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. 2018 Mar;22(3):1650-1665.
doi: 10.1111/jcmm.13442. Epub 2018 Jan 19.

GP73 Promotes Invasion and Metastasis of Bladder Cancer by Regulating the Epithelial-Mesenchymal Transition Through the TGF-β1/Smad2 Signalling Pathway

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Free PMC article

GP73 Promotes Invasion and Metastasis of Bladder Cancer by Regulating the Epithelial-Mesenchymal Transition Through the TGF-β1/Smad2 Signalling Pathway

Han-Jie Yang et al. J Cell Mol Med. .
Free PMC article

Abstract

This study investigated the effects of Golgi membrane protein 73 (GP73) on the epithelial-mesenchymal transition (EMT) and on bladder cancer cell invasion and metastasis through the TGF-β1/Smad2 signalling pathway. Paired bladder cancer and adjacent tissue samples (102) and normal bladder tissue samples (106) were obtained. Bladder cancer cell lines (T24, 5637, RT4, 253J and J82) were selected and assigned to blank, negative control (NC), TGF-β, thrombospondin-1 (TSP-1), TGF-β1+ TSP-1, GP73-siRNA-1, GP73-siRNA-2, GP73-siRNA-1+ TSP-1, GP73-siRNA-1+ pcDNA-GP73, WT1-siRNA and WT1-siRNA + GP73-siRNA-1 groups. Expressions of GP73, TGF-β1, Smad2, p-Smad2, E-cadherin and vimentin were detected using RT-qPCR and Western blotting. Cell proliferation, migration and invasion were determined using MTT assay, scratch testing and Transwell assay, respectively. Compared with the blank and NC groups, levels of GP73, TGF-β1, Smad2, p-Smad2, N-cadherin and vimentin decreased, and levels of WT1 and E-cadherin increased in the GP73-siRNA-1 and GP73-siRNA-2 groups, while the opposite results were observed in the WT1 siRNA, TGF-β, TSP-1 and TGF-β + TSP-1 groups. Cell proliferation, migration and invasion notably decreased in the GP73-siRNA-1 and GP73-siRNA-2 groups in comparison with the blank and NC groups, while in the WT1 siRNA, TGF-β, TSP-1 and TGF-β + TSP-1 groups, cell migration, invasion and proliferation showed the reduction after the EMT. These results suggest that GP73 promotes bladder cancer invasion and metastasis by inducing the EMT through down-regulating WT1 levels and activating the TGF-β1/Smad2 signalling pathway.

Keywords: Golgi membrane protein 73; TGF-β1/Smad2 signalling pathway; bladder cancer; epithelial-mesenchymal transition; invasion; metastasis.

Figures

Figure 1
Figure 1
Protein levels of GP73, TGF‐β1, Smad2, E‐cadherin and vimentin in bladder cancer tissues and in adjacent and normal bladder tissues detected by IHC assays (× 400). Note: GP73, Golgi membrane protein 73; TGF‐β1, transforming growth factor‐β1; IHC, immunohistochemistry.
Figure 2
Figure 2
Expression levels of GP73, E‐cadherin, N‐cadherin and vimentin in T24, 5637, RT4, 253J and J82 cells. Note: (A) mRNA expression for GP73, E‐cadherin, N‐cadherin and vimentin was detected by qRTpPCR; (B) protein expression of GP73, E‐cadherin, N‐cadherin and vimentin was detected by Western blotting; GP73, Golgi membrane protein 73; RTqPCR, reverse transcription quantitative polymerase chain reaction.
Figure 3
Figure 3
Protein levels of WT1 24 and 48 hrs after transfection of siRNA‐1, ‐2 and ‐3 against WT1, determined using Western blotting. Note: A, the protein levels of WT1; B, grey values of WT1 protein bands; 1, NC siRNA; 2, siRNA‐1 against WT1; 3, siRNA‐2 against WT1; 4, siRNA‐3 against WT1; WT1, Wilms' tumour gene 1; NC, negative control.
Figure 4
Figure 4
mRNA levels of GP73, TGF‐β1, Smad2, E‐cadherin, N‐cadherin and vimentin in 5637 and 253J cells 48 hrs after transfection. Note: (A) GP73 mRNA level in 5637 cells; (B) GP73 mRNA level in 253J cells; (C), mRNA levels of TGF‐β1, Smad2 and WT1 in 5637 cells; (D) mRNA levels of TGF‐β1, Smad2 and WT1 in 253J cells; (E) mRNA levels of vimentin, N‐cadherin and E‐cadherin in 5637 cells; (F), mRNA levels of vimentin, N‐cadherin and E‐cadherin in 253J cells; *, P < 0.05, compared with the blank and NC groups; #, P < 0.05, compared with the GP73‐siRNA‐1 group; NC, negative control; TSP‐1, thrombospondin‐1; GP73, Golgi membrane protein 73; WT1, Wilms' tumour gene 1; TGF‐β1, transforming growth factor‐β1.
Figure 5
Figure 5
Protein levels of GP73, WT1, TGF‐β1, Smad2, p‐Smad2, E‐cadherin, N‐cadherin and vimentin in 5637 and 253J cells 48 hrs after transfection. Note: (A) protein levels of GP73 and WT1 in 5637 cells; (B) protein levels of GP73 and WT1 in 253J cells; (C) protein levels of TGF‐β1, Smad2 and p‐Smad2 in 5637 cells; (D) protein levels of TGF‐β1, Smad2 and p‐Smad2 in 253J cells; (E) protein levels of vimentin, N‐cadherin and E‐cadherin in 5637 cells; (F), protein levels of vimentin, N‐cadherin and E‐cadherin in 253J cells; (G), protein band pattern of 5637 cells; (H), protein band pattern of 253J cells; *, P < 0.05, compared with the blank and NC groups; #, P < 0.05, compared with the GP73‐siRNA‐1 group; NC, negative control; TSP‐1, thrombospondin‐1; GP73, Golgi membrane protein 73; WT1, Wilms' tumour gene 1; TGF‐β1, transforming growth factor‐β1.
Figure 6
Figure 6
Proliferation of 5637 and 253J cells determined by MTT assay 48 hrs after transfection. Note: (A) cell growth curve for 5637 cells; (B) cell growth curve for 253J cells; *, P < 0.05 compared with the blank and negative control groups; #, P < 0.05 compared with the GP73‐siRNA‐1 group; NC, negative control; TSP‐1, thrombospondin‐1; GP73, Golgi membrane protein 73; MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide; TGF‐β, transforming growth factor‐β; OD, optical density.
Figure 7
Figure 7
Migration of 5637 and 253J cells detected by scratch tests 48 hrs after transfection (× 40). Note: (A) scratch test images of 5637 and 253J cells at 48 hrs after transfection; (B) statistical results for scratch healing rates of 5637 and 253J cells 48 hrs after transfection; *, P < 0.05 compared with the blank and NC groups; #, P < 0.05 compared with the GP73‐siRNA‐1 group; NC, negative control; TSP‐1, thrombospondin‐1; GP73, Golgi membrane protein 73; TGF‐β, transforming growth factor‐β.
Figure 8
Figure 8
Invasion of 5637 and 253J cells detected by Matrigel assays 48 hrs after transfection (× 200). Note: (A) invasion images of 5637 and 253J cells 48 hrs after transfection; (B) statistical results for the number of invasive 5637 and 253J cells 48 hrs after transfection; *, P < 0.05, compared with the blank and NC groups; #, P < 0.05, compared with the GP73‐siRNA‐1 group; NC, negative control; TSP‐1, thrombospondin‐1; GP73, Golgi membrane protein 73; TGF‐β, transforming growth factor‐β.

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