, and the GPX4 mRNA levels were assessed. (B) T24/R and 5637/R cells were transfected as indicated, and the protein levels of GPX4 were determined. (C) T24/R and 5637/R cells were transfected as indicated, and the cells were treated with the indicated concentrations of cisplatin for 24 h, and the cellular viabilities were assessed. (D) T24/R and 5637/R cells were treated as indicated, and cellular death was detected. (E) T24/R and 5637/R cells were treated as indicated, and the reactive oxygen species levels were assessed. (F) T24/R and 5637/R cells were treated as indicated, and the iron levels were determined. (G) T24/R and 5637/R cells were treated as indicated, and the glutathione levels were detected. (H) T24/R and 5637/R cells were treated as indicated, and lipid peroxidation levels were assessed. Data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01. GPX4, glutathione peroxidase 4; BC, bladder cancer; sh, short hairpin RNA; NC, negative control; ROS, reactive oxygen species; GSH, glutathione; MDA, malondialdehyde.

Figure 5.

Overexpression of GPX4 reverses the effects of silencing of SND1 on the sensitivity of BC cells to cisplatin. (A) T24/R and 5637/R cells were transfected as indicated for 24 h, and the mRNA levels of GPX4 were determined. (B) The protein levels of GPX4 were assessed. (C) T24/R and 5637/R cells were treated as indicated, and the cellular viabilities were detected. (D) Cellular death was assessed. (E) The reactive oxygen species levels were determined. (F) Th iron levels were detected. (G) The glutathione levels were assessed. (H) Malondialdehyde levels were determined. Data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01. GPX4, glutathione peroxidase 4; SND1, staphylococcal nuclease and tudor domain containing 1; BC, bladder cancer; sh, short hairpin RNA; NC, negative control; EV, empty vector; OV, overexpression; ROS, reactive oxygen species; GSH, glutathione; MDA, malondialdehyde.